BJ Structure Immediate Publications

Mechanism and specificity of the human paracaspase MALT1

2012-02-06T15:29:47Z

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB pathway, likely by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after Arg, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (k_cat/K_m values) of optimal peptidyl substrates are in the same order (10³ – 10⁴ M^-1 s^-1) as for a putative target protein CYLD. Thus, MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.

A naturally occurring nonapeptide functionally compensates the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

M Tan, W Yan, R Liu, M Wang, X Chen, X Zhou, E Wang — 2012-02-01T11:52:02Z

Aminoacyl-tRNA synthetases (aaRSs) establish the rules of the genetic code by catalyzing the formation of aminoacyl-tRNA. The quality control for aminoacylation reaction is achieved by editing activity, which is usually carried out by a discrete editing domain. For leucyl-tRNA synthetase (LeuRS), the connective peptide 1 (CP1) domain is the editing domain responsible for hydrolyzing mis-charged tRNA. The CP1 domain is universally present in LeuRSs except LeuRS from Mycoplasma mobile (MmLeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). We show here that the MmLinker, which is critical for aminoacylation activity of MmLeuRS, could confer remarkable tRNA charging activity to the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their coordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain, and shed light on future mechanism study.

Regulation of the inter subunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD{+} synthetase

W Chuenchor, T I. Doukov, M Resto, A Chang, B Gerratana — 2012-01-26T14:20:27Z

Glutamine-dependent NAD⁺ synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyzes the ATP-dependent formation of NAD⁺from NaAD⁺ at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 Å long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism, however, its regulatory mechanism is unclear. We report the structures of the inactive-glutaminase C176A variant in an apo form and in three synthetase ligand complexes with substrates (NaAD⁺/ATP), substrate analog (NaAD⁺/AMPCPP) and intermediate analogs (NaAD⁺/AMP/PP_i), as well as the structure of wild type mtuNadE in a products complex (NAD⁺/AMP/PPi/Glu). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: 1) at the entrance near the glutaminase active site, 2) in the middle of the tunnel and 3) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

A NOVEL LECTIN FROM AGROCYBE AEGERITA SHOWS HIGH BINDING SELECTIVITY FOR TERMINAL N-ACETYLGLUCOSAMINE

S Jiang, Y Chen, M Wang, Y Yin, Y Pan, B Gu, G Yu, Y Li, B Wong, Y Liang, h sun — 2012-01-23T15:09:25Z

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with N-acetylglucosamine (GlcNAc) coupled Sepharose 6B after (NH₄)₂SO₄precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by mass spectrometry against the translated transcriptome of A. aegerita. The molecular weight of AAL-2 was calculated to be 43.175 kDa from mass spectrometry (MS), which was consistent with the data calculated from the amino acid sequence. To analyze the sugar binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed and the result showed that AAL-2 bound with high selectivity to terminal, nonreducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal, nonreducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as wheat germ agglutinin (WGA) and Griffonia simplicifolia lectin-II (GSL-II). Isothermal titration calorimetry (ITC) further showed that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the current study, we also evaluated the antitumor activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumor growth and prolongation of survival time of tumor bearing mice in vivo.

Novel structural arrangement of nematode cystathionine beta-synthases: characterization of Caenorhabditis elegans CBS-1

R Vozdek, A Hnizda, J Krijt, M Kostrouchova, V Kozich — 2012-01-13T12:14:15Z

Cystathionine beta-synthases are eukaryotic pyridoxal 5'-phosphate (PLP)-dependent proteins that maintain cellular homocysteine homeostasis and produce cystathionine and hydrogen sulfide. In this study, we describe a novel structural arrangement of the cystathionine beta-synthase (CBS) enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyze CBS reactions, as demonstrated by analysis of truncated variants and active site-mutant proteins. In contrast to other metazoan enzymes, CBS-1 lacks the heme and the regulatory Bateman domain essential for activation by S-adenosylmethionine and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. This paper provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode cystathionine beta-synthases possess a structural feature that is unique among CBS protein.

Distinct roles in folding, CD81 receptor binding and viral entry for conserved histidines of HCV glycoprotein E1 and E2.

I Boo, K teWierek, F Douam, D LAVILLETTE, P Poumbourios, H Edelgard Drummer — 2012-01-12T14:09:38Z

The protonation of histidine in acidic environments underpins its role in regulating the function of pH-sensitive proteins. For pH-sensitive viral fusion proteins, His protonation in the endosome leads to the activation of their membrane fusion function. The hepatitis C virus (HCV) glycoprotein E1/E2 heterodimer mediates membrane fusion within the endosome but the roles of conserved histidines in the formation of a functional heterodimer and in sensing pH changes is unknown. We examined the functional roles of conserved histidines located within E1 and E2. The E1 mutations, H222A/R, H298R, and H352A, disrupted E1-E2 heterodimerization and reduced virus entry. Five of 6 histidines located within the E2 receptor-binding domain (RBD) were important for the E2 fold, their substitution with Arg or Ala causing aberrant heterodimerization and/or CD81 binding. Distinct roles in E1/E2 heterodimerization and in virus entry were identified for His-691 and His-693, respectively, within the membrane-proximal stem region. Viral entry and cell-cell fusion at neutral and low pH were enhanced with H445R, indicating that the protonation state of His-445 is a key regulator of HCV fusion. However, H445R did not overcome the block to virus entry induced by bafilomycin A1 indicating a requirement for an endosomal activation trigger in addition to acidic pH.

A novel, non-canonical mechanism of regulation of mammalian Ste20-related kinase 3 (MST3)

2012-01-09T16:14:27Z

The canonical pathway of regulation of the germinal centre kinase (GCK) III subgroup member, mammalian Sterile20-related kinase 3 (MST3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr¹⁷⁸-), induction of Ser-/Thr-protein kinase activity and nuclear localisation. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein, GOLGA2/gm130. Activation of MST3 by calyculin A (a protein Ser-/Thr- phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr¹⁷⁸-) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr³²⁸-) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr¹⁷⁸-) phosphorylation increased MST3 kinase activity but this activity was independent of MST3(Thr³²⁸-) phosphorylation. Interestingly, MST3(Thr³²⁸-) lies immediately C-terminal to a STRAD pseudokinase-like site recently identified as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr¹⁷⁸- /Thr³²⁸-) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr³²⁸-) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.

Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol monooxygenase

2012-01-06T10:56:42Z

Alkylglycerol monooxygenase (E.C. 1.14.16.5) forms a third, distinct class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a diiron centre, which comprises eight conserved histidines. Membrane enzymes containing this motif, including alkylglycerol monooxygenase, are especially labile and could not be purified to homogeneity in active form so far. To get a first insight on structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild type and mutant proteins containing a C-terminal myc tag together with fatty aldehyde dehydrogenase in Chinese hamster ovary cells. Among all acidic residues within the eight-histidine motif, only mutation of glutamate 137 to alanine led to an 18-fold increase in the Michaelis Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine essential for activity was identified. Nine membrane domains were predicted by 4 programs ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using Rosetta-Membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol monooxygenase.

Autoinhibition Mechanism of the Plasma Membrane Calcium Pump Isoforms 2 and 4 Studied Through Lipid-Protein Interaction

2012-01-04T12:04:22Z

The autoinhibition/activation of the plasma membrane calcium pump (PMCA) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae, was investigated using the phosphatidylcholine analogue [¹²⁵I]TID-PC/16, which was incorporated into mixtures of dimyristoyl-phosphatidylcholine and the nonionic detergent C₁₂E₁₀. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca²⁺-calmodulin of PMCA2 showed 30 times higher affinity than PMCA4. PMCA2 exhibited a lower level labeling in the autoinhibited conformation relative to PMCA4 indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid:protein stoichiometry show that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca²⁺transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behavior of annular lipids and PMCA.

Structure-function studies of a plant tyrosyl-DNA phosphodiesterase provide novel insights into DNA repair mechanisms of Arabidopsis thaliana

H Kim, S Na, S Lee, Y Jeong, H Hwang, J Hur, S Park, J Woo, S Kim — 2012-01-03T16:39:38Z

Tyrosyl-DNA phosphodiesterase 1 (TDP1), a member of the phospholipase D (PLD) superfamily, catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and the 3′-phosphate of DNA. We previously identified and characterized the AtTDP gene in Arabidopsis thaliana, an orthologue of yeast and human TDP1 genes (Lee et al (2010) Plant Physiology 154; 1460-1469). Sequence alignment of TDP1 orthologues revealed that AtTDP has both a conserved C-terminal TDP domain and, uniquely, an N-terminal SMAD/forkhead-associated (FHA) domain. To help understand the function of this novel enzyme, we analyzed the substrate saturation kinetics of fulllength AtTDP versus a truncated AtTDP mutant lacking the N–terminal FHA domain. The recombinant AtTDP protein hydrolyzed a single-stranded DNA substrate with K_m and k_cat / K_m values of 703 ± 137 nM and 1.5 x 10⁹ ± 0.04 x 10⁹ M^-1 min^-1, respectively. The AtTDP D1–122 protein (TDP domain) showed kinetic parameters that were equivalent to those of the fulllength AtTDP protein. A basic amino acid sequence (RKKVKP) within the AtTDP D123–605 protein (FHA domain) was necessary for nuclear localization of AtTDP. Analysis of active site mutations showed that a histidine and a lysine residue in each of the HKD motifs were critical for enzyme activity. Vanadates, inhibitors of phosphoryl transfer reactions, inhibited AtTDP enzymatic activity and retarded the growth of an Arabidopsis tdp mutant. Finally, we showed that expression of the AtTDP gene could complement a yeast tdp1Drad1D mutant, rescuing the growth inhibitory effects of vanadate analogs and camptothecin (CPT). Taken together, our data demonstrate the structure-based function of AtTDP through which AtTDP can repair DNA strand breaks in plants.

A proximal pair of positive charges provides the dominant ligand binding contribution to complement-like domains from the low density lipoprotein receptor-related protein (LRP)

P GW Gettins, K Dolmer — 2011-12-20T11:59:53Z

The low density lipoprotein receptor-related protein (LRP) can bind a wide range of structurally diverse ligands to regions composed of clusters of ~40 residue Ca²⁺-dependent, disulfide-rich, complement-like repeats (CR). While lysine residues from the ligands have been implicated in binding, there has been no quantitation of the energetic contributions of such interactions and hence of their relative importance in overall affinity, or of the ability of arginine or histidine to bind. We have used four representative CR domains from the principal ligand cluster of LRP to determine the energetics of interaction with well defined small ligands, that include methyl esters of lysine, arginine, histidine, and aspartic acid, as well as N-terminally blocked lysine methyl ester. We found that, not only lysine, but also arginine and histidine bound well, and when present with an additional proximal positive charge, accounted for about half the total binding energy of a protein ligand such as PAI-1. Two such sets of interactions, one to each of two CR domains could thus account for almost all the necessary binding energy of a real ligand such as PAI-1. For the CR domains, a central aspartate in the sequence DxDxD tightens Kd by ~20-fold, while DxDDD is no more effective. Together these findings establish the rules for determining binding specificity for protein ligands to LRP and to other LDL receptor family members.

Crystal structure of the Sox4 HMG/DNA complex suggests a mechanism for the positional interdependence in DNA recognition

R Jauch, C Keow Leng Ng, K Narasimhan, P R Kolatkar — 2011-12-19T14:39:29Z

It has recently been proposed that the sequence preferences of DNA-binding transcription factors can be well described by models that include the positional interdependence of the nucleotides of the target sites. Such binding models allow for multiple motifs to be invoked, such as principal and secondary motifs differing at two or more nucleotide positions. However, the structural mechanisms underlying the accommodation of such variant motifs by TFs remain elusive. Here we present the crystal structure of the high-mobility group (HMG) domain of Sox4 bound to DNA. By comparing this structure with previously solved structures of Sox17 and Sox2 we observed subtle conformational differences at the DNA binding interface. Furthermore, using quantitative electrophoretic mobility shift assays (EMSAs) we validated the positional interdependence of two nucleotides and the presence of a secondary Sox motif in the affinity landscape of Sox4. These data suggest that a concerted rearrangement of two interface amino acids enables Sox4 to accommodate primary and secondary motifs. The structural adoptions lead to altered dinucleotide preferences that mutually reinforce each other. These analyses underline the complexity of the DNA recognition by TFs and provide an experimental validation for the conceptual framework of positional interdependence and secondary binding motifs.

Solution structure of Pdp1 PWWP domain reveals its unique binding sites for methylated H4K20 and DNA

2011-12-13T12:30:41Z

Methylation of H4K20 plays an important role in the regulation of diverse cellular processes. In fission yeast, all the three states of H4K20 methylation are catalyzed by Set9. Pdp1 is a PWWP domain-containing protein, which associates with Set9 to regulate its chromatin localization and methyltransferase activity towards H4K20. The structure of Pdp1 PWWP domain, which is the first identified PWWP domain that binds to methyl-lysine at H4K20 site, was determined by solution NMR in the present study. Pdp1 PWWP domain adopts a classical PWWP fold, with a 5-strand antiparallel β-barrel followed by three α-helices. However, it differs significantly from other PWWP domains in some structural aspects that account in part for its molecular recognition. Moreover, we revealed a unique binding pattern of the PWWP domain in that the PWWP domain of Pdp1 bound not only to H4K20me3 but also to dsDNA via an aromatic cage and a positively charged area respectively. Electrophoretic Mobility Shift Assay (EMSA) illustrated the ability of Pdp1 PWWP domain to bind the nucleosome core particle and further mutagenesis experiments indicated the crucial role of this binding activity in histone H4K20 di- and tri- methylation in yeast cells. Our study may shed light on a novel mechanism of histone methylation regulation by the PWWP domain.

A Molten Globule-to-Ordered Structure Transition of Drosophila melanogaster Crammer Is Required for its Ability to Inhibit Cathepsin

T Tseng, C Cheng, D Chen, M Shih, Y Liu, S Danny Hsu, P Lyu — 2011-12-09T14:30:58Z

Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in long-term memory (LTM) formation. The mechanism by which the inhibitory activity is regulated remains unclear. Here we have shown that the oligomeric state of crammer is pH dependent. At neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer, not disulfide bonded dimer, is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, a condition that is similar to the environment in the lysosome where crammer is likely located. Upon binding to cathepsin L, however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that a cysteine-to-serine point mutation at position 72 (C72S) renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin.

Folded Functional Lipid-Poor Apolipoprotein A-I Obtained by Heating of High-Density Lipoproteins: Relevance to HDL Biogenesis

S Jayaraman, G Cavigiolio, O Gursky — 2011-12-08T11:25:28Z

High-density lipoproteins (HDL) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apolipoprotein A-I (apoA-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed protein:lipid weight ratio 3:1, with apoA-I, phosphatidylcholine and cholesterol ester at approximate molar ratios of 1:8:1. Compared to lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface, and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.

Structural determinants of rCNT2 sorting to the plasma membrane of polarized and non-polarized cells

I Pinilla-Macua, F Casado, M Pastor-Anglada — 2011-12-02T15:31:49Z

rCNT2 (Slc28a2) is the purine-preferring concentrative nucleoside transporter. It is expressed in both non polarized and polarized cells, where it is localized in the brush border membrane. Since no information about the domains implicated in the plasma membrane sorting of rCNT2 is available, this work aims at identifying structural and functional requirements for rCNT2 trafficking. The comprehensive topological mapping of the intracellular N terminal tail revealed two main features: 1) a glutamate enriched region (NPGLELME) between residues 21 and 28 seems to be implicated in the stabilization of rCNT2 in the cell surface, since mutagenesis of these conserved glutamates resulted in enhanced endocytosis. 2) Mutation of a potential Casein Kinase 2 (CK2) domain led to a loss of brush border-specific sorting. Although the shortest proteins assayed (-74AA, -48AA and -37AA) were accumulated intracellularly and lost their brush border membrane preference, they were still functional. A deeper analysis of CK2 implication in CNT2 trafficking, using a CK2 specific inhibitor (DMAT) and other complementary mutations mimicking the negative charge provided by phosphorylation (S46D and S46E), demonstrated an effect of this kinase on CNT2 activity. In summary, rCNT2 N-terminal tail contains dual sorting signals. An acidic region is responsible for its proper stabilization at the plasma membrane whereas the putative CK2 domain (Ser⁴⁶) is implicated in the apical sorting of the transporter.

Allosteric antibody inhibition of human Hepsin protease

2011-12-02T12:24:11Z

Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumor progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumors show this characteristic. To enable improved future patient treatment, we developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody hH35 potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around a3-helix, located far from the active center. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (Hepatocyte Growth Factor Activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding leading to complete enzyme inactivation.

An aspartate residue in the external vestibule of glycine transporter 2 (GLYT2) controls cation access and transport coupling

2011-12-01T15:14:33Z

Synaptic glycine levels are controlled by glycine transporters (GLYTs). GLYT1 is the main regulator of synaptic glycine concentrations, which catalyzes Na⁺/Cl^-/glycine cotransport with a 2:1:1 stoichiometry. By contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na⁺. Using Molecular Interaction Potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na⁺ interactions. Replacement of D471 in this region reduced Na⁺ affinity and Na⁺ cooperativity of transport, an effect not produced in the homologous position (D295) in GLYT1. Unlike the GLYT1D295 mutation, this D471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [³H]glycine accumulation. The homologous D471 and D295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na⁺ and Li⁺-induced conformational changes. While these two cations had opposite effects on GLYT1 they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. Based on these findings, we propose a role for D471 in controlling cation access to GLYT2 Na⁺ sites, ion coupling during transport and the subsequent conformational changes.

The crystal structure of human UDP-glucose pyrophosphorylase reveals a latch effect that influence enzymatic activity

Q Yu, X Zheng — 2011-12-01T14:41:46Z

UDP-glucose pyrophosphorylase (UGPase) is highly conserved among eukaryotes. UGPase reversely catalyzes the formation of UDP-glucose and is critical in carbohydrate metabolism. Previous studies have mainly focused on the UGPases from plants, fungi and parasites and indicate that the regulatory mechanisms responsible for the enzyme activity vary among different organisms. Here the crystal structure of human UGPase (hUGPase) was determined and shown to form octamers through end-to-end and side-by-side interactions. The observed latch loop in hUGPase distinctly differs from yeast UGPase (yUGPase), which could explain why hUGPase and yUGPase possess different enzymatic activities. Mutagenesis studies showed that both dissociation of octamers and mutations of the latch loop can significantly affect the UGPase activity. Moreover, this latch effect is also evolutionarily meaningful in UGPase from different species.

FUNCTIONAL MAPPING OF THE DISPARATE ACTIVITIES OF THE YEAST MOONLIGHTING PROTEIN HAL3

J Abrie, A González, E Strauss, J Ariño — 2011-11-29T15:03:13Z

The Saccharomyces cerevisiae Hal3 protein is a moonlighting protein, able to function both as an inhibitory subunit of the Ppz1 protein phosphatase and as a constituent protomer of an unprecedented heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC), the third enzyme of the CoA biosynthetic pathway. We initiated here the dissection of the structural elements required for both disparate cellular tasks by using a combination of biochemical and genetic approaches. We show that the conserved Hal3 core (the PPCDC domain) is necessary for both functions, as determined by in vitro and in vivo assays. The Hal3 N-terminal domain is not functional by itself, although in vitro experiments indicate that when this domain is combined with the core it has a relevant function in Hal3’s heteromeric PPCDC activity. Both the N-terminal and the acidic C-terminal domain also appear to be important for Hal3’s Ppz1 regulatory function, although our results indicate that the C-terminal domain fulfils the key role in this regard. Finally, we show that the introduction of two key Asn and Cys residues, essential for monofunctional PPCDC activity but absent in Hal3, is not sufficient to convert it to such a homomeric PPCDC, and that additional modifications of Hal3’s PPCDC domain aimed at increasing its resemblance to known PPCDC also fails to introduce this activity. This suggests that Hal3 has undergone significant evolutionary drift from ancestral PPCDC proteins. Taken together, our work highlights specific structural determinants that could be exploited for full understanding of Hal3’s cellular functions.

Interfacial water molecules in SH3 interactions: A revised paradigm for polyproline recognition.

J Martin-Garcia, J Ruiz-Sanz, I Luque — 2011-11-24T14:16:22Z

In spite of its biomedical relevance, polyproline recognition is still not fully understood. The disagreement between the current description of SH3 complexes and their thermodynamic behavior calls for a revision of the SH3 binding paradigm. Recently, Abl-SH3 was demonstrated to recognize its ligands by a dual binding mechanism involving a robust network of water-mediated hydrogen bonds that complements the canonical hydrophobic interactions. The systematic analysis of the SH3 structural database presented here reveals that this dual binding mode is universal to SH3 domains. Tightly bound, buried interfacial water molecules were found in all SH3 complexes studied mediating the interaction between the peptide ligand and the domain. Moreover, structural waters were also identified in a high percentage of free SH3 domains. A detailed analysis of the pattern of water-mediated interactions enabled the identification of conserved hydration sites in the polyproline-recognition region and the establishment of relationships between hydration profiles and the sequence of both, ligands and SH3 domains. Water-mediated interactions were also systematically observed in WW, UEV and EVH-1 structures. These results clearly indicate that the current description of proline-rich sequence recognition by protein-protein interaction modules is incomplete and insufficient for a correct understanding of these systems. A new binding paradigm is required that includes interfacial water molecules as relevant elements in polyproline recognition.

Oxidation of Met1606 in von Willebrand Factor is a Risk Factor for Thrombotic and Septic Complications in Chronic Renal Failure

2011-11-18T11:49:19Z

Chronic Kidney Disease (CKD) is a life-threatening pathology, often requiring haemodialysis (HD) and characterized by high oxidative stress, inflammation and perturbation of vascular endothelium. HD-patients have increased levels of von Willebrand factor (vWF), a large protein (~240kDa) released as ultra large polymers (UL-vWF, M.W.~20000-50000kDa) from vascular endothelial cells and megakaryocytes and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13, a zinc-protease cleaving vWF at the single Tyr1605-Met1606 bond, and by leukocyte serine proteases, released by activated polymorphonuclear cells during bacterial infections. Previous studies showed that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF, which are not only more prothrombotic than shorter vWF oligomers but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD-patients have greatly increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In this study, we first purified vWF from HD-patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers, which are also more resistant to ADAMTS-13. Using targeted mass spectrometry techniques, we estimated that HD-vWF contains <10% of Met1606 in the sulphoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate more efficiently platelets and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD.