MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'BJ Structure' source. Mon, 06 Feb 2012 13:31:36 +0100 http://www.medworm.com/index.php?rid=5635194&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20112210 We report the structures of the inactive-glutaminase C176A variant in an apo form and in three synthetase ligand complexes with substrates (NaAD+/ATP), substrate analog (NaAD+/AMPCPP) and intermediate analogs (NaAD+/AMP/PPi), as well as the structure of wild type mtuNadE in a products complex (NAD+/AMP/PPi/Glu). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: 1) at the entrance near the glutaminase active site, 2) in the middle of the tunnel and 3) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5635194 Thu, 26 Jan 2012 05:00:00 +0100 5635194 http://www.medworm.com/index.php?rid=5592693&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111478 In this study, we describe a novel structural arrangement of the cystathionine beta-synthase (CBS) enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyze CBS reactions, as demonstrated by analysis of truncated variants and active site-mutant proteins. In contrast to other metazoan enzymes, CBS-1 lacks the heme and the regulatory Bateman domain essential for activation by S-adenosylmethionine and only forms monomers. We determined the tissue and subcellula... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5592693 Fri, 13 Jan 2012 05:00:00 +0100 5592693 http://www.medworm.com/index.php?rid=5568550&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111509 Alkylglycerol monooxygenase (E.C. 1.14.16.5) forms a third, distinct class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a diiron centre, which comprises eight conserved histidines. Membrane enzymes containing this motif, including alkylglycerol monooxygenase, are especially labile and could not be purified to homogeneity in active form so far. To get a first insight on structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild type and mutant proteins containing a C-terminal myc tag together with fatty aldehyde dehydrogenase in Chinese hamster ov... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5568550 Fri, 06 Jan 2012 05:00:00 +0100 5568550 http://www.medworm.com/index.php?rid=5500912&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111885 Methylation of H4K20 plays an important role in the regulation of diverse cellular processes. In fission yeast, all the three states of H4K20 methylation are catalyzed by Set9. Pdp1 is a PWWP domain-containing protein, which associates with Set9 to regulate its chromatin localization and methyltransferase activity towards H4K20. The structure of Pdp1 PWWP domain, which is the first identified PWWP domain that binds to methyl-lysine at H4K20 site, was determined by solution NMR in the present study. Pdp1 PWWP domain adopts a classical PWWP fold, with a 5-strand antiparallel β-barrel followed by three α-helices. However, it differs significantly from other PWWP domains in some structural aspects that account in part for its molecular recognition. Moreover, we revealed a unique ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5500912 Tue, 13 Dec 2011 05:00:00 +0100 5500912 http://www.medworm.com/index.php?rid=5473459&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111317 Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumor progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumors show this characteristic. To enable improved future patient treatment, we developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody hH35 potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody bi... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5473459 Fri, 02 Dec 2011 05:00:00 +0100 5473459 http://www.medworm.com/index.php?rid=5464996&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111598 UDP-glucose pyrophosphorylase (UGPase) is highly conserved among eukaryotes. UGPase reversely catalyzes the formation of UDP-glucose and is critical in carbohydrate metabolism. Previous studies have mainly focused on the UGPases from plants, fungi and parasites and indicate that the regulatory mechanisms responsible for the enzyme activity vary among different organisms. Here the crystal structure of human UGPase (hUGPase) was determined and shown to form octamers through end-to-end and side-by-side interactions. The observed latch loop in hUGPase distinctly differs from yeast UGPase (yUGPase), which could explain why hUGPase and yUGPase possess different enzymatic activities. Mutagenesis studies showed that both dissociation of octamers and mutations of the latch loop can significantly af... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5464996 Thu, 01 Dec 2011 05:00:00 +0100 5464996 http://www.medworm.com/index.php?rid=5447460&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111089 In spite of its biomedical relevance, polyproline recognition is still not fully understood. The disagreement between the current description of SH3 complexes and their thermodynamic behavior calls for a revision of the SH3 binding paradigm. Recently, Abl-SH3 was demonstrated to recognize its ligands by a dual binding mechanism involving a robust network of water-mediated hydrogen bonds that complements the canonical hydrophobic interactions. The systematic analysis of the SH3 structural database presented here reveals that this dual binding mode is universal to SH3 domains. Tightly bound, buried interfacial water molecules were found in all SH3 complexes studied mediating the interaction between the peptide ligand and the domain. Moreover, structural waters were also identified in a high ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5447460 Thu, 24 Nov 2011 05:00:00 +0100 5447460 http://www.medworm.com/index.php?rid=5417169&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111747 In this study we have obtained the 41 transcripts encoding proteins by RT-PCR using RNA template from Drosophila S2 cells, an embryonic cell line. This observation suggests that all the annotated DmGSTs in the proteome are expressed in the late embryonic stages of Drosophila melanogaster. To avoid confusion in naming these numerous DmGSTs, we have designated them following the universal GST nomenclature as well as previous designations that fit within this classification. Furthermore, in the cell line, we identified an apparent processed pseudogene, gste8; in addition to 2 isoforms from Delta class that have been previously published. Only about one third of the expressed DmGSTs could be purified by conventional GSH affinity chromatography. The diverse kinetic properties as well as physiol... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5417169 Tue, 15 Nov 2011 05:00:00 +0100 5417169 http://www.medworm.com/index.php?rid=5374775&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110721 Glucokinase is an enzyme central to glucose metabolism that displays positive cooperativity to substrate glucose. Small molecule glucokinase activators (GKAs) modulate GK catalytic activity and glucose affinity and are currently being pursued as a treatment for type 2 diabetes. GK progress curves monitoring product formation are linear up to 1 mM glucose, but biphasic at 5 mM, with the transition from the lower initial velocity to the higher steady-state velocity being described by a rate constant kact. In the presence of a liver-specific GKA (Compound A), progress curves at 1 mM glucose are similar to those at 5 mM, reflecting activation of GK by Compound A. We show that GK regulatory protein (GKRP) is a slow, tight-binding inhibitor of GK. Analysis of progress curves indicate that this i... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5374775 Tue, 01 Nov 2011 04:00:00 +0100 5374775 http://www.medworm.com/index.php?rid=5343381&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111397 In conclusion, the crystal RrgB-fiber is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=5343381 Fri, 21 Oct 2011 04:00:00 +0100 5343381 http://www.medworm.com/index.php?rid=5343382&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111659 The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni2+ ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni2+ insertion into the apo-enzyme. Crystals of apo-HpUreE and its Ni2+ and Zn2+ bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 Å (apo), 1.59 Å (Ni) and 2.52 Å (Zn) resolution, show the conserved proximal and solvent-exposed His102 residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apo-protein are disordered in the crystal, but acqui... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5343382 Thu, 20 Oct 2011 04:00:00 +0100 5343382 http://www.medworm.com/index.php?rid=5268377&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111472 Recently, it was shown that a deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) reduced the numbers of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice. Although these results indicate that ChGn-1 regulates the number of CS chains, the mechanism mediating this regulation is not clear. ChGn-1 is thought to initiate CS biosynthesis by transferring the first N-acetylgalactosamine (GalNAc) to the tetrasaccharide in the protein linkage region of CS. However, in vitro chondroitin polymerization does not occur on the non-reducing terminal GalNAc-linkage pentasaccharide structure. Here, we showed that several different heteromeric enzyme complexes comprised of different combinations of four chondroitin synthase family members synthesized more CS chains w... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5268377 Tue, 27 Sep 2011 04:00:00 +0100 5268377 http://www.medworm.com/index.php?rid=5246010&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111263 Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its pyridine dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN, that plays a central role by degrading NAD into adenine and nicotinamide riboside, the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN that determined the enzyme is a Zn2+-dependent 5’-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5246010 Tue, 20 Sep 2011 04:00:00 +0100 5246010 http://www.medworm.com/index.php?rid=5233097&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110567 17beta-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyzes the interconversion of inactive 17-keto-steroids and their active 17beta-hydroxy counterparts. 17beta-HSDcl belongs to the short-chain dehydrogenase/ reductase superfa mily (SDR). It is currently the only fungal HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provided insights into the evolution of the HSD enzymes. The crystal structures of the 17beta-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active s... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5233097 Mon, 19 Sep 2011 04:00:00 +0100 5233097 http://www.medworm.com/index.php?rid=5233099&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111278 Mycophenolic acid (MPA) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMP dehydrogenase (IMPDH). The MPA biosynthetic cluster of P. brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA-resistance. Surprisingly, all members of subgenus Penicillium contain genes encoding IMPDHs of both the A and B type, regardless of their ability to produce MPA. Duplication of the IMPDH gene occurred prior to and independent of the acquisition of the MPA biosynthetic cluster. Both P. brevicompactum IMPDHs are MPA-resistant while the IMPDHs from a nonproducer are MPA-sensitive. Resistance comes with a catalytic cost: while P. brevicompactum IMPDH-B is <1000-fold more resistant to MPA than a... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5233099 Thu, 15 Sep 2011 04:00:00 +0100 5233099 http://www.medworm.com/index.php?rid=5233098&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110380 Prx4 (peroxiredoxin 4) is the only peroxiredoxin located in the ER (endoplasmic reticulum) and a proposed scavenger for H2O2. In this work we presented crystal structures of human Prx4 in three different redox forms and characterized the reaction features of Prx4 with H2O2. Prx4 exhibits a toroid-shaped decamer constructed of five catalytic dimers. Structural analysis revealed conformational changes around helix α2 and the C-terminal reigon with a YF motif from the partner subunit, which are required for inter-chain disulfide formation between Cys87 and Cys208, a critical step of the catalysis. The structural explanation for the restricting role of the YF motif on the active site dynamics is provided in detail. Prx4 has a high reactivity to H2O2, but is susceptible to over-oxidation... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5233098 Thu, 15 Sep 2011 04:00:00 +0100 5233098 http://www.medworm.com/index.php?rid=5233100&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101481 Although the central a-helical Y(X)4Lf-motif (X: variable; f: hydrophobic) of the translational regulator 4E-BP is the core binding region for the mRNA cap-binding protein eIF4E, the functions of its N- and C-terminal flexible regions for interaction with eIF4E remain to be elucidated. To identify the role of C-terminal region in such interaction, the binding features of full-length and sequential C-terminal deletion mutants of 4E-BPn (n=1-3) subtypes were investigated by surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC). Consequently, the conserved PGVTS/T motif within the C-terminal region was shown to act as the second binding region and to play an important role in the tight binding to eIF4E. The 4E-BP subtypes increased the association constant with e... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5233100 Tue, 13 Sep 2011 04:00:00 +0100 5233100 http://www.medworm.com/index.php?rid=5203892&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111107 Relaxin-like factor (RLF), also known as insulin-like peptide 3 (INSL3), is a novel member of the insulin-relaxin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. Here we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS analysis of the trypsinised sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C-, and A-domains previously deduced from the cDNA. Moreover, the N-terminal peptide was 4 amino acid residues longer than pre... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5203892 Wed, 07 Sep 2011 04:00:00 +0100 5203892 http://www.medworm.com/index.php?rid=5190983&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110869 Cellulases participate in a number of biological events such as plant cell wall remodeling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally-compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited both by their low catalytic efficiency and stability. In this work, we conducted a detailed functional and structural characterization of the thermostable cellulase 5A from Bacillus subtilis (BsCel5A), which consists of a GH5 catalytic domain fused to a family 3 carbohydrate-binding module (CBM3). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5190983 Wed, 31 Aug 2011 23:00:00 +0100 5190983 http://www.medworm.com/index.php?rid=5155312&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111103 Geobacter sulfurreducens (Gs) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes for over one hundred cytochromes and despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA has a crucial role in the conversion of electronic energy into proton motive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In this work, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained i... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5155312 Tue, 23 Aug 2011 23:00:00 +0100 5155312 http://www.medworm.com/index.php?rid=5155313&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110699 Ubiquitin (Ub) C-terminal hydrolases (UCHs) are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among four UCHs yet identified, neither UCH-L3 nor UCH-L1 can catalyze the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it presents in the PA700 complex of the proteasome. Here we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the Lys48-linked di-ubiquitin (K48-diUb) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually < 14 residues), such as that of UCH-L5 or BAP1, is able to cleave both small and... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5155313 Thu, 18 Aug 2011 23:00:00 +0100 5155313 http://www.medworm.com/index.php?rid=5116899&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110440 Glucokinase (GK) is activated by glucose binding to the substrate site, is inhibited by GK regulatory protein (GKRP) but stimulated by GK activator drugs (GKAs). To further explore the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady state kinetics of the enzyme and tryptophan fluorescence (TF). TF studies of the normal binary GK/glucose complex corroborate recent crystallography showing that it exists in a closed conformation greatly different from the open conformation of the ligand free structure but indistinguishable from the ternary GK/glucose/GKA complex. GKAs did activate and GKRP did inhibit normal GK while its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhanc... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5116899 Tue, 09 Aug 2011 23:00:00 +0100 5116899 http://www.medworm.com/index.php?rid=5094105&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110788 PRSS3/mesotrypsin is an atypical isoform of trypsin, the upregulation of which has been implicated in promoting tumor progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small molecule trypsin inhibitors have low affinity and little selectivity, while protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. Here, we use mutagenesis of a mesotrypsin substrate, the Kunitz domain of the amyloid precursor protein (APPI), and of a poor mesotrypsin inhibitor, bovine pancreatic trypsin inhibitor (BPTI), to dissect mesotrypsin specificity at the key P2' position. We find that bulky and charged residues strongly disfavor binding, while acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexe... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5094105 Mon, 01 Aug 2011 23:00:00 +0100 5094105 http://www.medworm.com/index.php?rid=5017642&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110865 Trypanosoma brucei (Tb) sterol C24-methyl transferase (24-SMT) synthesizes an unconventional 24-alkyl sterol product set consisting of ∆24(25), ∆24(28)- and ∆25(27)-olefins. The C-methylation reaction requires Si(β)-face C24-methyl addition coupled to reversible migration of positive charge from C24 to C25. The hydride shifts responsible for charge migration in formation of multiple ergostane olefin isomers catalyzed by TbSMT were examined by incubation of a series of sterol acceptors paired with AdoMet. Data obtained with zymosterol compared to the corresponding [24-2H]- and [27-13C]- derivatives revealed isotopic sensitive branching in the hydride transfer reaction on the path to form 24-methyl-∆24(25)-olefin product (kinetic isotope effect, kH/kD = 1.2... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5017642 Thu, 07 Jul 2011 23:00:00 +0100 5017642 http://www.medworm.com/index.php?rid=5006159&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110376 The betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in the infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulphur of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported before either for a cysteine residue nor for a low-molecular-weight thiol. The Michael addition of the cysteine thiolate in the “resting” conformation to the double bond of ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=5006159 Wed, 06 Jul 2011 23:00:00 +0100 5006159 http://www.medworm.com/index.php?rid=4997065&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110948 The incorporation of copper into biological macromolecules such as Cu-Zn superoxide dismutase (SOD1) is essential for the viability of most organisms. However, copper is toxic and therefore the intracellular free copper concentration is kept to an absolute minimum. Several proteins, termed metallochaperones, are charged with the responsibility of delivering copper from membrane transporters to its intracellular destination. The Copper Chaperone for SOD1 (CCS) is the major pathway for SOD1 copper loading. We have determined the first solution structure of hCCS by Small Angle X-ray Scattering (SAXS) in conjunction with size exclusion chromatography (SEC). Our findings highlight the importance of this combined on-line chromatographic technology with SAXS, which has allowed us to unambiguously... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4997065 Thu, 30 Jun 2011 23:00:00 +0100 4997065 http://www.medworm.com/index.php?rid=4974502&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110890 The capsule of Campylobacter jejuni strain 81-176 comprises the unusual 6-deoxy-alpha-D-altro-heptose, whose biosynthesis and function are not known. In this work, we characterized enzymes of the capsular cluster, WcbK and WcaG, to determine their role in 6-deoxy-altro-heptose synthesis. These enzymes are similar to the Yersinia pseudotuberculosis GDP-manno-heptose dehydratase/reductase DmhA/DmhB, that we characterized previously. Capillary electrophoresis and mass spectrometry analyses showed that WcbK is a GDP-manno-heptose dehydratase whose product can be reduced by WcaG, and that WcbK/WcaG can use the substrate GDP-mannose, although with lower efficiency than heptose. Comparison of kinetic parameters for WcbK and DmhA indicated that the relaxed substrate specificity of WcbK comes at th... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4974502 Mon, 27 Jun 2011 23:00:00 +0100 4974502 http://www.medworm.com/index.php?rid=4974501&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110753 We report here the crystal structures of human GSTk (hGSTk) in apo form and in complex with S-hexylglutathione (GTX) and the steady-state kinetic studies, revealing insights into the catalytic mechanism of hGSTk and other GSTks. The substrate binding induces conformational change of the active site from an “open” conformation in the apo form to a “closed” conformation in the GTX-bound complex, facilitating formations of the GSH-binding site (G site) and the hydrophobic substrate-binding site (H site). The conserved Ser16 at the G site functions as the catalytic residue in the deprotonation of the thiol group and the conserved Asp69, Ser200, Asp201, and Arg202 form a network of interactions with the g-glutamyl carboxylate to stabilize the thiolate anion. The H si... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4974501 Mon, 27 Jun 2011 23:00:00 +0100 4974501 http://www.medworm.com/index.php?rid=4951174&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110390 Chitinolytic β-N-acetyl-D-hexosaminidase is a branch of GH20 β-N-acetyl-D-hexosaminidase that is only distributed in insects and microorganisms, and is therefore a potential target for the action of insecticides. O-(2-acetamido-2-deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate (PUGNAc) was initially identified as an inhibitor against GH20 β-N-acetyl-D-hexosaminidases. So far no crystal structure of PUGNAc in complex with any GH20 β-N-acetyl-D-hexosaminidase has been reported. We showed here the sensitivities of chitinolytic β-N-acetyl-D-hexosaminidases towards PUGNAc can vary in 100 folds, with the order being OfHex1 (insect β-N-acetyl-D-hexosaminidase from Ostrinia furnacalis)< SmCHB (bacterial chitobiase from Serratia marcescens)< SpHex... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4951174 Mon, 20 Jun 2011 23:00:00 +0100 4951174 http://www.medworm.com/index.php?rid=4931919&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110681 Proteins SP-B and SP-C are essential to promote formation of surface active films at the respiratory interface, but their mechanism of action is still under investigation. We have analyzed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of fluorescent probes Nile Red (permeable) and FM®1-43 (impermeable) to membranes. We have also analyzed the effect of single or combined proteins on membrane permeation by the soluble fluorescent dye calcein. Fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast to Nile Red, very similar in all the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other ha... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4931919 Wed, 15 Jun 2011 23:00:00 +0100 4931919 http://www.medworm.com/index.php?rid=4931920&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110500 Sil1 functions as a nucleotide exchange factor (NEF) for Bip in eukaryotic cells. In order to understand how Sil1 functions as a NEF, we analyzed the crystal structure of the yeast Bip-Sil1 complex at a resolution of 2.3Å. In the complex, the Sil1 molecule acts as a “molecular clamp” which binds to the IIb lobe of the Bip ATPase domain. Sil1 binding causes lobe IIb to rotate ~13.5° away from the ADP-binding pocket and lobe Ib to rotate in the opposite direction for ~3.7°. These conformational changes in Bip open up the nucleotide-binding pocket in the ATPase domain and simultaneously disrupt the hydrogen bonds between Bip and bound ADP, thus providing a mechanism for ADP release. We also demonstrate that Sil1 mutations that disrupt binding to the Bip ATPa... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4931920 Tue, 14 Jun 2011 23:00:00 +0100 4931920 http://www.medworm.com/index.php?rid=4900977&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110801 We report here a 2.5 Å resolution crystal structure of the ATD of GluA1. Comparative analyses of the structure of GluA1-ATD and other subunits sheds light on our understanding of how ATD drives subfamily-specific assembly of AMPARs. In addition, analysis of the crystal lattice of GluA1-ATD suggests a novel mechanism by which the ATD might participate in inter-tetramer AMPAR clustering, as well as in trans-synaptic protein-protein interactions. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=4900977 Sun, 05 Jun 2011 23:00:00 +0100 4900977 http://www.medworm.com/index.php?rid=4882605&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101784 Crucial to glucose homeostasis in man, the human pyruvate dehydrogenase complex (hPDC) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1-E3) and an accessory subunit, E3 binding protein (E3BP). Its icosahedral E2/E3BP 60-meric ‘core’ provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme cooperativity. Current core models indicate either a 48E2+12E3BP or 40E2+20E3BP subunit composition. Here, we demonstrate clear differences in subunit content and organisation between recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. Our data provide a rational... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4882605 Mon, 30 May 2011 23:00:00 +0100 4882605 http://www.medworm.com/index.php?rid=4827463&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110345 Macrophage infectivity potentiators (Mips) are a subset of immunophilins associated with virulence in a range of micro-organisms. These proteins possess prolyl-peptide isomerase activity and are inhibited by drugs including rapamycin and tacrolimus. We determined the structure of the Mip homologue (BpML1) from the human pathogen and biowarfare threat Burkholderia pseudomallei by nuclear magnetic resonance and X-ray crystallography. The crystal structure suggests that key catalytic residues in the BpML1 active site have unexpected conformational flexibility consistent with a role in catalysis. The structure further revealed BpML1 binding to a helical peptide, in a manner resembling the physiological interaction of human TGFb receptor 1 with the human immunophilin FKBP12. Furthermore, the st... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4827463 Sun, 15 May 2011 23:00:00 +0100 4827463 http://www.medworm.com/index.php?rid=4811822&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110292 A hyperthermophophilic, membrane-related β-1,4 endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolyzing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65~2.01 Å. The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid re... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4811822 Tue, 10 May 2011 23:00:00 +0100 4811822 http://www.medworm.com/index.php?rid=4794141&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110304 Genomes of all free-living organisms encode the enzyme deoxyuridine triphosphate pyrophosphatase (dUTPase) which plays a key role in preventing uracil incorporation into DNA. Here, we present the results of the biochemical and structural characterization of the Saccharomyces cerevisiae dUTPase DUT1. The hydrolysis of dUTP by DUT1 was strictly dependent on a divalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+, or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve suggesting a positive cooperativity between the subunits. The crystal structure of DUT1 was solved at 2 Å resolution ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4794141 Thu, 05 May 2011 23:00:00 +0100 4794141 http://www.medworm.com/index.php?rid=4752786&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101804 Active sex hormones such as testosterone and progesterone are metabolized to tetrahydrosteroids in liver to terminate hormone action. One main metabolic pathway, the 5beta-pathway, involves 5beta-steroid reductase (AKR1D1), which catalyzes the reduction of the 4-ene structure, and ketosteroid reductases (AKR1C1-AKR1C4), which catalyze the subsequent reduction of the 3-oxo group. The activities of the four human AKR1C enzymes on 5beta-dihydrotestosterone, 5beta-pregnane-3,20-dione, and 20alpha-hydroxy-5beta-pregnan-3-one, the intermediate 5beta-dihydrosteroids on the 5beta-pathway of testosterone and progesterone metabolism were investigated. Product characterization by LC-MS revealed that the reduction of the 3-oxo group of the three steroids predominantly favored the formation of the corr... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4752786 Mon, 25 Apr 2011 23:00:00 +0100 4752786 http://www.medworm.com/index.php?rid=4731874&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110410 Microbial motility frequently depends on flagella or type IV pili. Using recently developed archaeal genetic tools, archaeal flagella and its assembly machinery have been identified. Archaeal flagella are functionally similar to bacterial flagella and their assembly systems are homologous to type IV pili assembly systems of Gram negative bacteria. Therefore, elucidating their biochemistry may result in insights in both archaea and bacteria. FlaI, a critical cytoplasmic component of the archaeal flagella assembly system in Sulfolobus acidocaldarius, is a member of the type II/IV secretion system ATPase superfamily, and is proposed to be bi-functional in driving flagella assembly and movement. Here we show that purified FlaI is a Mn2+-dependent ATPase that binds MANT-ATP with a high a... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4731874 Tue, 19 Apr 2011 23:00:00 +0100 4731874 http://www.medworm.com/index.php?rid=4731875&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101712 Abeta immunotherapy has been revealed as a hopeful tool in Alzheimer’s disease treatment. In contrast to complete antibodies, the administration of single-chain variable Fragments (scFv) produces neither meningoencephalitis nor cerebral hemorrhage. Here, the recombinant expression of scFv-h3D6, a derivative of an antibody specific for Abeta oligomers, is presented, as well as the subsequent proof of its capability to recover the toxicity induced by the Abeta1-42 peptide in the SH-SY5Y neuroblastoma cell-line. To gain insight into the conformational changes underlying the prevention of Abeta toxicity by this antibody fragment, the conformational landscape of scFv-h3D6 upon temperature perturbation is also described. Heating the native state does not lead to any extent of unfolding bu... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4731875 Sun, 17 Apr 2011 23:00:00 +0100 4731875 http://www.medworm.com/index.php?rid=4657789&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110091 The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts all-trans retinyl ester (atRE) to 11-cis retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome–based floatation assay, we present evidence that the RPE65 membrane binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and circular dichroism spectroscopy, we showed that upon membrane binding, RPE65 undergoes conformational chan... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4657789 Tue, 29 Mar 2011 23:00:00 +0100 4657789 http://www.medworm.com/index.php?rid=4635818&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110056 Cytotoxicity, a major obstacle in therapeutic application of antimicrobial peptides, is controlled by leucine zipper like sequences in melittin and other naturally-occurring antimicrobial peptides. Magainin 2 shows significantly lower cytotoxicity than many naturally-occurring antimicrobial peptides and lacks this structural element. To investigate the consequences of introducing a leucine zipper sequence in magainin 2, a novel analogue (Mag-mut) was designed by rearranging only the positions of its hydrophobic amino acids to include this structural element. Both magainin 2 and Mag-mut showed appreciable similarities in their secondary structures in presence of negatively charged lipid vesicles, in localizing and permeabilizing the selected bacteria and exhibiting bactericidal activities. ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4635818 Fri, 25 Mar 2011 00:00:00 +0100 4635818 http://www.medworm.com/index.php?rid=4605110&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110002 In this report, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from Mycobacterium tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analog of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active site subpocket inducing significant structural re... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4605110 Thu, 17 Mar 2011 00:00:00 +0100 4605110 http://www.medworm.com/index.php?rid=4559217&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101859 Organophosphorus insecticides and nerve agents irreversibly inhibit serine hydrolase superfamily enzymes. One enzyme of this superfamily, the industrially important (for β- lactam antibiotic synthesis) acetyl xylan esterase/cephalosporin acetyl hydrolase (AXE/CAH) from the biotechnologically valuable organism Bacillus pumilus, exhibits low sensitivity to the organophosphate paraoxon, reflected in a high KI for it (∼5 mM) and in a long t1/2 (∼1 min) for enzyme-paraoxon (E-DEP) covalent adduct formation. The crystal structure of the E-DEP complex determined at 2.7 Å-resolution reveals strain in the active Ser181-bound organophosphate as a likely cause for the limited paraoxon sensitivity. The strain results from active site size limitation imposed by bulky... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4559217 Mon, 07 Mar 2011 00:00:00 +0100 4559217 http://www.medworm.com/index.php?rid=4544343&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102097 The compatible solute ATP-binding cassette (ABC) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared to its 70% sequence-identical paralog OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine, respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with substrate-binding pocket located at the inter-domain cleft. Upon substrate-binding, the two domains ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4544343 Thu, 03 Mar 2011 00:00:00 +0100 4544343 http://www.medworm.com/index.php?rid=4476870&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101661 All the peroxisomal β-oxidation pathways characterized thus far house at least one multifunctional enzyme (MFE) catalyzing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for D. melanogaster MFE-2 (DmMFE-2), complements the S. cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4476870 Mon, 14 Feb 2011 00:00:00 +0100 4476870 http://www.medworm.com/index.php?rid=4463921&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102049 ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2) and Insulin Regulated aminopeptidase (IRAP) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding onto MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorogenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the primary specificity (S1) pocket. Molecular modeling of the three S1 pockets reveals substrat... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4463921 Fri, 11 Feb 2011 00:00:00 +0100 4463921 http://www.medworm.com/index.php?rid=4436282&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101405 We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally-useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using Principal Component Analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA < DhlA~D... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4436282 Fri, 04 Feb 2011 00:00:00 +0100 4436282 http://www.medworm.com/index.php?rid=4398739&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101771 Calsequestrin is a Ca2+ buffering protein localized in the muscle sarcoplasmic reticulum (SR), however it is unknown if Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference to Ca2+ over other ions. Therefore, a major objective of this study was to determine how CASQ2 selects Ca2+ over other metal ions, by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline-earth (divalent) and transition metals (polyvalent). We additionally investigated how Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) mutations affect CASQ2 structure and its molecular behavior when exposed to different metal ions. Our results show that alkali and alkaline-earth metals can initiate si... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4398739 Wed, 26 Jan 2011 00:00:00 +0100 4398739 http://www.medworm.com/index.php?rid=4398738&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101442 Human sperm-specific glyceraldehyde-3-phosphate dehydrogenase (hGAPDS) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. Here, we have over-expressed in E. coli a highly-soluble form of hGAPDS truncated at the N-terminus (hGAPDS∆N), and crystallized the homo-tetrameric enzyme in two ligand complexes. The hGAPDS∆N-NAD+-phosphate structure maps the two anion recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognised Pi site identified in other organisms. The hGAPDS∆N-NAD+-glycerol structure shows serendipi... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4398738 Wed, 26 Jan 2011 00:00:00 +0100 4398738 http://www.medworm.com/index.php?rid=4398740&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101984 We present a detailed biochemical analysis of two chimeric CPR composed of the association of human or yeast FMN with the alternate connecting/FAD domains. Despite the assembly of domains having relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimeric enzymes while the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimerogenesis are a decrease of the internal electron transfer rate between both flavins correlated with changes in the geometry of chimeric CPR in solution. Results presented herein highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the imp... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4398740 Tue, 25 Jan 2011 00:00:00 +0100 4398740 http://www.medworm.com/index.php?rid=4360088&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101593 The interaction between streptavidin and biotin is one of the strongest non-covalent interactions in nature. Streptavidin is a widely used tool and a paradigm for protein-ligand interactions. We recently developed a streptavidin mutant, termed traptavidin, possessing 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. Here, we determined crystal structures of apo-traptavidin and biotin-traptavidin at 1.5 Å resolution. In apo-streptavidin the L3/4 loop, near biotin’s valeryl tail, is typically disordered and open, but closes upon biotin binding. In contrast, this L3/4 loop was shut in both apo-traptavidin and biotin-traptavidin. The reduced flexibility of L3/4 and decreased conformational change on biotin binding provide an explanation for trapt... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4360088 Tue, 18 Jan 2011 00:00:00 +0100 4360088 http://www.medworm.com/index.php?rid=4360087&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102014 The oxyanion derivatives of the chalcogens tellurium and selenium are toxic to living organisms even at very low levels. Bacteria have developed mechanisms to overcome their toxicity by methylating them. The structure of the TehB from Escherichia coli has been determined in the presence of the co-factor analogues S-adenosyl-homocysteine (SAH) and sinefungin (non-hydrolysable form of S-adenosyl-L-methionine) at 1.48 Å and 1.9 Å respectively. Interestingly, our kinetic data show that TehB does not discriminate between selenium or tellurite oxyanions, making it a very powerful detoxifying protein. Analysis of the active site has identified three conserved residues that are capable of binding and orientating the metals for nucleophilic attack, His176, Arg177 and Arg184. Mutagenes... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4360087 Tue, 18 Jan 2011 00:00:00 +0100 4360087 http://www.medworm.com/index.php?rid=4321983&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101797 Factor VIII functions as a cofactor for factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates activity of the factor VIIIa-factor IXa complex approximately 100,000-fold and the major phospholipid-binding motif of factor VIII is thought to be on the C2 domain. We prepared a factor VIII C2 domain construct (fVIII-C2) from E. coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and fluorescence resonance energy transfer, revealed that fVIII-C2 membrane affinity was approximately 40-fold lower than intact factor VIII. In contrast to the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiologic NaCl. fVIII-C2 binding was also not... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4321983 Fri, 07 Jan 2011 00:00:00 +0100 4321983 http://www.medworm.com/index.php?rid=4317766&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101643 We report here the x-ray structure of the entire extra-cellular region of PrkC from Staphylococcus aureus. This structure reveals that the extra-cellular region of PrkC, EC-PrkC, is a linear modular structure, composed of three PASTA domains and an unpredicted C-terminal domain, which presents the typical features of adhesive proteins. Using several solution techniques, we also evidenced that EC-PrkC shows no tendency to dimerise even in the presence of high concentrations of muropeptides. X-ray structural results obtained here provide molecular clues into the mechanism of muropeptide-induced PrkC activation. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=4317766 Thu, 06 Jan 2011 00:00:00 +0100 4317766 http://www.medworm.com/index.php?rid=4313449&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101471 Reversible phosphorylation of the C-terminal domain (CTD) of eukaryotic RNA polymerase II largest subunit represents a critical regulatory mechanism during the transcription cycle and mRNA processing. Ssu72 is an essential phosphatase conserved in eukaryotes that dephosphorylates phosphorylated Ser5 (phos.Ser5) of the CTD heptapeptide. Its function is implicated in transcription initiation, elongation and termination as well as RNA processing. Here we report the high resolution x-ray crystal structures of Drosophila melanogaster Ssu72 phosphatase in the apo form and in complex with an inhibitor mimicking the transition state of phosphoryl transfer. Ssu72 facilitates dephosphorylation of the substrate through a phosphoryl-enzyme intermediate, as visualized in the complex structure of Ssu72 ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4313449 Wed, 05 Jan 2011 00:00:00 +0100 4313449 http://www.medworm.com/index.php?rid=4313448&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101334 The genetic element pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. This plasmid/virus hybrid infects several species of the hyperthermophilic, acidophilic crenarchaeon Sulfolobus. The open reading frame c68 of pSSVx encodes for a 7.7 kDa protein that does not show significant sequence homology to any protein with known 3D structure. EMSA experiments, DNA footprinting and CD analyses indicate that recombinant C68, purified from Escherichia coli, binds to two different operator sites, that are located upstream of its own promoter. The three-dimensional structure, solved by a single wavelength anomalous diffraction experiment on a selenomethionine derivative, shows that the protein assumes a swapped-hairpin fold, which is a distinctive fold... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4313448 Wed, 05 Jan 2011 00:00:00 +0100 4313448 http://www.medworm.com/index.php?rid=4308864&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101096 Sclerostin is an extracellular negative regulator of bone formation that is a recognised therapeutic target for osteoporosis therapy. Here, we perform DNA aptamer selection against sclerostin, then characterise aptamer-sclerostin binding and ability to inhibit sclerostin function in cell culture. We show that a selected DNA aptamer was highly selective for binding to sclerostin with affinities in the nanomolar range as determined by solid-phase assays and by isothermal titration calorimetry. Binding between sclerostin and the aptamer was exothermic and enthalpically driven. Circular dichroism confirmed that the aptamer had temperature-dependent parallel G-quadruplex characteristics. The aptamer was stabilised with 3' inverted thymidine to investigate efficacy at inhibiting sclerostin funct... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4308864 Tue, 04 Jan 2011 00:00:00 +0100 4308864 http://www.medworm.com/index.php?rid=4277306&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101813 Allostery is vital to the function of many proteins. In some cases, rather than a direct steric effect, mutual modulation of ligand binding at spatially-separated sites may be achieved through a change in protein dynamics. Thus changes in vibrational modes of the protein, rather than conformational changes, allow different ligand sites to communicate. Evidence for such an effect has been found in TRAP, a regulatory protein found in species of Bacillus. TRAP is part of a feedback system to modulate expression of the trp operon, which carries genes involved in tryptophan synthesis. Negative feedback is thought to depend on binding of tryptophan-bound, but not unbound TRAP, to a specific mRNA leader sequence. We find that, contrary to expectations, at low temperatures TRAP is able to bind RNA... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4277306 Tue, 21 Dec 2010 00:00:00 +0100 4277306 http://www.medworm.com/index.php?rid=4274811&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101667 The FeII/2-oxoglutarate (2OG)-dependent dioxygenase superfamily comprises proteins that couple a wide range of oxidation reactions to decarboxylation of 2-oxoglutarate to succinate. A member of this class of mononuclear non-heme iron proteins is the E. coli DNA/RNA repair enzyme AlkB. In this work, we describe the magnetic and optical properties of the yet uncharacterized human AlkB homologue ALKBH4. Through EPR and UV-Vis spectroscopy studies, we address the iron binding environment of the proposed catalytic centre of wild-type ALKBH4 and an FeII binding mutant. We could observe a novel unusal ferric high spin EPR active species in presence of sulfide with gmax 8.2. The FeII site was probed with NO. An intact histidine-carboxylate site is necessary for productive iron binding. We also rep... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4274811 Mon, 20 Dec 2010 00:00:00 +0100 4274811 http://www.medworm.com/index.php?rid=4263981&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100507 Sodium-coupled neutral amino acid transporter 2 (SNAT2[1]) belongs to the SLC38 family of solute transporters. Transport of 1 amino acid molecule into the cell is driven by the co-transport of 1 Na+ ion. The functional significance of the C-terminus of SNAT2, which is predicted to be located in the extracellular space, is currently unknown. Here, we removed 13 amino acid residues from the SNAT2 C-terminus and studied the effect of the deletion on transporter function. The truncation abolished amino acid transport currents at negative membrane potentials (< 0 mV), as well as substrate uptake. However, transport currents were observed at positive membrane potentials, demonstrating that transport was accelerated while the driving force decreased. Membrane expression levels were norm... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4263981 Thu, 16 Dec 2010 00:00:00 +0100 4263981 http://www.medworm.com/index.php?rid=4199968&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100887 S100A11 is a dimeric, EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2), and facilitate membrane vesiculation events. In contrast to other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an “open” conformation that is very similar to S100A11 in its calcium-bound state (Ca2+-S100A11). To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were repl... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4199968 Thu, 25 Nov 2010 00:00:00 +0100 4199968 http://www.medworm.com/index.php?rid=4196100&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101357 In eukaryotes, disulphide bonds are formed in the endoplasmic reticulum, facilitated by the Ero1 oxidase/protein disulphide isomerase (PDI) system. Mammals have two ERO1 genes, encoding Ero1α and Ero1β proteins. Ero1β is constitutively expressed in professional secretory tissues and induced during the unfolded protein response. We show here that recombinant human Ero1β is twice as active as Ero1α in enzymatic assays. Ero1β oxidizes PDI more efficiently than other PDI family members and drives oxidative protein folding preferentially via the active site in the a’ domain of PDI. Our data reveal that Ero1β oxidase activity is regulated by long-range disulphides and that Cys130 plays a critical role in the feedback regulation. Compared wi... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4196100 Tue, 23 Nov 2010 00:00:00 +0100 4196100 http://www.medworm.com/index.php?rid=4196099&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101895 Ure2, the protein determinant of the Saccharomyces cerevisiae prion [URE3], has a natively disordered N-terminal domain that is important for prion formation in vivo and amyloid formation in vitro; the globular C-domain has a glutathione transferase-like fold. Here, we swapped the position of the N- and C- regions, with or without an intervening peptide linker, to create the Ure2 variants CLN-Ure2 and CN-Ure2, respectively. The native structural content and stability of the variants were the same as wild-type Ure2, as indicated by enzymatic activity, far-UV circular dichroism and equilibrium denaturation. CLN-Ure2 was able to form amyloid-like fibrils, but with a significantly longer lag time than wild-type Ure2; and the two proteins were unable to cross-seed. Under the same conditions, CN... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4196099 Tue, 23 Nov 2010 00:00:00 +0100 4196099 http://www.medworm.com/index.php?rid=4182399&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101648 Organophosphylates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine. Engineering of human butyrylcholinesterase by substitution of histidine for glycine at position 117 led to the creation of OP hydrolase activity. But the lack of structural information and poor understanding of the hydrolytic mechanism of G117H has hampered further improvements in the catalytic activity. We solved the crystallographic structure of the G117H mutant with a variety of ligands in the active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from His... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4182399 Fri, 19 Nov 2010 00:00:00 +0100 4182399 http://www.medworm.com/index.php?rid=4132308&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100892 Only recently extracellular RNases of the RNase A superfamily, with the characteristic CKxxNTF sequence signature, have been identified in fish. This has led to the recognition that these RNases are present in the whole vertebrate sub-phylum. In fact, they comprise the only enzyme family unique to vertebrates. Four RNases from zebrafish (Danio rerio) have been previously reported, with a very low RNase activity, some endowed, like human angiogenin, with powerful angiogenic and bactericidal activities. Here we report the three-dimensional structure, the thermodynamic behaviour, and the biological properties of a novel zebrafish RNase, ZF-RNase-5. The investigation of its structural and functional properties, extended to all other sub-family members, provides an inclusive description of the ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4132308 Thu, 04 Nov 2010 00:00:00 +0100 4132308 http://www.medworm.com/index.php?rid=4116855&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101286 Human glutaredoxin 5 (GLRX5) is an evolutionarily conserved thiol-disulfide oxidoreductase that has a direct role in the maintenance of normal cytosolic and mitochondrial iron homeostasis and its expression affects haem biosynthesis and erythropoiesis. We have crystallised the human GLRX5 bound to two [2Fe2S] clusters and four glutathione (GSH) molecules. The crystal structure revealed a tetrameric organisation with the [2Fe2S] clusters buried in the interior and shielded from the solvent by the conserved β1-α2 loop, Phe69 and the GSH molecules. Each [2Fe2S] cluster is ligated by the N-terminal active site cysteine (Cys67) thiols contributed by two protomers and two cysteine thiols from two GSH. The two subunits coordinating the cluster are in a more extended conformation com... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4116855 Thu, 28 Oct 2010 23:00:00 +0100 4116855 http://www.medworm.com/index.php?rid=4072827&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101017 CYP101D2 from Novosphingobium aromaticivorans DSM12444 is closely related to CYP101D1 from the same bacterium and to P450cam (CYP101A1) from Pseudomonas putida. All three are capable of oxidising camphor stereoselectively to 5-exo-hydroxycamphor. The crystal structure of CYP101D2 reveals that the likely ferredoxin binding site on the proximal face is largely positively charged, similar to that of CYP101D1. However, both the native and camphor-soaked forms of CYP101D2 have open conformations with an access channel. In the active site of the camphor-soaked form the camphor carbonyl interacts with the heme iron-bound water. Two other potential camphor binding sites were also identified from electron densities in the camphor-soaked structure: one is located in the access channel, flanked by th... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4072827 Thu, 14 Oct 2010 23:00:00 +0100 4072827 http://www.medworm.com/index.php?rid=4039726&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101185 Carbohydrate-active enzymes face large substrate diversity in a highly selective manner with only a limited number of available folds. They are therefore subjected to multiple divergent and convergent evolutionary events. This and their frequent modularity render their functional annotation in genomes difficult in a number of cases. A classification of polysaccharide lyases (the enzymes that cleave polysaccharides using an elimination instead of a hydrolytic mechanism) is presented thoroughly for the first time. Based on the analysis of a large panel of experimentally characterized polysaccharide lyases, we examined the correlation of various enzyme properties with the three levels of the classification: fold, families and subfamilies. The resulting hierarchical classification, which shoul... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4039726 Wed, 06 Oct 2010 23:00:00 +0100 4039726 http://www.medworm.com/index.php?rid=4035577&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101241 In most eubacteria, fungi, apicomplexa, plants, and some metazoans, the active form of vitamin B6, pyridoxal 5-phosphate (PLP) is de novo synthesized from three substrates, ribose 5-phosphate (R5P), dihydroxyacetone phosphate (DHAP), and ammonia hydrolyzed from glutamine by a complexed glutaminase. Of the three active sites of deoxyxylulose 5-phosphate (DXP)-independent PLP synthase (Pdx1), the R5P isomerization site has been assigned, but the sites for DHAP isomerization and PLP formation remain unknown. Here, we present the crystal structures of yeast Pdx1/Snz1, in apo-, glyceraldehyde 3-phosphate (G3P)- and PLP-bound forms, at 2.30, 1.80, and 2.20 Å, respectively. Structural and biochemical analysis enabled us to assign the PLP-formation site, a G3P-binding site and a G3P-transfe... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4035577 Mon, 04 Oct 2010 23:00:00 +0100 4035577 http://www.medworm.com/index.php?rid=4011339&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100744 We have incorporated cytochrome P450 3A4 (CYP3A4) and NADPH-cytochrome P450 reductase (CPR) into liposomes with a high lipid:protein ratio by an improved method. In the purified proteoliposomes CYP3A4 binds testosterone with Kd,app = 36 (±6) μM and Hill coefficient = 1.5 (±0.3), and 75 (±4) % of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its FeII-CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14 (±2) s-1. Measurements of the first electron transfer were... BJ Structure journals http://www.medworm.com/rss/comments.php?id=4011339 Tue, 28 Sep 2010 23:00:00 +0100 4011339 http://www.medworm.com/index.php?rid=3997818&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101054 This study shows the importance of both the metal ion composition and a hydrogen bond network that connects the metal ion centre with the substrate binding pocket using residues R254 and Y257 in the mechanism and substrate specificity of this enzyme. For the Co(II)-derivative of OpdA two protonation equlibria (pKa1 ~5; pKa2 ~10) have been identified as relevant for catalysis, and a terminal hydroxide acts as the likely hydrolysis-initiating nucleophile. In contrast, the Zn(II)- and Cd(II)-derivatives only have one relevant protonation equilibrium (pKa ~4-5), and the μOH is the proposed nucleophile. The observed mechanistic flexibility may reconcile contrasting reaction models that were published previously and may be beneficial for the rapid adaptation of OP-degrading enzymes to cha... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3997818 Thu, 23 Sep 2010 23:00:00 +0100 3997818 http://www.medworm.com/index.php?rid=3997819&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101338 Modification of cellular proteins with N-acetylglucosamine (O-GlcNAc) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and O-GlcNAcase, which recognize their target proteins via as yet unidentified mechanisms. Here, we uncover the first insights into the mechanism of substrate recognition by human O-GlcNAcase. The structure of a novel bacterial O-GlcNAcase orthologue reveals a putative substrate binding groove, conserved with metazoan O-GlcNAcases. Guided by this structure, conserved amino acids lining this groove in human O-GlcNAcase were mutated and the activity on three different substrate proteins (TAB1, FoxO1 and CREB) was tested in an in vitro de-glycosylation assay. The... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3997819 Wed, 22 Sep 2010 23:00:00 +0100 3997819 http://www.medworm.com/index.php?rid=3992499&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100642 To produce functional haemoglobin, nascent a-globin (ao) and b-globin (bo) chains must each bind a single haem molecule (to form ah and bh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. The a-haemoglobin stabilizing protein (AHSP) has previously been shown to bind ah and regulate redox activity of the haem iron. Here, we use a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with ao that inhibits ao aggregation and promotes ao folding in the absence of haem. These findings indicate that AHSP may function as an ao-specific chaperone, and suggest and important role for ao... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3992499 Tue, 21 Sep 2010 23:00:00 +0100 3992499 http://www.medworm.com/index.php?rid=3924162&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101079 Inositol dehydrogenase from Bacillus subtilis (BsIDH) is a NAD+-dependent enzyme that catalyses the oxidation of the axial hydroxyl group of myo-inositol to form scyllo-inosose. We have determined the crystal structures of wild type BsIDH and of the inactive K97V mutant in apo-, holo- and ternary complexes with inositol and inosose. BsIDH is a tetramer, with a novel arrangement consisting of 2 long continuous β-sheets, formed from all 4 monomers, in which the central 2 strands are crossed over to form the core of the tetramer. Each subunit in the tetramer consists of two domains, an N-terminal Rossmann fold domain containing the cofactor-binding site, and a C-terminal domain containing the inositol-binding site. Structural analysis allowed us to determine residues important i... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3924162 Tue, 31 Aug 2010 23:00:00 +0100 3924162 http://www.medworm.com/index.php?rid=3924161&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100921 Three mutations of the β4GALT7 gene corresponding to A186D, L206P and R270C were identified in patients with the progeroid form of the Ehlers-Danlos syndrome and described to be associated with reduction or loss of activity of the β1,4-galactosyltransferase7. But the molecular bases of the reduction or loss of activity remain to be determined. In the present study, wild-type, A186D, L206P and R270C β1,4-GalT7s were expressed in CHO618 cells as membrane proteins and in Escherichia coli as soluble maltose binding-fusion proteins. Ability of expressed proteins to transfer galactose from donor to acceptor susbtrate was systematically characterized by kinetic analysis. The physicochemical properties of soluble proteins were explored by isothermal titration calorimetry which... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3924161 Tue, 31 Aug 2010 23:00:00 +0100 3924161 http://www.medworm.com/index.php?rid=3911056&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101043 In this study we report on the properties of a series of site-directed mutants of two adjacent tyrosines involved in iron coordination, and demonstrate that, in contrast to mutation of equivalent residues of human transferrin N-lobe, the mutant FbpAs retain significant iron binding affinity regardless of the nature of the replacement amino acid. The Y195A and Y196A FbpAs are not only capable of binding iron but are proficient in mediating periplasm to cytoplasm iron transport in a reconstituted FbpABC pathway in a specialized E. coli reporter strain. This indicates that their inability to mediate iron acquisition from transferrin is due to their inability to compete for iron with receptor bound transferrin. Wild-type iron-loaded protein could be crystalized in a closed or open state depend... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3911056 Thu, 26 Aug 2010 23:00:00 +0100 3911056 http://www.medworm.com/index.php?rid=3906845&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100813 BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of energy-coupling factor (ECF) transporters. These systems are composed of ABC ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN), and a core transporter component (BioY) in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET approach using living recombinant E. coli cells. To this end, the donor-acceptor pair of Cerulean and yellow fluorescent protein, respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activi... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3906845 Wed, 25 Aug 2010 23:00:00 +0100 3906845 http://www.medworm.com/index.php?rid=3898770&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100584 This study provides evidence that lipin proteins function as oligomeric complexes and that the three mammalian lipin isoforms can form combinatorial units. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=3898770 Mon, 23 Aug 2010 23:00:00 +0100 3898770 http://www.medworm.com/index.php?rid=3898769&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101246 In this study, promiscuous TtHICDH was evolved to an enzyme showing distinct IPMDH activity by directed evolution using a DNA shuffling technique. Through 5 repeats of DNA shuffling/screening, variants that allow Escherichia coli C600 (leuB-) to grow on a minimal medium in 2 days were obtained, one of which, LR5-1, with 8 amino acid replacements, was found to possess a 65-fold increased kcat/KM value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y caused a further increase in kcat/KM value and partial recovery of the decreased thermotolerance of LR5-1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5-1 effectively increased IPMDH activity: replacements around the substrate-binding site contributed to the improved reco... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3898769 Mon, 23 Aug 2010 23:00:00 +0100 3898769 http://www.medworm.com/index.php?rid=3888505&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100641 Of the four human IgG antibody subclasses IgG1-IgG4, IgG4 is of interest in that this does not activate complement. The solution structures of antibodies are critical to understanding function and therapeutic applications. Thus IgG4 was studied by synchrotron X-ray scattering. The Guinier X-ray radius of gyration RG increased from 5.0 nm to 5.1 nm with increase of concentration. The distance distribution function P(r) revealed a single peak at 0.3 mg/ml, which unusually became resolved into two peaks that shifted to smaller r values when the concentration increased to 1.3 mg/ml, even though the maximum dimension of IgG4 was unchanged at 17 nm. This indicated a small concentration dependence of the IgG4 solution structure. By analytical ultracentrifugation no concentration dependence in the... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3888505 Thu, 19 Aug 2010 23:00:00 +0100 3888505 http://www.medworm.com/index.php?rid=3864784&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100788 The structure of the glycosaminoglycan (GAG) chain of recombinantly expressed decorin proteoglycan is examined using a combination of the intact chain analysis and the domain compositional analysis. The GAG has a number averaged molecular weight of 22,000 Da as determined by polyacrylamide gel electrophoresis (PAGE). Nuclear magnetic resonance (NMR) spectroscopic analysis using two-dimensional correlation spectroscopy indicates that the ratio of glucuronic acid to iduronic acid in decorin peptidoglycan is 5 to 1. GAG domains terminated with a specific disaccharide obtained by enzymatic degradation of decorin GAG with highly-specific endolytic and exolytic lyases are analyzed by PAGE and further depolymerized with the enzymes. The disaccharide compositional profiles of the resulting domains... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3864784 Thu, 12 Aug 2010 23:00:00 +0100 3864784 http://www.medworm.com/index.php?rid=3848187&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100890 In mammalian cells MonoCarboxylate Transporters (MCTs) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4 and embigin for MCT2. In rat and rabbit erythrocytes MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT-PCR we have shown that Xenopus laevis oocytes contain endogenous basigin but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. By contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3848187 Sun, 08 Aug 2010 23:00:00 +0100 3848187 http://www.medworm.com/index.php?rid=3801793&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100759 Component C3 is the central protein of the complement system. During complement activation, the thioester group in C3 is slowly hydrolysed to form C3u, then the presence of C3u enables the rapid conversion of C3 to functionally-active C3b. C3u shows functional similarities to C3b. To clarify this mechanism, the self-association properties and solution structures of C3 and C3u were determined using analytical ultracentrifugation and X-ray scattering. Sedimentation coefficients identified two different dimerisation events in both proteins. A fast dimerisation was observed in 50 mM NaCl that was removed in 137 mM NaCl. Low amounts of a slow dimerisation was observed for C3u and C3 in both buffers. The X-ray radius of gyration RG values were unchanged for both C3 and C3u in 137 mM NaCl, but de... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3801793 Wed, 28 Jul 2010 23:00:00 +0100 3801793 http://www.medworm.com/index.php?rid=3801792&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100314 Src family kinases (SFKs) are central regulators of many signaling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein–protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix (ALG-2 [apoptosis-linked gene 2]-interacting protein X) as a novel Hck (hemopoietic cell kinase) SH3 domain interactor. The Alix–Hck-SH3 interaction was confirmed in vitro by a GST (glutathione S-transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cellulo. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 with a dissociation constant of 34.5 µM. Hete... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3801792 Wed, 28 Jul 2010 23:00:00 +0100 3801792 http://www.medworm.com/index.php?rid=3794020&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100351 β-Glucosidase from Kluyveromyces marxianus (KmBglI) belongs to the glycoside hydrolase family 3 (GH3). The enzyme is particularly unusual in that a PA14 domain (pf07691), for which a carbohydrate-binding role has been claimed, is inserted into the catalytic core sequence. Here, we determined the enzymatic properties and crystal structure of KmBglI in complex with glucose at 2.55 Å resolution. A striking characteristics of KmBglI was that the enzyme activity is essentially limited to disaccharides, and when trisaccharides were used as the substrates the activity was drastically decreased. This chain length specificity is in sharp contrast to the preferred action on oligosaccharides of barley β-D-glucan glucohydrolase (ExoI), which does not have a PA14 domain insertion. ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3794020 Mon, 26 Jul 2010 23:00:00 +0100 3794020 http://www.medworm.com/index.php?rid=3783675&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100415 In this study, formation of structurally-related lipids by oxidation of docosahexanoic acid (DHA)-containing phospholipids is demonstrated using lipidomic approaches. Precursor scanning reverse phase LC/MS/MS identified a new family of lipids that comprise phospholipid-esterified hydroxydocosahexanoic acid (HDOHE). Two diacyl and two plasmalogen phosphatidylethanolamines containing predominantly the 14-HDOHE positional isomer (18:0p/14-HDOHE-PE, 18:0a/14-HDOHE-PE, 16:0a/14-HDOHE-PE, 16:0p/14-HDOHE-PE) were structurally characterized using MS/MS and by comparison with biogenic standards. An involvement of 12-LOX was indicated since purified recombinant human 12-LOX also generated the 14-HDOHE isomer from DHA. Pharmacological studies using inhibitors and recombinant platelet 12-LOX indicate ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3783675 Thu, 22 Jul 2010 23:00:00 +0100 3783675 http://www.medworm.com/index.php?rid=3771514&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100651 Central nervous system (CNS) epinephrine is implicated in a wide range of physiological and pathological conditions. Phenylethanolamine N-methyltransferase (PNMT) catalyses the final step in the biosynthesis of epinephrine, the conversion of norepinephrine to epinephrine by methylation. To help elucidate the role of CNS epinephrine, and to develop potential drug leads, potent, selective, and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits, and the ability to sample chemical diversity more easily. Described here is the application of fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commerci... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3771514 Mon, 19 Jul 2010 23:00:00 +0100 3771514 http://www.medworm.com/index.php?rid=3757178&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100866 NrfA is a pentaheme cytochrome present in a wide-range of γ-, δ- and ε-proteobacteria. Its nitrite and nitric oxide reductase activities have been studied extensively and contribute to respiratory nitrite ammonification and nitric oxide detoxification respectively. Sulphite is a third substrate for NrfA that may be encountered in the microoxic environments where nrfA is expressed. Consequently, we have performed quantitative kinetic and thermodynamic studies of the interactions between sulphite and E. coli NrfA to provide a biochemical framework from which to consider their possible cellular consequences. A combination of voltammetric, spectroscopic and crystallographic analyses define dissociation constants for sulphite binding to NrfA in oxidised (ca. 54 µM), ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3757178 Wed, 14 Jul 2010 23:00:00 +0100 3757178 http://www.medworm.com/index.php?rid=3757177&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100609 Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) of the insulin receptor kinase (IRK) superfamily that share a –YXXXYY- autophosphorylation motif within their activation loops (A-loops). A common activation and regulatory mechanism is believed to exist for members of this superfamily typified by IRK and IGF1RK (insulin-like growth factor receptor kinase-1). Chromosomal translocations involving ALK were first identified in anaplastic large-cell lymphoma, a subtype of non-Hodgkin’s lymphoma, where aberrant fusion of the ALK kinase domain with the nucleophosmin (NPM) dimerization domain results in autophosphosphorylation and ligand-independent activation. Activating mutations within the full-length ALK kinase domain, most commonly R1275Q and F1174L, recently ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3757177 Wed, 14 Jul 2010 23:00:00 +0100 3757177 http://www.medworm.com/index.php?rid=3729356&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100289 The E. coli glutaredoxins 1 and 3 (Grx1 and Grx3) are structurally similar (37% sequence identity), yet have different activities in vivo. Unlike Grx3, Grx1 efficiently reduces protein disulfides in proteins like ribonucleotide reductase (RR), while being poor at reducing S-glutathionylated proteins. An E. coli strain lacking genes encoding thioredoxins 1 and 2 and Grx1 is not viable on either rich or minimal medium, however, a M43V mutation in Grx3 restores growth under these conditions and results in a Grx1-like protein [Ortenberg, R., et al. (2004) PNAS 101, 7439-44]. To uncover the structural basis of this change in activity, we have compared wild type and mutant Grx3 using CD and NMR spectroscopy. Ligand-induced stability measurements demonstrate that the Grx3(M43V/C65Y) mutant has ac... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3729356 Mon, 05 Jul 2010 23:00:00 +0100 3729356 http://www.medworm.com/index.php?rid=3670756&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100181 In this study, Ser313 was mutated to Ala, Cys, Phe, and Thr. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313 with Cys or Phe abolished MTX transport, whereas residual activity was preserved for the Ala and Thr mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased Vmax values without changes in Kts for MTX compared to wild type RFC. S313A and S313T differentially impacted binding of 10 diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing Cys-less TMD1-6 (N6) and TMD7-12 (C6) half-molecules with Cys insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6-C6 and N6-N6 cross-links were seen for all Cys pairs. From the N6 ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3670756 Wed, 16 Jun 2010 23:00:00 +0100 3670756 http://www.medworm.com/index.php?rid=3670755&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100315 UDP-GlcNAc is an essential precursor for glycoprotein and glycolipid synthesis. A functional nucleotidyltransferase gene from Arabidopsis encoding a 58.3 kDa N-acetylglucosamine-1-phosphate uridylyltransferase (GlcNAc1pUT-1) was identified. In the forward reaction the enzyme catalyzes the formation of UDP-N-acetylglucosamine and PPi, from the respective monosaccharide 1-phosphate and UTP. The enzyme can utilize the 4-epimer, UDP-GalNAc as a substrate as well. The enzyme requires divalent ions (Mg2+ or Mn2+) for activity and is highly active between pH 6.5-8.0, and at 30-37 ºC. The apparent Km values for the forward reaction were 337 µM (GlcNAc-1-P) and 295 µM (UTP) respectively. Another GlcNAc1pUT-2, which shares 86% amino-acid sequence identity with GlcN... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3670755 Wed, 16 Jun 2010 23:00:00 +0100 3670755 http://www.medworm.com/index.php?rid=3647852&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100649 The disintegrin and metalloprotease ADAM12 has important functions in normal physiology as well as in diseases, such as cancer. Little is known about how ADAM12 confers its protumorigenic effect; however, its proteolytic capacity is likely a key component. Thus, selective inhibition of ADAM12 activity may be of great value therapeutically and as an investigative tool to elucidate its mechanisms of actions. We have previously reported the inhibitory profile of tissue inhibitors of metalloproteinases (TIMPs) against ADAM12, demonstrating in addition to TIMP-3, a unique ADAM inhibitory activity of TIMP-2. These findings strongly suggest that it is feasible to design a TIMP mutant selectively inhibiting ADAM12. With this purpose, we characterized the molecular determinants of the ADAM12&#x2013... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3647852 Wed, 09 Jun 2010 23:00:00 +0100 3647852 http://www.medworm.com/index.php?rid=3602874&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091645 Clinically approved inhibitors of HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature, infectious viral particle. Advances in chemical synthesis have led to the development of new chemical libraries with high diversity using rapid in-solution syntheses. These libraries have been previously shown to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44,000 compounds from such a library to identify inhibitors of HIV-1 protease. One compound was identified inhibits wild type protease, as well as a drug-resistant protease with 6 mutations. Moreover, analysis of ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3602874 Wed, 26 May 2010 23:00:00 +0100 3602874 http://www.medworm.com/index.php?rid=3598741&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091855 The mitochondrial import receptor Tom70 interacts with chaperone-preprotein complexes through two domains: one that binds Hsp70/Hsc70 and Hsp90, and a second that binds preproteins. The oligomeric state of Tom70 has been controversial with evidence for both monomeric and homodimeric forms. Here, we report that the functional state of human Tom70 appears to be a monomer with mechanistic implications for its function in mitochondrial protein import. Based on analytical ultracentrifugation, cross-linking, size exclusion chromatography and multi-angle light scattering, we found that the soluble cytosolic fragment of human Tom70 exists in equilibrium between monomer and dimer. A point mutation introduced at the predicted dimer interface increased the percentage of monomeric Tom70. Although chap... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3598741 Tue, 25 May 2010 23:00:00 +0100 3598741 http://www.medworm.com/index.php?rid=3569515&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100240 Hgt1p, a high affinity glutathione transporter from Saccharomyces cerevisiae belongs to the recently described family of oligopeptide transporters (OPT), majority of whose members have still unknown substrate specificity. To obtain insights into substrate recognition and translocation, we have subjected all 21 residues of transmembrane helix 9 (TMD9) to alanine scanning mutagenesis. F523 was found to be critical for glutathione recognition, since F523A mutants showed a 4-fold increase in Km without affecting expression or localization. F523 and the previously identified polar residue Q526 were on the same face of the helix suggesting a joint participation in glutathione recognition, while two other polar residues, S519 and N522 of TMD9, although also oriented in the same face, did not appe... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3569515 Sun, 16 May 2010 23:00:00 +0100 3569515 http://www.medworm.com/index.php?rid=3551638&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100233 The metal ion coordination sites of many metalloproteins have been characterised by a variety of spectroscopic techniques and small-molecule model systems, revealing many important insights into the structural determinants of metal ion coordination. However, our understanding of this fundamentally and practically important phenomenon remains frustratingly simplistic; in many proteins it is essentially impossible to predict metal ion specificity and the effects of remote ‘outer-shell’ residues on metal ion coordination strength are also poorly defined. This is exemplified by our inability to explain why metalloenzymes with identical metal ion coordination spheres, such as the closely related orthologs of bacterial phosphotriesterase (PTE) from Agrobacterium radiobacter and Pse... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3551638 Mon, 10 May 2010 23:00:00 +0100 3551638 http://www.medworm.com/index.php?rid=3541838&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091857 Resveratrol has demonstrated cancer chemopreventive activity in animal models and some clinical trials are underway. In addition, resveratrol was shown to promote cell survival, increase lifespan and mimic caloric restriction, thereby improving health and survival of mice on high-calorie diet. All of these effects are potentially mediated by the pleiotropic interactions of resveratrol with different enzyme targets including cyclooxygenases-1 and -2 (COX-1 and COX-2), NAD+-dependent histone deacetylase SIRT1, and quinone reductase 2 (QR2). Nonetheless, the health benefits elicited by resveratrol as a direct result of these interactions with molecular targets have been questioned since it is rapidly and extensively metabolized to sulfate and glucuronide conjugates, resulting in low pl... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3541838 Thu, 06 May 2010 23:00:00 +0100 3541838 http://www.medworm.com/index.php?rid=3541837&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100337 The human cysteine peptidase cathepsin K is a key enzyme in bone homeostasis and other physiological functions. Here we investigate the mechanism of cathepsin K action at physiological plasma pH and its regulation by modifiers that bind outside of the active site. We show that at physiological plasma pH the enzyme fluctuates between multiple conformations that are differently susceptible to macromolecular inhibitors and can be manipulated by varying the ionic strength of the medium. The behavior of the enzyme in vitro can be described by the presence of two discrete conformations with distinctive kinetic properties and different susceptibility to inhibition by the substrate benzoyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin. We identify and characterize sulfated glycosaminoglycans as natu... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3541837 Thu, 06 May 2010 23:00:00 +0100 3541837 http://www.medworm.com/index.php?rid=3537546&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100386 Protein physicochemical properties must undergo complex changes during evolution, as a response to modifications in the organism environment, as a result of the proteins taking up new roles or because of the need to cope with the evolution of molecular interacting partners. Recent work has emphasized the role of stability and stability/function trade-offs in these protein adaptation processes. Here, on the other hand, we report that combinations of a few conservative, high-frequency-of-fixation mutations in the thioredoxin molecule lead to largely independent changes in both, stability and the diversity of catalytic mechanisms revealed by single-molecule atomic-force spectroscopy. Furthermore, the changes found are evolutionary significant, as they combine typically hyperthermophilic stabi... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3537546 Wed, 05 May 2010 23:00:00 +0100 3537546 http://www.medworm.com/index.php?rid=3493711&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100489 The two closely related eukaryotic AAA+ proteins, RuvBL1 (TIP49) and RuvBL2 (TIP48), are essential components of large multi-protein complexes involved in diverse cellular processes. While the molecular mechanisms of RuvBL1 and RuvBL2 remain unknown, oligomerization is likely to be important for their function together or individually and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1/2 complex and the role of the ATPase-insert domain (Domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexameris... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3493711 Wed, 21 Apr 2010 23:00:00 +0100 3493711 http://www.medworm.com/index.php?rid=3490013&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091843 This study investigates the aggregation state of purified full-length PKD2L1-CRD as well as truncations of CRDs from PKD2 channels. Far- and Near-UV CD spectroscopy show that the full-length PKD2L1 CRD (PKD2L1-198) and the truncated PKD2 CRD (PKD2-244) are α-helical with no β-sheet, the α-helix content agrees with sequence-based predictions, and some of its aromatic residues are in an asymmetric environment created at least by partially structured regions. Additionally, the CRD truncations exhibit an expected biochemical function by binding Ca2+ in a physiologically relevant range with Kds of 2.8 μM for PKD2-244 and 0.51 μM for PKD2L1-198. Complimentary biophysical and biochemical techniques establish that truncations of the PKD2 and PKD2L1 CRDs ar... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3490013 Tue, 20 Apr 2010 23:00:00 +0100 3490013 http://www.medworm.com/index.php?rid=3373611&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100056 Human angiotensin-converting enzyme (ACE; EC 3.4.15.1) is an important drug target due to its role in the regulation of blood pressure via the renin-angiotensin-aldosterone system. Somatic ACE comprises two homologous domains, the differing substrate preferences of which present a new avenue for domain-selective inhibitor design. We have co-crystallised lisW-S, a C-domain-selective derivative of the drug lisinopril, with human testis ACE and determined a structure by X-ray crystallography to a resolution of 2.30 Å. In this structure, lisW-S is seen to have a similar binding mode to its parent compound lisinopril, but the P2′ Trp moiety takes a different conformation to that seen in other inhibitors having a Trp in this position. We have further examined the domain-specific in... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3373611 Wed, 17 Mar 2010 00:00:00 +0100 3373611 http://www.medworm.com/index.php?rid=3326314&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091939 This study has identified an aromatic ‘zipper’ in the H-site contributing a network of aromatic π-π interactions. Several residues of the cluster directly interact with the hydrophobic substrate while others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Tyr-119-Glu mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the ‘zipper’ that contributes to the H-site remodeling, thus illustrating a role in co-substrate binding modulation. Phe-123-Ala structure shows molecular rearrangement of the H-site in one subunit but not the other, explaining weakened hydrophobic substrate binding and kinetic cooperativity effects of Phe-123 ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3326314 Wed, 03 Mar 2010 00:00:00 +0100 3326314 http://www.medworm.com/index.php?rid=3163184&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091530 The production of selective protein kinase inhibitors is often frustrated by the similarity of the enzyme active sites. For this reason, it is challenging to design inhibitors that discriminate between the three Aurora kinases, which are important targets in cancer drug discovery. We have used a triple point mutant of Aurora-A (AurAx3) that mimics the active site of Aurora-B to investigate the structural basis of MLN8054 selectivity. The bias toward Aurora-A inhibition by MLN8054 is fully recapitulated by AurAx3 in vitro. X-ray crystal structures of the complex suggest that the basis for the discrimination is electrostatic repulsion due to the Thr271Glu substitution, which we have confirmed using a single point mutant. The activation loop of Aurora-A in the AurAx3/MLN8054 complex exhibits ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3163184 Tue, 12 Jan 2010 00:00:00 +0100 3163184 http://www.medworm.com/index.php?rid=3108061&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091501 Hemocyanins are multimeric oxygen transport proteins which bind oxygen to type 3 copper sites. Arthropod hemocyanins are composed of 75 kDa subunits, while molluscan hemocyanins contain 350-400 kDa subunits with 7-8 different 50 kDa “functional units” (FU‑a to FU‑h), each with an active site. FU‑h possesses a tail of 100 amino acids not present in the other FUs. Here we show by X-ray crystallography of FU-h of keyhole limpet hemocyanin isoform 1 (KLH1) that the structure of the tail domain is cupredoxin-like but contains no copper. The copper-free domain #3 in arthropod hemocyanin subunits has recently also been reinterpreted as being cupredoxin-like. We propose that the cupredoxin-like domain in both hemocyanin types once served to upload copper to the a... BJ Structure journals http://www.medworm.com/rss/comments.php?id=3108061 Mon, 21 Dec 2009 00:00:00 +0100 3108061 http://www.medworm.com/index.php?rid=2962569&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091499 Fungal aryl-alcohol oxidase (AAO) provides H2O2 for lignin biodegradation. AAO is active on benzyl alcohols that are oxidized to aldehydes. However, the H2O2 formed from some of them was more than stoichiometric with respect to the aldehyde detected. This was due to a double reaction that involves aryl-aldehyde oxidase activity. The latter was investigated using different benzylic aldehydes, whose oxidation to acids was demonstrated by GC-MS. The steady and pre-steady state kinetic constants together with the chromatographic results revealed a strong influence of substrate electron withdrawing/donating substituents on activity, being the highest on p-nitrobenzaldehyde and halogenated aldehydes and the lowest on methoxylated aldehydes. Moreover, activity was correlated to the aldehyde hydra... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2962569 Thu, 05 Nov 2009 00:00:00 +0100 2962569 http://www.medworm.com/index.php?rid=2958298&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091167 Current drug therapies against Trypanosoma cruzi, causative agent of Chagas disease, have limited effectiveness and are highly toxic. T. cruzi-specific metabolic pathways that utilise trypanothione for the reduction of peroxides are being explored as potential novel therapeutic targets. We solved the X-ray crystal structure of one of the T. cruzi enzymes involved in peroxide reduction, the glutathione peroxidase-like enzyme TcGPXI. We also characterized the wild-type, Cys48Gly and Cys96Gly variants of TcGPXI by NMR spectroscopy and biochemical assays. Our data show that residues Cys48 and Cys96 are required for catalytic activity. In solution, the TcGPXI molecule readily forms a Cys48-Cys96 disulfide bridge and the polypeptide segment containing Cys96 lacks regular secondary structure. NMR... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2958298 Tue, 03 Nov 2009 00:00:00 +0100 2958298 http://www.medworm.com/index.php?rid=2942973&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091209 Protein-bile acid interactions are crucial microscopic events at the basis of both physiological and pathological biochemical pathways. Bile acid binding proteins are intracellular transporters able to bind ligands with different stoichiometry, selectivity, and cooperativity. The molecular determinants and energetics of interaction are the observables that connect the microscopic to the macroscopic frameworks. This paper addresses the study and proposes a mechanism for the multi-site interaction of bile acids with chicken ileal bile acid binding protein (I-BABP) with the aim of elucidating the determinants of ligand binding in comparison to homologous proteins from different species and tissues. A thermodynamic binding model describing two independent consecutive binding sites is derived f... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2942973 Thu, 29 Oct 2009 00:00:00 +0100 2942973 http://www.medworm.com/index.php?rid=2934086&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091422 The eukaryotic transcription elongation factor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) sensitivity inducing factor (DSIF) is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF together with the negative elongation factor NELF, leads to promoter-proximal pausing of RNA polymerase II. Here we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176 to 273) at a resolution of 1.55 Å. The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hS... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2934086 Tue, 27 Oct 2009 00:00:00 +0100 2934086 http://www.medworm.com/index.php?rid=2926775&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091441 The side chains of Asn-191 and Asn-300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagines by Ala. The rate constants for the overall hydride transfer to and from C2 of mannitol which were estimated as ~5 × 102 s-1 and ∼1.5 × 103 s-1 in the wild-type enzyme, respectively, were selectively slowed, between ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2926775 Mon, 26 Oct 2009 00:00:00 +0100 2926775 http://www.medworm.com/index.php?rid=2919485&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091254 Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature mid-point (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (∆Heq) to its ability to function over various temperature ranges. We show here that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independen... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2919485 Wed, 21 Oct 2009 23:00:00 +0100 2919485 http://www.medworm.com/index.php?rid=2845796&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091207 Human kinesin Eg5 plays an essential role in mitosis by separating duplicated centrosomes and establishing the bipolar spindle. Eg5 is an interesting drug target for the development of cancer chemotherapy, with 7 inhibitors already in clinical trials. Here, we report the crystal structure of the Eg5 motor domain complexed with a potent antimitotic inhibitor S-trityl-L-cysteine to 2.0 Å resolution. The Eg5-STLC complex crystallises in space group P32 with 3 molecules per asymmetric unit. Two of the molecules reveal the final inhibitor-bound state of Eg5, whereby Loop L5 has swung downwards to close the inhibitor-binding pocket, helix α-4 has rotated by about 15° and the neck-linker has adopted a docked conformation. The third molecule, however, revealed an unprecedented... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2845796 Tue, 29 Sep 2009 23:00:00 +0100 2845796 http://www.medworm.com/index.php?rid=2795921&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090888 N-acetyl-L-glutamate (NAG), the essential allosteric activator of the first urea cycle enzyme, carbamoyl-phosphate synthetase (CPSI), is a key regulator of this crucial cycle for ammonia-detoxification in animals (including humans). Automated cavity searching and flexible docking have allowed identification of the NAG site in the crystal structure of human CPSI C-terminal domain. The site, a pocket lined by invariant residues and located between the central β-sheet and two α-helices, opens at the β-sheet C-edge and is roofed by a 3-residue lid. It can tightly accommodate one extended NAG molecule having the δ-COO- at the pocket entry, the α-COO- and acetamido groups tightly hydrogen-bonded to the pocket, and the terminal methyl of the acetamido substituen... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2795921 Mon, 14 Sep 2009 23:00:00 +0100 2795921 http://www.medworm.com/index.php?rid=2792711&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091079 Multinuclear Cu(I) clusters are common in nature, but little is known about their formation or transfer between proteins. CopZ and CopA from Bacillus subtilis, which are involved in a copper-efflux pathway, both readily accommodate multinuclear Cu(I) clusters. Using the luminescence properties of a multinuclear Cu(I)-bound form of the two N-terminal soluble domains of CopA (CopAab) we have investigated thermodynamic and kinetic properties of cluster formation and loss. We demonstrate that Cu(I)-bound forms of dimeric CopZ containing more than 1 Cu(I) per CopZ monomer can transfer Cu(I) to apo-CopAab leading to the formation of luminescent dimeric CopAab. Kinetic studies demonstrated that transfer is a first order process and that the rate determining steps for transfer from CopZ to CopAab ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2792711 Sun, 13 Sep 2009 23:00:00 +0100 2792711 http://www.medworm.com/index.php?rid=2729447&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090723 On average, each human gene has about four single nucleotide polymorphisms (SNPs) in the coding region, half of which are non-synonymous or missense SNPs (nsSNP). Current attention is focused on those that are known to perturb function and are strongly linked to disease. However, the vast majority of SNPs have not been investigated for the possibility of causing disease. We set out to assess the fraction of nsSNPs that encode proteins that have altered stability and activity, for this class of variants would be candidates to perturb cellular function. We tested the thermostability and, where possible, the catalytic activity for the most common variant (wild-type) and minor variants (total of 46 SNPs) for 16 human enzymes for which the 3D structures were known. There were significant differ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2729447 Mon, 24 Aug 2009 23:00:00 +0100 2729447 http://www.medworm.com/index.php?rid=2722949&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090732 T cell receptor (TCR) recognition of antigenic peptides bound and presented by major histocompatibility complex (MHC) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide/MHC molecules weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Further, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. Here, we studied how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the HTLV-1 Tax11-19 peptide (Tax) enhanced binding by th... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2722949 Thu, 20 Aug 2009 23:00:00 +0100 2722949 http://www.medworm.com/index.php?rid=2719187&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090540 We report here biochemical and structural studies on caspase-6. As an effector caspase, caspase-6 is a constitutive dimer independent of the maturation state of the enzyme. The ligand-free structure shows caspase-6 in a partially mature but latent conformation. The cleaved inter-domain linker remains partially inserted in the central groove of the dimer as observed in other caspases. However, in contrast to previous structures, not only is the catalytic machinery mis-aligned, but several structural elements required for substrate recognition are missing. Most importantly, residues forming a short anti-parallel β-sheet abutting the substrate in other caspase structures are part of an elongation of the central helix. Despite the dramatic structural changes that would be required to ad... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2719187 Thu, 20 Aug 2009 23:00:00 +0100 2719187 http://www.medworm.com/index.php?rid=2711558&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090543 Synopsis Steroid hormone receptor maturation is a multi-step process that involves several TPR proteins that bind to the maturation complex via the C-termini of hsp70 and hsp90. We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat shock proteins in the TPR/hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5 although mutation studies produced no evidence that they bound to the domain’s hsp90-binding groove. However,... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2711558 Mon, 17 Aug 2009 23:00:00 +0100 2711558 http://www.medworm.com/index.php?rid=2673852&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090626 We report here that the C-terminal domain of Neisseria gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460 to 658 exhibits Mn2+ dependent endonuclease activity. Sedimentation velocity,sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal binding motif, DMHA(X)2E(X) 4E and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore, suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein but have MutL proteins that harbor the D[M/Q]HA... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2673852 Wed, 05 Aug 2009 23:00:00 +0100 2673852 http://www.medworm.com/index.php?rid=2644545&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090780 It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins (EtuA and EtuB) are found encoded on the genome clustered with the genes for ethanol utilisation. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates, and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, whi... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2644545 Mon, 27 Jul 2009 23:00:00 +0100 2644545 http://www.medworm.com/index.php?rid=2629395&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090870 Chromodomain helicase DNA-binding protein 4 (CHD4) ATPase is a major subunit of the repressive NuRD (nucleosome remodeling and deacetylase) complex, which is involved in transcriptional regulation and development. CHD4 contains two plant homeodomain (PHD) fingers of unknown function. Here we show that the second PHD finger (PHD2) of CHD4 recognizes the amino-terminus of histone H3 and that this interaction is facilitated by acetylation or methylation of Lys9 (H3K9ac and H3K9me, respectively) but is inhibited by methylation of Lys4 (H3K4me) or acetylation of Ala1 (H3A1ac). An 18 μM binding affinity toward unmodified H3 rises to 0.6 μM for H3K9ac and to 0.9 μM for H3K9me3, while dropping to 2.0 mM for H3K4me3, as measured by tryptophan fluorescence and NMR. A peptide lib... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2629395 Tue, 21 Jul 2009 23:00:00 +0100 2629395 http://www.medworm.com/index.php?rid=2613394&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090786 The social amoeba Dictyostelium discoideum has become established as a simple model for the examination of cell-cell interactions and early studies suggested that shifts in glycosylation profiles take place during its life cycle. In the present study, we have applied HPLC and mass spectrometric methods to show that the major N-glycans in axenic cultures of the AX3 strain are oligomannosidic forms, most of which carry core fucose and/or intersecting and bisecting N-acetylglucosamine residues, including the major structure with the composition Man8GlcNAc4Fuc1. The postulated α1,3-linkage of the core fucose which correlates with the cross-reactivity of Dictyostelium glycoproteins with an anti-horseradish peroxidase antiserum; a corresponding core α1,3-fucosyltransferase activity... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2613394 Thu, 16 Jul 2009 23:00:00 +0100 2613394 http://www.medworm.com/index.php?rid=2605360&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090565 In this study we show that the b’ domain of human PDI is in conformational exchange but that its structure is stabilised by the addition of peptide ligands or by binding the x-linker region. The location of the ligand binding site in b’ was mapped by NMR chemical shift perturbation and found to consist primarily of residues from the core β-sheet and helices 1 and 3. This site is where the x-linker region binds in the x-ray structure of b’x and we show that peptide ligands can compete with x binding at this site. The finding that x binds in the principal ligand binding site of b’ further supports the hypothesis that x functions to gate access to this site and so modulates PDI activity. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=2605360 Tue, 14 Jul 2009 23:00:00 +0100 2605360 http://www.medworm.com/index.php?rid=2601198&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090913 This study provides structural information on HSA-lysophospholipids interaction and may facilitate the understanding of the transport and distribution of lysophospholipids. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=2601198 Mon, 13 Jul 2009 23:00:00 +0100 2601198 http://www.medworm.com/index.php?rid=2575823&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090738 Pup from Mycobacterium tuberculosis (Mtb) is the first ubiquitin-like protein identified in non-eukaryotic cells. Though different ubiquitin-like proteins from eukaryotes share low sequence similarity, their three-dimensional (3D) structures exhibit highly conserved typical ubiquitin-like fold. Interestingly, our studies reveal that Pup not only shares low sequence similarity but also presents a totally distinguished structure compared with other ubiquitin-like superfamily proteins. Diverse structure predictions combined with CD and NMR spectroscopic studies all demonstrate that Pup is an intrinsically disordered protein. Moreover, {1H}-15N nuclear Overhauser effect (NOE) data and chemical shift index (CSI) analyses indicate that there is a residual secondary structure at the C-terminus of... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2575823 Mon, 06 Jul 2009 23:00:00 +0100 2575823 http://www.medworm.com/index.php?rid=2569155&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090702 Substrate specificity is critically important for enzyme catalysis. In the adrenaline synthesising enzyme phenylethanolamine N-methyltransferase (PNMT), minor changes in substituents can convert substrates into inhibitors. Here we report the crystal structures of 6 human PNMT complexes including the first structure of the enzyme in complex with its physiological ligand R-noradrenaline: determining this structure required rapid soak methods because of the tendency for noradrenaline to oxidize. Comparison of the PNMT:noradrenaline complex with the previously determined PNMT:octopamine complex demonstrates that these two substrates form almost equivalent interactions with the enzyme and show that octopamine is a valid model substrate for PNMT. The crystal structures illustrate the adaptabilit... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2569155 Wed, 01 Jul 2009 23:00:00 +0100 2569155 http://www.medworm.com/index.php?rid=2514958&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090134 In this report, we demonstrate that Pygopus can interact with the histone acetyltransferase (HAT) cyclic AMP responsive element binding protein (CREB) binding protein (CBP). The interaction is via the N-terminal homology domain (NHD) of Pygopus which binds to two regions in the HAT domain of CBP. Transfected and endogenous hPygo2 and CBP proteins co-immunoprecipitate in human embryonic kidney (HEK293) cells and both proteins co-localize in SW480 colorectal cancer cells. The interaction with CBP also enhances both DNA-tethered and TCF/LEF1-dependent transcriptional activity of Pygopus. Furthermore, immunoprecipitated Pygopus protein complexes displayed CBP-dependent histone acetyltransferase activity. Our data support a model in which the NHD region of Pygopus is required to augment T-cell ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2514958 Wed, 24 Jun 2009 23:00:00 +0100 2514958 http://www.medworm.com/index.php?rid=2503386&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090224 Cytosolic glutathione transferases (GSTs) are a multifunctional group of enzymes widely distributed in nature and involved in cellular detoxification processes. The three-dimensional structure of Glycine max GSTU4-4 (GmGSTU4-4) complexed with glutathione (GSH) was determined by the molecular replacement method at 2.7 Å resolution. The bound GSH is located in a region formed by the beginning of α-helices H1, H2 and H3 in the N-terminal domain of the enzyme. Significant differences in the GSH binding site (G-site) as compared to the structure determined in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH) were found. These differences were identified in the hydrogen-bonding and electrostatic interaction pattern and, consequently, GSH was found bound in two different conformat... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2503386 Thu, 18 Jun 2009 23:00:00 +0100 2503386 http://www.medworm.com/index.php?rid=2503387&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090853 Cation diffusion facilitator transporters are found in all three kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include: Zn2+, Co2+, Fe2+, Cd2+, Ni2+, Mn2+, however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ (Lin et al, (2008) J. Biol. Chem. 283: 33865-33873). Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amin... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2503387 Wed, 17 Jun 2009 23:00:00 +0100 2503387 http://www.medworm.com/index.php?rid=2503388&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090010 Tissue kallikrein 1 (KLK1) is a member of the tissue kallikrein family of serine proteases and the primary kinin generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki = 0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface plasmon resonance biosensor assay when KLK1 was complexed to known active site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were tested at concentration of 1 µM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva, and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2503388 Mon, 15 Jun 2009 23:00:00 +0100 2503388 http://www.medworm.com/index.php?rid=2503390&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090198 Complement plays crucial roles in the immune system, but incorrect regulation causes inflammation and targeting of self-tissue, leading to diseases such as systemic lupus erythematosus, rheumatoid arthritis and age-related macular degeneration. In vivo, the initiating complexes of the classical and lectin pathways are controlled by SERPING1 (C1-inhibitor), which inactivates their C1s and MASP-2 protease components. Glycosaminoglycans (GAGs) and dextran sulfate (DXS) are able to significantly accelerate SERPING1-mediated inactivation of the key effector enzyme of the classical C1 complex, C1s, though the mechanism is poorly understood. Here it was shown that C1s can bind to DXS and heparin and that these polyanions could enhance C1s proteolytic activity at low concentrations and inhibit it ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2503390 Thu, 11 Jun 2009 23:00:00 +0100 2503390 http://www.medworm.com/index.php?rid=2503389&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090405 This study defines the highly conserved segment spanning Glu96-Asp111 in FV as multifunctional. Of the three amino acids evaluated, Asp111 is essential and likely functions through direct and indirect effects on Ca2+ and Cu2+ interactions. Glu96 and Asp102 individually influence FV/FVa by more subtle effects possibly at the metal ion-dependent subunit interface. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=2503389 Thu, 11 Jun 2009 23:00:00 +0100 2503389 http://www.medworm.com/index.php?rid=2503391&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090534 Small RNAs modulate gene expression by forming a ribonucleoprotein complex with Argonaute proteins and direct them to specific complementary sites in target nucleic acids. However, the interactions required for the recruitment of target nucleic acid to the ribonucleoprotein complex are poorly understood. Here, we have investigated this question by using let-7a, Argonaute2 and a fully complementary mRNA target. Importantly, we have found recombinant Argonaute2 is sufficient to direct let-7a guided cleavage of mRNA. Thus this model system has allowed us to investigate the mechanistic basis of silencing in vitro and in vivo. Current models suggest that Argonaute proteins bind to both the 5’ and 3’ termini of the guide RNA. Here, we find that the termini of the let-7a microRN... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2503391 Tue, 09 Jun 2009 23:00:00 +0100 2503391 http://www.medworm.com/index.php?rid=2467841&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090180 Arabinanases are glycosidases that hydrolyze alpha-(1→5)- arabinofuranosidic linkages found in the backbone of the pectic polysaccharide arabinan. Here we describe the biochemical characterization and the enzyme-substrate crystal structure of an inverting family 43 arabinanase, from Geobacillus stearothermophilus T-6 (AbnB). Based on viscosity and reducing power measurements, and based on products analysis for the hydrolysis of linear arabinan by AbnB, the enzyme work in an endo mode of action. Isothermal titration calorimetry studies of a catalytic mutant with various arabinooligosaccharides suggested that the enzyme active site can accommodate at least five arabinose units. The crystal structure of AbnB was determined at 1.06 Å resolution revealing a single five-bladed-beta... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2467841 Mon, 08 Jun 2009 04:00:00 +0100 2467841 http://www.medworm.com/index.php?rid=2455839&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090782 Archaeal and eukaryotic RNAPs are comprised of twelve subunits with discrete functions. Two of the subunits, F/E, have been hypothesised to associate with RNAP in a reversible manner during the transcription cycle. We have characterised the molecular interactions between F/E and the RNAP core. F/E binds to RNAP with submicromolar affinity but is not in a dynamic exchange with unbound F/E. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=2455839 Wed, 03 Jun 2009 04:00:00 +0100 2455839 http://www.medworm.com/index.php?rid=2446195&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090434 This study focuses on the metal-binding properties of UreE from Helicobacter pylori (HpUreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. Isothermal titration calorimetry (ITC) showed that HpUreE binds one equivalent of Ni2+ (Kd = 0.15 mM) or Zn2+ (Kd = 0.49 mM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn2+ and Ni2+, which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal ion specific binding. The formation of a HpUreE-HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2446195 Fri, 29 May 2009 04:00:00 +0100 2446195 http://www.medworm.com/index.php?rid=2439764&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090474 In this study, we identified FAM20B as a kinase that phosphorylates the xylose residue in the linkage region. Overexpression of fam20b increased the amount of both chondroitin sulfate and heparan sulfate in HeLa cells, while the RNA interference of fam20b resulted in a reduction of their amount in the cells. Gel filtration analysis of the glycosaminoglycan chains synthesized in the overexpressing cells revealed that the glycosaminoglycan chains had similar length to those in mock-transfected cells. These results suggest that FAM20B regulates the number of glycosaminoglycan chains by phosphorylating the xylose residue in the glycosaminoglycan-protein linkage region of proteoglycans. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=2439764 Thu, 28 May 2009 04:00:00 +0100 2439764 http://www.medworm.com/index.php?rid=2422655&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090422 The homeostasis of metal ions in cells is the result of the contribution of several cellular pathways that involve transient, often weak, protein-protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by coordinating residues of both interacting partners. Here we addressed the interaction between the human copper(I)-chaperone HAH1 and one metal-binding domain of one of its partners, namely the P-type copper-transporting ATPase ATP7A. The adduct was structurally characterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cadmium(II). Further insight was obtained through molecular modelling techniques and site-directed mutagenesis. It is found that ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2422655 Tue, 19 May 2009 04:00:00 +0100 2422655 http://www.medworm.com/index.php?rid=2422654&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082242 We have designed and chemically synthesised an artificial β-defensin based on a minimal template derived from the comparative analysis of over 80 naturally occurring sequences. This molecule has the disulfide-bridged, β-sheet core structure of natural β-defensins and shows a robust, salt-sensitive antimicrobial activity against bacteria and yeast, as well as a chemotactic activity against immature dendritic cells. An SAR study using two truncated fragments or a Cys→Ser point-mutated analogue, in which one or two of the three disulphide bridges were absent, indicated that altering the structure resulted in a different type of membrane interaction and a switch to different modes of action towards both microbial and host cells, and that covalent dimerisation could ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2422654 Tue, 19 May 2009 04:00:00 +0100 2422654 http://www.medworm.com/index.php?rid=2422656&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082330 Eosinophil cationic protein (ECP) is an eosinophil secretion protein with antipathogen activities involved in the host immune defense system. The bactericidal capacity of ECP relies on its action on both the plasma membrane and the bacterial wall. In a search for the structural determinants of ECP antimicrobial activity, we have identified an N-terminal domain (residues 1-45) that retains most of ECP’s membrane-destabilizing and antimicrobial activities. Two sections of this domain, ECP(1-19) and ECP(24-45), have also been evaluated. All three peptides bind and partially insert into lipid bilayers, inducing aggregation of lipid vesicles and leakage of their aqueous content. In such an environment, the peptides undergo conformational change, significantly increasing their α-he... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2422656 Mon, 18 May 2009 04:00:00 +0100 2422656 http://www.medworm.com/index.php?rid=2413009&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090528 The 26S proteasome is a 2,500,000-dalton protease complex that degrades polyubiquitylated proteins by a mechanism that requires ATP hydrolysis. It also degrades short non-ubiquitylated peptides and certain unstructured proteins by an energy-independent mechanism that requires bound ATP to maintain its component subcomplexes, the 20S proteasome and PA700, in a functionally assembled state. Proteolysis of both types of substrates requires PA700-induced opening of reversible gates at substrate access pores of the 20S proteasome. Here we demonstrate that the rate of peptide substrate hydrolysis, a functional monitor of gate opening, is regulated variably by multiple effectors. ATPγS and other nonhydrolyzable ATP analogs increased peptide substrate hydrolysis by intact 26S proteasome. T... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2413009 Wed, 13 May 2009 04:00:00 +0100 2413009 http://www.medworm.com/index.php?rid=2413008&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090379 Alzheimer’s disease (AD) is linked to amyloid beta peptide (Aβ) misfolding. Studies demonstrate that the level of soluble Aβ oligomeric forms correlates better with the progression of the disease than the level of fibrillar forms. Conformation-dependent antibodies have been developed to detect either Aβ oligomers or fibrils, suggesting that structural differences between these forms of Aβ exist. Using conditions which yield well-defined Aβ(1-42) oligomers or fibrils, we studied the secondary structure of these species by attenuated total reflection-FTIR spectroscopy. Whereas fibrillar Aβ was organized in a parallel β-sheet conformation, oligomeric Aβ displayed distinct spectral features, which were attributed to an anti-paralle... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2413008 Wed, 13 May 2009 04:00:00 +0100 2413008 http://www.medworm.com/index.php?rid=2413010&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090265 Galectin-1 (gal-1) mediates cell-cell and cell-extracellular matrix adhesion, essentially by interacting with β-galactoside-containing glycans of cell surface glycoconjugates. While most structural studies with gal-1 have investigated its binding to simple carbohydrates, in particular lactose and N-acetyllactosamine, this view is limited because gal-1 functions at the cell surface by interacting with more complex glycans that are heterogeneous in size and composition. Here, we used NMR spectroscopy to investigate the interaction of human gal-1 to a large (120 kDa), complex glycan [GRG: α-(1→2)-L-rhamnosyl-α-(1→4)-D-galacturonosyl] that contains non-randomly distributed, mostly terminal β(1→4)-linked galactose side-chains. We used 15N-1H HSQC... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2413010 Tue, 12 May 2009 04:00:00 +0100 2413010 http://www.medworm.com/index.php?rid=2413011&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090069 Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a moderately potent and poorly selective inhibitor of CK2, one of the most pleiotropic Ser/Thr protein kinases, implicated in neoplasia and in other global diseases. By virtual screening of the MMS database we have now identified quinalizarin (1,2,5,8-tetrahydroxy-anthraquinone) as an inhibitor of CK2 more potent and selective than emodin. CK2 inhibition by quinalizarin is competitive with respect to ATP, with a Ki value around 50 nM. Tested at 1 μM concentration on a panel of 75 protein kinases, quinalizarin drastically inhibits only CK2, with a promiscuity score (11.1) which is the lowest ever reported so far for a CK2 inhibitor. Especially remarkable is the ability of quinalizarin to discriminate between CK2 and a number of kina... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2413011 Mon, 11 May 2009 04:00:00 +0100 2413011 http://www.medworm.com/index.php?rid=2396664&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090427 The IKK complex is a key regulatory component of NF-κB activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO, the regulatory subunit of the IKK complex plays a pivotal role in this process by integrating upstream signals, in particular the recognition of poly-ubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a te... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2396664 Thu, 07 May 2009 04:00:00 +0100 2396664 http://www.medworm.com/index.php?rid=2379968&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090268 Protein arginine N-methyltransferases (PRMTs) methylate arginine residues within proteins using S-adenosyl-L-methionine (AdoMet) to form S-adenosyl-L-homocysteine and methylarginine residues. All PRMTs produce ω-NG-monomethylarginine (MMA) residues and either asymmetric ω-NG-,NG--dimethylarginine (aDMA) or symmetric ω-NG-,N’G--dimethylarginine (sDMA) residues, referred to as Type I or Type II activity, respectively. Here we report methylation activity from PRMT2 and compare it to PRMT1 activity using ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), gel electrophoresis, and thin layer chromatography. We show that PRMT2 is a Type I enzyme and that the ratio of aDMA to MMA produced by PRMTs 1 and 2 is dependent on the substrate reg... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2379968 Thu, 30 Apr 2009 04:00:00 +0100 2379968 http://www.medworm.com/index.php?rid=2374151&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081395 The enzyme kinetics of the amide ligase MurE from Pseudomonas aeruginosa were determined using the synthesized nucleotide substrate UDP-MurNAc-Ala-Glu. When coupled to a competitive bio-panning using a M13 phage display library encoding ~ 2.7 x 109 random peptide permutations and the specific substrates meso-A2pm and ATP, a peptide inhibitor of MurE was identified. The MurEp1 dodecamer selected and synthesized inhibited MurE ATPase activity with an IC50 value of 500 μM. The inhibition was shown to be time-dependent and reversed by the addition of meso-A2pm or UDPMurNAc- Ala-Glu during the pre-incubation step. Kinetic analysis defined MurEp1 as a mixed inhibitor against both substrates with Ki values of 160 and 80 μM, respectively. MurEp1 was found to interfere in meso-A2pm an... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2374151 Tue, 28 Apr 2009 04:00:00 +0100 2374151 http://www.medworm.com/index.php?rid=2374152&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090175 Receptor associated protein (RAP) is a three domain 38 kDa ER-resident protein that is a chaperone for the low density lipoprotein receptor-related protein (LRP). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple complement-like repeats (CR) that span the principal ligand binding region, cluster II, of LRP. We found that D3 binds with low nanomolar affinity to all (CR)2 species examined. Addition of a third CR domain increases the affinity for D3 slightly. pH change from 7.4 to 5.5 gave only a 6-fold increase in Kd for D3 at 37&#x00B0... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2374152 Mon, 27 Apr 2009 04:00:00 +0100 2374152 http://www.medworm.com/index.php?rid=2363588&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081968 The aromatic substrate prenyltransferase (AS-PT) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane α-helices. Para-hydroxybenzoic acid prenyltransferases (PPTs) are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in this study as representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three of s... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2363588 Fri, 24 Apr 2009 04:00:00 +0100 2363588