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. Wed, 17 Mar 2010 16:26:58 +0100 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 http://www.medworm.com/index.php?rid=2339874&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090128 Despite their widely varying physiological functions in carbonyl metabolism, Candida tenuis xylose reductase (AKR2B5) and many related enzymes of the aldo-keto reductase protein superfamily utilise 9,10 phenanthrenequinone (PQ) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80, His113) of AKR2B5 in the conversion of the reactive α-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by Ala) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 103 –... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2339874 Wed, 15 Apr 2009 04:00:00 +0100 2339874 http://www.medworm.com/index.php?rid=2339873&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090091 This study provides structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment or to design BChE-based catalytic bioscavengers. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=2339873 Wed, 15 Apr 2009 04:00:00 +0100 2339873 http://www.medworm.com/index.php?rid=2323263&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082170 In this study, we determined the crystal structure of APA and all sugar moieties of the N196-glycan were clearly observed in the electron-density map. Although the sugar moieties of the glycan generally have high structural flexibility, most sugar moieties of the N196-glycan were well organized in the deep cleft of the subunit interface, and mediated many inter- and intra-subunit H-bonds. Analytical ultracentrifugation and guanidinium chloride (GdmCl) unfolding experiments revealed that the presence of the N196-glycan was important for stabilizing the hexameric state and overall stability of APA, respectively. Our results could provide a structural basis for studying not only other glycoproteins that carry an immature N-glycan, but also the structural role of N-glycans that are located in ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2323263 Thu, 09 Apr 2009 04:00:00 +0100 2323263 http://www.medworm.com/index.php?rid=2323265&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082428 Multiheme cytochromes that could form protein “nanowires” were identified in Geobacter sulfurreducens genome and represent a new type of multiheme cytochromes. The sequences of these proteins, two with 12-hemes (GSU1996, GSU0592) and one with 27-hemes (GSU2210) suggest that they are formed by domains homologous to triheme cytochromes c7. While all three hemes have bis-His coordination in cytochromes c7, in each domain of the above polymers the heme equivalent to heme IV has His-Met coordination. We previously determined the structure and measured the macroscopic redox potential of one representative domain (domain C) of a dodecaheme cytochrome (GSU1996). In the present study, the microscopic redox properties of the individual heme groups of domain C were determined using NMR ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2323265 Wed, 08 Apr 2009 04:00:00 +0100 2323265 http://www.medworm.com/index.php?rid=2323267&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090336 Citramalate synthase (LiCMS) catalyzes the first reaction of the isoleucine biosynthesis pathway in Leptospira interrogan, the pathogen of leptospirosis. The catalytic reaction is regulated through feedback inhibition by its end-product isoleucine. To understand the molecular basis of the high selectivity of inhibitor and the mechanism of feedback inhibition, we determined the crystal structure of the regulatory domain of LiCMS (LiCMSC) in complex with Ile and performed biochemical study of the inhibition of LiCMS using mutagenesis and kinetics methods. LiCMSC forms a dimer of dimers in both the crystal structure and solution, and the dimeric LiCMSC is the basic functional unit. LiCMSC consists of six β-strands forming two anti-parallel β-sheets and two α-helices and a... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2323267 Tue, 07 Apr 2009 04:00:00 +0100 2323267 http://www.medworm.com/index.php?rid=2323266&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082238 The ClpB chaperone forms a hexamer ring and rescues aggregated proteins in cooperation with the DnaK system. Each subunit of ClpB has two nucleotide-binding modules, AAA-1 and AAA-2, and an 85-Å-long coiled-coil. The coiled-coil consists of two halves: wing-1, leaning toward AAA-1, and wing-2, leaning away from all the domains. The coiled-coil is stabilized by leucine zipper-like interactions between Leu and Ile residues of two amphipathic α-helices that twist around each other to form each wing. To destabilize the two wings, we developed a series of mutants by replacing these residues with Ala. As the number of replaced residues increased, the chaperone activity was lost and the hexamer became unstable. The mutants, which had a stable hexameric structure but lost the chapero... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2323266 Tue, 07 Apr 2009 04:00:00 +0100 2323266 http://www.medworm.com/index.php?rid=2295036&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090206 IscA/SufA paralogs are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. While deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant can be partially restored by supplementing with the branched-chain amino acids (BCAA) and thiamin. We further demonstrate that the iscA/sufA double mutant fails to assemble the [4Fe-4S] cluster in dihydroxyacid dehydratase (IlvD) of the BCAA biosynthesis pathway under aerobic conditions. In vitro studies show that the iron-bound IscA/SufA can efficiently provide iron for the [4Fe-4S] cluster assembly in IlvD and restore the enzyme activi... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2295036 Tue, 24 Mar 2009 04:00:00 +0100 2295036 http://www.medworm.com/index.php?rid=2283093&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081991 CCN family 2/connective tissue growth factor (CCN2/CTGF) is a multicellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF interactive proteins capable of modulating its action in cartilage, we carried out a yeast-two hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. Here we report on the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, as a CCN2/CTGF-binding protein. Among the 4 domains of CCN2/CTGF, the IG... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2283093 Thu, 19 Mar 2009 04:00:00 +0100 2283093 http://www.medworm.com/index.php?rid=2257116&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082019 Polyadenylate polymerase (PAP) is the template-independent RNA polymerase responsible for synthesis of the 3' poly(A) tails of mRNA. To investigate the role of proton transfer in the catalytic mechanism of PAP, the pH dependence of the steady state kinetic parameters of yeast PAP were determined for the forward (adenylyltransfer) and reverse (pyrophosphorolysis) reactions. The results indicate that productive formation of an enzyme-RNA-MgATP complex is pH independent over a broad pH range, but that formation of an active enzyme-RNA-MgPPi complex is strongly pH dependent, consistent with the production of a proton on the enzyme in the forward reaction. The pH dependence of the maximum velocity of the forward reaction suggests two protonic species are involved in enzyme catalysis. Optimal en... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2257116 Thu, 12 Mar 2009 04:00:00 +0100 2257116 http://www.medworm.com/index.php?rid=2257118&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090110 O-GlcNAcylation is an essential, dynamic and inducible posttranslational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. Here, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the h... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2257118 Tue, 10 Mar 2009 04:00:00 +0100 2257118 http://www.medworm.com/index.php?rid=2235419&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082171 Carbohydrates are crucial in living cells, playing myriads of functional roles that range from being structural or energy-storage devices to molecular labels that, through non-covalent interaction with proteins, impart exquisite selectivity in processes such as molecular trafficking and cellular recognition. The molecular bases that govern the recognition between carbohydrates and proteins have not been fully understood yet. In this work, we have obtained a surface area-based model for the formation heat capacity of protein-carbohydrate complexes, which includes separate terms for the contributions of the two molecular types. The carbohydrate’s model, which was calibrated using carbohydrate dissolution data, indicates that the heat capacity contribution of a given group surface depe... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2235419 Tue, 03 Mar 2009 05:00:00 +0100 2235419 http://www.medworm.com/index.php?rid=2202356&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081588 Lipocalins are a broad family of proteins identified initially in eukaryotes and more recently in gram-negative bacteria. The functions of lipocalin or lipid binding proteins are often elusive and very diverse. We have recently determined the structure of GrlR which plays a key role in the regulation of locus of enterocyte effacement (LEE) proteins. GrlR adopts a lipocalin-like fold which comprises of eight stranded β-barrel followed by an α-helix at the C-terminus. GrlR has a highly hydrophobic cavity region and could be a potential transporter of lipophilic molecules. To verify this hypothesis, we carried out structure based analysis on GrlR, determined the structure of lipid-GrlR complex and measured the binding of lipid to recombinant GrlR by isothermal titration calorime... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2202356 Thu, 19 Feb 2009 05:00:00 +0100 2202356 http://www.medworm.com/index.php?rid=2178523&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082077 Mutations in the human porphobilinogen deaminase gene cause the inherited defect, acute intermittent porphyria. Here we report the structure of the human ubiquitous porphobilinogen deaminase mutant, Arg167Gln, that has been determined by X-ray crystallography and refined to 2.8 Å resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P21212 with two molecules in the asymmetric unit (a = 81.0 Å, b = 104.4 Å, c = 109.7 Å). Phases were obtained by molecular replacement using the E. coli porphobilinogen deaminase structure as a search model. The human enzyme is composed of three domains each of approximately 110 amino acids and possesses a dipyrromethane cofactor at the active site which is located between domains 1 and 2. An ordered sulphate... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2178523 Wed, 11 Feb 2009 05:00:00 +0100 2178523 http://www.medworm.com/index.php?rid=2178522&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082296 In this study, we performed a biochemical characterization of the interaction of divalent cations with the vaccinia virus D10 protein. Synergistic activation of the enzyme was observed in the presence of magnesium and manganese ions, suggesting the existence of two metal ion binding sites on the D10 protein. Moreover, dual ligand titration experiments using fluorescence spectroscopy demonstrated the presence of two metal ion binding sites on the enzyme. A three-dimensional structural model of the active site of the enzyme was generated which highlighted the importance of three glutamate residues involved in the coordination of two metal ions and a water molecule. Mutational analyses confirmed the role of two glutamate residues for the binding of metal ions. We demonstrate that one metal io... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2178522 Wed, 11 Feb 2009 05:00:00 +0100 2178522 http://www.medworm.com/index.php?rid=2161327&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082407 Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of the low-density lipoprotein receptor (LDLR) through an as yet undefined mechanism, leading to a reduction in cellular LDL-cholesterol (LDLc) and a concomitant increase in serum LDLc. Central to the function of PCSK9 is a direct protein-protein interaction formed with the LDLR. Here we investigate a strategy to modulate LDL uptake by blocking this interaction using specific antibodies directed against PCSK9. Studies using surface plasmon resonance demonstrated that direct binding of PCSK9 to the LDLR could be abolished with three different anti-PCSK9 antibodies. Two of these antibodies were raised against peptide epitopes in a region of the catalytic domain of PCSK9 that is involved in the interaction with the LDL... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2161327 Thu, 05 Feb 2009 05:00:00 +0100 2161327 http://www.medworm.com/index.php?rid=2161326&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082204 CD1e displays unique features in comparison to other CD1 proteins. CD1e accumulates in Golgi compartments of immature dendritic cells and is directly transported to lysosomes, where it is cleaved in a soluble form. In these latter compartments, CD1e participates in the processing of glycolipid antigens. Here we show that the N-terminal end of the membrane-associated molecule begins at aminoacid 20, while the soluble molecule consists of aminoacids 32 to 333. Thus, immature CD1e includes an N-terminal propeptide which is cleaved in acidic compartment and so, absent on the mature endosomal form. Mutagenesis experiments demonstrated that the propeptide controls the assembly of the CD1e α chain with β2-microglobulin while propeptide-deleted CD1e molecules are immunologically acti... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2161326 Thu, 05 Feb 2009 05:00:00 +0100 2161326 http://www.medworm.com/index.php?rid=2161325&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081449 The P22 tailspike protein (TSP) is a well-established model system for studying the folding and assembly of oligomeric proteins, and previous works have documented both in vivo and in vitro folding intermediates using this protein. Especially important is the C-terminus of P22 TSP, which plays a critical role in the assembly and maturation of the protrimer intermediate to its final trimeric form. In this paper, we show that by grafting the C-terminus of TSP onto the monomeric maltose binding protein (MBP), the resulting chimera (MBP-537) is a trimeric protein. Moreover, Western blot studies (using an anti-TSP antibody) indicate that the TSP C-terminus in the MBP-537 chimera has the same conformation as the native TSP. The oligomerization of the MBP-537 chimera appears to involve hydrophobi... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2161325 Thu, 05 Feb 2009 05:00:00 +0100 2161325 http://www.medworm.com/index.php?rid=2145265&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082353 Insulin-like peptide 5 (INSL5) is a two-chain peptide hormone related to insulin and relaxin. It was recently discovered through searches of expressed sequence tag databases and although the full biological significance of INSL5 is still being elucidated, high expression in peripheral tissues such as the colon as well as in the brain and hypothalamus, suggests roles in gut contractility and neuroendocrine signaling. INSL5 activates the relaxin family peptide receptor 4 with high potency, and appears to be the endogenous ligand for this receptor, based on overlapping expression profiles and their apparent co-evolution. Here we have used solution state NMR to characterize the three-dimensional structure of synthetic human INSL5. The structure reveals an insulin/relaxin-like fold with three h... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2145265 Fri, 30 Jan 2009 05:00:00 +0100 2145265 http://www.medworm.com/index.php?rid=2135388&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082081 Heparan sulphate (HS) proteoglycans are key regulators of vital processes in the body. HS chains with distinct sequences bind to various protein ligands such as growth factors and morphogens and thereby functions as important regulators of protein gradient formation and signal transduction. HS is synthesized through the concerted action of many different ER and Golgi-resident enzymes. In higher organisms many of these enzymes occur in multiple isoforms that differ in substrate specificities and spatial and temporal expression. In order to investigate how the structural complexity of HS has evolved we have focused our studies on the sea anemone Nematostella vectensis, which belongs to the Anthozoa, which are considered to have retained many ancestral features. Members of all enzyme families... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2135388 Mon, 26 Jan 2009 05:00:00 +0100 2135388 http://www.medworm.com/index.php?rid=2117198&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082082 In this study, we conducted a systematic analysis 20 residues in IIIS6 of the BgNav channel using alanine scanning mutagenesis. Our results show that alanine substitutions of four residues, I1514, G1516, F1518 and N1522, altered sodium channel sensitivity to pyrethroid insecticides. Whereas the G1516A, F1518A and N1522A substitutions reduced sodium channel sensitivity to all seven pyrethroids examined including four type I (lacking the α-cyano group at the phenoxybenzyl alcohol) and three type II (containing the α-cyano group) pyrethroids, the I1514A substitution enhanced sodium channel sensitivity to only four type I and type II pyrethroids that contain the phenoxybenzyl alcohol. We also show that alanine/lysine substitutions of L1521 and S1517 affected the action of batrach... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2117198 Tue, 20 Jan 2009 05:00:00 +0100 2117198 http://www.medworm.com/index.php?rid=2102361&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082061 Peroxiredoxin 6 (Prdx6), a bifunctional protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, is responsible for the metabolism of lung surfactant phospholipids. We propose that the aiPLA2 activity of the enzyme is regulated through phosphorylation. Incubation of isolated rat alveolar type II cells (AECII) with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) agonist, had no effect on Prdx6 expression but led to ~75% increase in aiPLA2 activity that was abolished by pretreatment of cells with the mitogen activated protein kinase (MAPK) inhibitors, SB202190 or PD98059. Prdx6 phosphorylation after incubation of AECII with TPA was demonstrated by autoradiography following immunoprecipitation with either anti-phosphothreonine or phosphoserine antibodie... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2102361 Wed, 14 Jan 2009 05:00:00 +0100 2102361 http://www.medworm.com/index.php?rid=2098583&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080921 S-acylation (commonly known as palmitoylation) is a widespread posttranslational modification that consists of the addition of a lipid molecule to cysteine residues of a protein through a thioester bond. This modification is predominantly mediated by a family of proteins referred to as Palmitoyltransferases (PATs). Most PATs are polytopic membrane proteins, with 4-6 transmembrane domains, a conserved DHHC motif and variable C-and N-terminal regions, that are likely responsible for conferring localisation and substrate specificity. There is very little additional information on the structure-function relations of PATs. Swf1 and Pfa3 are yeast members of the DHHC family of proteins. Swf1 is responsible for the S-acylation of several transmembrane SNARES and other integral membrane proteins. ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=2098583 Tue, 13 Jan 2009 05:00:00 +0100 2098583 http://www.medworm.com/index.php?rid=1999698&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081739 In this study, the residues N341, Y60 and F351, which are likely to influence THF binding, were mutated to Ala, Ala and Gly, respectively, to elucidate the role of these residues in THF-dependent and independent reactions catalyzed by SHMT. The N341A and Y60A bsSHMT mutants were inactive for the THF-dependent activity while the mutations had no effect on THF-independent activity. However, mutation of F351 to Gly did not have any effect on either of the activities. The crystal structures of the Gly binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT whereas Y60A and F351G bsSHMTs exist as a mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all the three mutants obtained in the presence of L-allo-Thr were similar t... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1999698 Mon, 01 Dec 2008 05:00:00 +0100 1999698 http://www.medworm.com/index.php?rid=1993368&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081093 Human β1,4-galactosyltransferase7 (β1,4-GalT7) is involved in the biosynthesis of the tetrasaccharide linker protein region (GlcAβ1-3Galβ1-3Galβ1-4Xylβ1) of proteoglycans, by catalysing the transfer of galactose from the uridine 5’-diphosphogalactose, to xylose residue. This reaction is rate-limiting in the glycosaminoglycan biosynthesis. In the present study, we established a large scale production system of the β1,4-GalT7 fused with the maltose-binding protein to study substrate recognition. Calorimetric binding studies showed that the binding of the donor substrate UDP-Gal largely promoted binding of the acceptor substrate. To identify the structural basis governing substrate recognition, we used a fragment-based approach involving... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1993368 Tue, 25 Nov 2008 05:00:00 +0100 1993368 http://www.medworm.com/index.php?rid=1985745&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080992 The chaperone/usher pathway controls assembly of fibres of adhesive organelles of Gram-negative bacteria. The final steps of fibre assembly and fibre translocation to the cell surface are coordinated by outer membrane proteins, ushers. Ushers consist of several soluble periplasmic domains and a single transmembrane β-barrel. Here we report isolation and structural-functional characterization of a novel middle domain of the Caf1A usher (UMD) from Yersinia pestis. The isolated UMD is a highly soluble monomeric protein capable of autonomous folding. A 2.8 Å resolution crystal structure of UMD revealed that this domain has an immunoglobulin-like fold similar to that of donor strand complemented Caf1 fibre subunit. Moreover these proteins displayed significant structural similarit... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1985745 Tue, 25 Nov 2008 05:00:00 +0100 1985745 http://www.medworm.com/index.php?rid=1918607&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081462 In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine-replacements affecting the final 6 amino-acids of TonB2. Deletions of more than two amino-acids of the TonB2-C-terminus abolished ferric-anguibactin transport, while replacement of the last three residues resulted in a protein with wild type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121-206) has an αββαβ structure, while residues 76-120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain albeit with two ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1918607 Thu, 30 Oct 2008 04:00:00 +0100 1918607 http://www.medworm.com/index.php?rid=1896834&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081557 This study reports that diamide, a sulfhydryl group oxidant, induces disulfide cross-linking of poorly-glycosylated band 3 and that the oligomerised band 3 fraction is selectively tyrosine (Tyr-) phosphorylated both in glucose-6-phosphate dehydrogenase (G6PD) deficient and control erythrocytes. This phenomenon is irreversible in G6PD deficient RBCs while it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size exclusion separation of Triton X-100 extracted membrane proteins, Syk was found only in the high molecular weight (MW) fraction containing oligomerised / phosphorylated band 3. Src family inhibitors efficiently abro... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1896834 Wed, 22 Oct 2008 04:00:00 +0100 1896834 http://www.medworm.com/index.php?rid=1892560&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081427 The inducible form of nitric oxide synthase (NOS2) plays an important role in sepsis incurred as a result of infection with gram-negative bacteria that elaborate endotoxin. The high mobility group A1 (HMGA1) architectural transcription factor facilitates NOS2 induction by binding a specific AT-rich Oct sequence in the core NOS2 promoter via AT-hook motifs. The small-molecule, minor groove binder (MGB) netropsin selectively targets AT-rich DNA sequences and can interfere with transcription factor binding. Therefore we hypothesized that netropsin would improve survival from murine endotoxemia by attenuating NOS2 induction through interference with HMGA1-DNA binding to the core NOS2 promoter. Netropsin improved survival from endotoxemia in wild type mice, yet not in NOS2-deficient mice, suppo... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1892560 Tue, 21 Oct 2008 04:00:00 +0100 1892560 http://www.medworm.com/index.php?rid=1881280&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081726 The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the peptides’ helical conformation and tendency to aggregate in solution. We have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37, and orang-utan LL-37, using several complementary biophysical and biochemical methods. CD and ATR-FTIR spectroscopy on model membranes indicate that RL-37,... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1881280 Thu, 16 Oct 2008 04:00:00 +0100 1881280 http://www.medworm.com/index.php?rid=1871438&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081247 In this study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an “alanine face” enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit are mutated to alanine, a “glycine face” enzyme in which the same residues are mutated to glycine, and an “all alanine” helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations cause a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine face mutant enzyme is 30 s-1, the glycine face mutant enzyme is 17 s-1, and the all alanine helix enzyme is 16 s-1... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1871438 Mon, 13 Oct 2008 04:00:00 +0100 1871438 http://www.medworm.com/index.php?rid=1848777&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081385 The failure of most non-ionic detergents to release patches of detergent-resistant membrane (DRM) at 37˚C undermines the claim that DRMs consist of lipid nanodomains that exist in an Lo (liquid ordered) phase on the living cell surface. We show here that inclusion of cations (Mg2+, K+) to mimic the intracellular environment stabilises membranes during solubilization sufficiently to allow the isolation of DRMs at 37˚C, using either Triton X-100 or Brij 96. These DRMs are sensitive to chelation of cholesterol, maintain outside-out orientation of membrane glycoproteins, have prolonged (18 hour) stability at 37˚C, and are vesicles or sheets up to 150-200 nm diameter. DRMs containing glycosylphosphatidylinositol (GPI)-anchored proteins PrP and Thy-1 can be separated by immu... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1848777 Fri, 03 Oct 2008 04:00:00 +0100 1848777 http://www.medworm.com/index.php?rid=1848776&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081284 Seven different forms of the enzyme catechol O-methyltransferase (COMT) were found in isolated rat hepatocytes by two-dimensional gel electrophoresis and immunoblotting: five small variants (S-COMT), and two large variants (L-COMT). The identities of these COMT forms were verified by tryptic fingerprinting, using MALDI-TOF mass spectrometry, and by amino acid sequencing, using electrospray ionization with ion trap tandem mass spectrometry (ESI-IT-MS/MS). Analysis of tissue distributions showed that the S-COMT forms were highly expressed in liver and kidney whereas L-COMT was expressed more strongly in other tissues. Both of the two L-COMT forms were found in all the examined tissues except the heart, which expressed only the most acidic form, and the kidney, which expressed only the most b... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1848776 Fri, 03 Oct 2008 04:00:00 +0100 1848776 http://www.medworm.com/index.php?rid=1776521&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071702 In this study, we investigate the contributions of residues Cys10 and His106 to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving population of at least 2 partially-folded intermediates, one of which is dimeric. Mutation of residues Cys10 and His106 leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His106 plays an important role in the catalytic mechanism of the enzyme, while Cys10 is involved in binding of the substrate GSH. Engineering of the Cys10 site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol-disulfide ox... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1776521 Tue, 09 Sep 2008 04:00:00 +0100 1776521 http://www.medworm.com/index.php?rid=1765564&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081403 Obtustatin and viperistatin represent the shortest known snake venom monomeric disintegrins. We have produced recombinant full-length wild-type and site-directed mutants of obtustatin to assess the role of the 21KTS23 tripeptide and C-terminal residues for specific inhibition of the a1b1 integrin. Threonine-22 appeared to be the most critical residue for disintegrin activity, whereas substitution of flanking lysine or serine for alanine resulted in less pronounced decreased of the disintegrin's anti-α1β1 integrin activity. The triple mutant 21AAA23 was devoid of blocking activity towards α1β1 integrin-mediated cell adhesion. The potency of recombinant KTS-disintegrins also depended on the residue C-terminally adjacent to the active motif. Substitution of wild-ty... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1765564 Fri, 05 Sep 2008 04:00:00 +0100 1765564 http://www.medworm.com/index.php?rid=1756213&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081355 We report the structure of the Fc fragment of rabbit IgG at 1.95 angstrom resolution. Rabbit IgG was the molecule for which Porter established the four-chain, Y-shaped structure of the antibody molecule, and crystals of the Fc (“Fragment crystallisable”) were first reported almost 50 years ago in this journal (Porter R.R., 1959, Biochem. J., 73, 119-126). This high-resolution analysis, apparently of the same crystal form, reveals several features of IgG-Fc structure that have not previously been described. More of the lower hinge region is visible in this structure than in others, demonstrating not only the acute bend in the IgG molecule that this region can mediate, as seen in receptor complexes, but also that this region has a tendency to adopt a bent structure even in the ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1756213 Wed, 03 Sep 2008 04:00:00 +0100 1756213 http://www.medworm.com/index.php?rid=1684210&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081235 Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease resistant fragments of PrPSc using proteinase K (PK), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrPSc in its full-length form. Here we show that thermolysin can degrade PrPC while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML prions, the majority of PK-sensitive disease-related PrP ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1684210 Wed, 06 Aug 2008 04:00:00 +0100 1684210 http://www.medworm.com/index.php?rid=1647124&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080892 The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone versus time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism, in which the enzymatic form Eox is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1647124 Wed, 23 Jul 2008 04:00:00 +0100 1647124 http://www.medworm.com/index.php?rid=1647123&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080632 Protein glycation is involved in structure and stability changes that impair protein functionality, being associated with several human diseases, like diabetes and amyloidotic neuropathies (Alzheimer, Parkinson and Andrade’s syndrome). To understand the relationship of protein glycation with protein dysfunction, unfolding and β-fibber formation, numerous studies have been made in vitro. All these previous experiments were conducted in non-physiologic or pseudo physiological conditions that bear little to no resemblance to what may happen in a living cell. In vivo, glycation occurs in a crowded and organized environment, where proteins are exposed to a steady state of glycation agents, namely methylglyoxal, while in vitro, a bolus of a suitable glycation agent is added to dilu... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1647123 Wed, 23 Jul 2008 04:00:00 +0100 1647123 http://www.medworm.com/index.php?rid=1635975&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080389 The modification of proteins by reducing sugars through the process of non-enzymatic glycation is one of the principal mechanisms by which hyperglycaemia may precipitate the development of diabetic complications. Fructosamine 3-kinase (Fn3K) and fructosamine 3-kinase-related-protein (Fn3KRP) are two recently discovered enzymes that may play roles in metabolising early glycation products. However, while the activity of these enzymes towards various glycated substrates has been established, very little is known about their structure-function relationships or their respective mechanisms of action. Furthermore, their only structural similarities noted to date with members of other kinase families has been with the bacterial aminoglycoside kinases. Here, we employed affinity labelling with the ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1635975 Fri, 18 Jul 2008 04:00:00 +0100 1635975 http://www.medworm.com/index.php?rid=1635974&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080543 β2-Microglobulin-related (Aβ2M) amyloidosis is a frequent and serious complication in patients on long-term dialysis. Partial unfolding of β2-microglobulin (β2-m) may be essential to its assembly into Aβ2M amyloid fibrils in vivo. Although sodium dodecyl sulfate around the critical micelle concentration induces partial unfolding of β2-m to an α-helix-containing aggregation-prone amyloidogenic conformer and subsequent amyloid fibril formation in vitro, the biological molecules with similar activity under near-physiological conditions are still unclear. The effect of various non-esterified fatty acids (NEFAs), which are representative anionic amphipathic compounds in the circulation, on the growth of Aβ2M amyloid fibrils at a neutral pH... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1635974 Fri, 18 Jul 2008 04:00:00 +0100 1635974 http://www.medworm.com/index.php?rid=1603729&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081182 The final 25 amino acids of the ectodomain of the P2X receptors, immediately prior to the second transmembrane segment (pre-TM2: Arg304 to Ile328 in rat P2X2), are highly conserved. Whole-cell patch clamp recordings showed that single cysteine substitutions in the N-terminal half of pre-TM2 (Arg304 to Ile314) led to loss of function at Arg304, Leu306, Lys308, and Ile312. Cysteine substitutions within this region also resulted in a significant reduction in the apparent molecular mass of receptors, due to loss of complex glycosylation at the nearby acceptor site Asn298, which was not seen for the C-terminal portion of pre-TM2 (Asp315 to Ile328). The reduction in complex glycosylation was not due to reduced cell-surface presentation, demonstrating that glycosylation at Asn298 was acting as a ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1603729 Thu, 10 Jul 2008 04:00:00 +0100 1603729 http://www.medworm.com/index.php?rid=1599731&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080779 Deubiquitinating enzymes (DUBs) are a family of proteases responsible for the specific removal of ubiquitin attached to target proteins and thus control the free cellular pools of this molecule. DUB activity is usually assayed using full-length ubiquitin, and these enzymes generally show low activity on small substrates that constitute the P4-P1 LRGG C-terminal motif of ubiquitin. To gain insight into the C-terminal recognition region of ubiquitin by DUBs we synthesized positional scanning libraries of fluorogenic tetrapeptides and tested them on three examples of human DUBs (OTU-1, Iso-T and UCH-L3) and one viral ubiquitin specific protease – Plpro from West Nile virus. In most cases the results show flexibility in the P4 position, very high specificity for Arg in P3 position and G... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599731 Fri, 04 Jul 2008 04:00:00 +0100 1599731 http://www.medworm.com/index.php?rid=1599730&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081000 Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonisation of the lungs of immunocompromised patients. Glucosamine-6-phosphate N-acetyltransferase (GNA1) catalyses the acetylation of glucosamine-6-phosphate (GlcN-6P) to N-acetylglucosamine-6-phosphate (GlcNAc-6P), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. Here, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kine... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599730 Fri, 04 Jul 2008 04:00:00 +0100 1599730 http://www.medworm.com/index.php?rid=1599732&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080728 Recent clinical data indicates that the emergence of mutant drug-resistant kinase alleles may be particularly relevant for targeted kinase inhibitors. In order to explore the how different classes of targeted therapies impact upon resistance mutations we performed EGFR resistance mutation screens with erlotinib, lapatinib and CI-1033. Distinct mutation spectra were generated with each inhibitor and were reflective of their respective mechanisms of action. Lapatinib yielded the widest variety of mutations, whilst mutational variability was lower in the erlotinib and CI-1033 screens. Lapatinib was uniquely sensitive to mutations of residues located deep within the selectivity pocket, whilst mutation of either G796 or C797 resulted in a dramatic loss of CI-1033 potency. The clinically observe... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599732 Mon, 30 Jun 2008 04:00:00 +0100 1599732 http://www.medworm.com/index.php?rid=1599734&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080785 In Bacillus megaterium, the synthesis of vitamin B12 (cobalamin) and siroheme diverge at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD+ as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 Å resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional siroheme synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give siroheme. Guided by the structure, a number of active site residues within SirC were investigated by site-directed mutagenesis. No active s... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599734 Thu, 26 Jun 2008 04:00:00 +0100 1599734 http://www.medworm.com/index.php?rid=1599733&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080580 Glucoamylase hydrolyzes starch and polysaccharides to β-D-glucose. Rhizopus oryzae glucoamylase (RoGA) consists of two functional domains, an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. The crystal structures of SBD from RoGA (RoGACBM21) and the complexes with β-cyclodextrin (SBD-βCD) and maltoheptaose (SBD-G7) were determined. Two carbohydrate binding sites, I (Trp47) and II (Tyr32), were resolved and their binding was cooperative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagines also participate in the sugar binding. A conformational change in Tyr32 was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599733 Thu, 26 Jun 2008 04:00:00 +0100 1599733 http://www.medworm.com/index.php?rid=1599735&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071499 The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates like amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, we have investigated the influence of additives like SDS, CTAB and DTT on the slow aggregation of hen eggwhite lysozyme at pH 12.2. For this purpose, techniques like steady state & time-resolved fluorescence anisotropy of covalently labeled dansyl probe,... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599735 Fri, 13 Jun 2008 04:00:00 +0100 1599735 http://www.medworm.com/index.php?rid=1599736&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080255 In this study we further characterized the G-protein binding features of RGL2 and also demonstrated that RGL2 has guanine nucleotide exchange activity toward the small GTPase RalA. We found that RGL2/Rlf properties are well conserved between human and mouse species. Both RGL2 and Rlf have a putative PKA phosphorylation site at the C-terminal of the domain that regulate the interaction with small GTPases. We demonstrated that RGL2 is phosphorylated by PKA and phosphorylation reduces the ability of RGL2 to bind H-Ras. As RGL2 and Rlf are unique in the RalGDS family in having a PKA site in the Ras Binding Domain, these results indicate that Ras may distinguish between the different RalGDS family members by their phosphorylation by PKA. (Source: BJ Structure) BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599736 Mon, 09 Jun 2008 04:00:00 +0100 1599736 http://www.medworm.com/index.php?rid=1599738&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080375 The subclass B2 CphA β-lactamase from Aeromonas hydrophila is a Zn(II) containing enzyme that hydrolyse specifically carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His118, Asp120, His196 and His263) and in substrate specificity (Val67, Thr157, Lys224 and Lys226), site-directed mutants were generated and characterised. Our results confirm that the first zinc ion is in interaction with Asp120 and His263, and thus is located in the “cysteine” zinc binding site. His118 and His196 residues seem to be in interaction with the second zinc ion as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val67 plays a significant role in the binding of biapenem and benzylpenici... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599738 Fri, 23 May 2008 04:00:00 +0100 1599738 http://www.medworm.com/index.php?rid=1599737&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080242 We report here the crystal structures of the catalytic domain of LiCMS and its complexes with substrates and the kinetic and mutagenesis studies of LiCMS, which together reveal the molecular basis of the high substrate specificity and the catalytic mechanism of LiCMS. The catalytic domain consists of a TIM barrel flanked by an extended C-terminal region. It forms a homodimer in the crystal structure and the active site is located at the center of the TIM barrel near the C-terminal ends of the β-strands and composed of conserved residues of the β-strands of one subunit and the C-terminal region of the other. The substrate specificity of LiCMS towards pyruvate against other α-keto acids is dictated primarily by residues Leu81, Leu104, and Tyr144 which form a hydrophobic ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599737 Fri, 23 May 2008 04:00:00 +0100 1599737 http://www.medworm.com/index.php?rid=1599740&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080544 Small heat shock protein α-crystallin isolated from the eye lens exists as a large (700 kDa) heteropolymer composed of two subunits, αA and αB, of 20 kDa each. While trace amounts of αA-crystallin are found in other tissues, non-lenticular distribution of α-crystallin is dominated by the αB homopolymer. In most vertebrate lens, the molar ratio of αA to αB is generally 3:1. However, the importance of 3:1 ratio of αA to αB subunits in the eye lens is not known. Here in, we have investigated the physiological significance of 3:1 by determining the secondary/tertiary structure, hydrophobicity and chaperone-like activity of αA- and αB-homopolymers and heteropolymers with different ratios of αA to α... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599740 Wed, 14 May 2008 04:00:00 +0100 1599740 http://www.medworm.com/index.php?rid=1599739&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080714 Hydroxycinnamoyl-CoA Hydratase-Lyase (HCHL) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy 3-methoxybenzaldehyde) and is exploited in whole-cell systems for the bioconversion of ferulic acid to natural-equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA, then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active site residues identified using the crystal structure of HCHL revealed that whilst Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599739 Wed, 14 May 2008 04:00:00 +0100 1599739 http://www.medworm.com/index.php?rid=1599741&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080353 Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyzes the formation of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active s... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599741 Mon, 21 Apr 2008 04:00:00 +0100 1599741 http://www.medworm.com/index.php?rid=1599742&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080440 Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyzes both aliphatic and aromatic nitriles. Substituting Tyr-142 in the wild-type enzyme with the aromatic amino acid Phe did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (Ala, Val, and Leu) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile, and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated phi-electron system of the aromatic ring of Tyr-142 or substrate as an electron acceptor. Moreover, the mutants containing charged amino acids such as Asp, Glu, Arg, and Asn at position 142 displayed no activity toward any nitrile, possibly owing to t... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599742 Tue, 15 Apr 2008 04:00:00 +0100 1599742 http://www.medworm.com/index.php?rid=1599747&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071073 In this study, we combine steady-state enzyme kinetic and X-ray structural analyses of eleven single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu{super201}···Asp{super305} and Asp{super248}···Asp{super305}, reduced the steady-state catalysis dramatically. The roles of these two COOH···COOH bonds on catalysis could be partly replaced by COOH···H{sub2}O bonds, but not by COOH···CONH{sub2} bonds, reminiscent of the low-barrier Asp···Asp H-bond in the active site of pepsin-like aspartic peptida... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599747 Thu, 20 Mar 2008 04:00:00 +0100 1599747 http://www.medworm.com/index.php?rid=1599746&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20070819 A new GH12 family xyloglucan-specific endo-β-1,4-glucanase (EC 3.2.1.151) from Aspergillus niger, AnXEG12A, was overexpressed, purified and characterized. Whereas seven xyloglucanases from GH74 and two xyloglucanases from GH5 have been characterized previously, this is only the third characterized example of a GH12 family xyloglucanase. GH12 enzymes are structurally and mechanistically distinct from GH74 enzymes. While over 100 GH12 sequences are now available, little is known about the structural and biochemical basis of xyloglucan binding and hydrolysis by GH12 enzymes. Comparison of the AnXEG12A cDNA sequence with the genome sequence of A. niger showed the presence of two introns, one in the coding region and the second one in the 333&#x0... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599746 Thu, 20 Mar 2008 04:00:00 +0100 1599746 http://www.medworm.com/index.php?rid=1599745&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071021 Osteopontin (OPN) is a highly phosphorylated glycoprotein present in many tissues and body fluids. In urine OPN is a potent inhibitor of nucleation, growth and aggregation of calcium oxalate crystals suggesting a role in the prevention of renal stone formation. The role of OPN in nephrolithiasis is however somewhat unclear as it may also be involved in urinary stone formation, and it has been identified among the major protein components of renal calculi. Most likely the function of OPN in urine is dependent upon the highly anionic character of the protein. Besides a very high content of aspartic- and glutamic acid residues, OPN is subjected to significant posttranslational modification (PTM), such as phosphorylation, sulphation and glycosylation, which may function as regulatory sw... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599745 Thu, 20 Mar 2008 04:00:00 +0100 1599745 http://www.medworm.com/index.php?rid=1599744&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071476 In this study, the effect of glutathione on the Cys-redox state of Tim9 was investigated, and the standard redox potential of Tim9 was determined to be about -0.31V at pH 7.4, 25°C, with both the wild type and Tim9F43W mutant proteins using reverse-phase HPLC and fluorescence approaches. The results show that reduced Tim9 can be oxidized by glutathione under cytosolic concentrations. Next, we studied the rate of mitochondrial import and oxidative folding of Tim9 under identical conditions. The rate of import was about three-fold slower than that of oxidative folding of Tim9, resulting in about 20% of the precursor protein being imported into an excess amount of mitochondria. A similar correlation between import and oxidative folding was obtained fo... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599744 Thu, 20 Mar 2008 04:00:00 +0100 1599744 http://www.medworm.com/index.php?rid=1599743&cid=s_37621_60_f&fid=37621&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071372 Antifreeze proteins (AFPs) are produced by many organisms that inhabit ice-laden environments. They facilitate survival at sub-zero temperatures by binding to and inhibiting the growth of ice crystals in solution. The Antarctic bacterium Marinomonas primoryensis produces an exceptionally large (> 1MDa) hyperactive Ca{super2+}-dependent AFP. We have cloned, expressed and characterized a 322-amino-acid region of the protein where the antifreeze activity is localized, which shows similarity to the Repeats-in-ToXin family of proteins. The recombinant protein requires Ca{super2+} for structure and activity, and is capable of depressing the freezing point of a solution in excess of 2 °C at a concentration of 0.5 mg/mL, ... BJ Structure journals http://www.medworm.com/rss/comments.php?id=1599743 Thu, 20 Mar 2008 04:00:00 +0100 1599743