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10 pages, 7 figures, 3 tables.-- PMID: 9521103 [PubMed].-- PMCID: PMC2143928. ; Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor alpha convertase, TACE, involved in the release of the inflammatory cytokine, TNFalpha. The structure of adamalysin II in noncovalent complex with two small-molecule right-hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X-ray diffraction data up to 2.6 and 2.8 A resolution. The inhibitors bind to the S'-side of the proteinase, inserting between two protein segments, establishing a mixed parallel-antiparallel three-stranded beta-sheet and coordinate the central zinc ion in a bidentate manner via their two C-terminal oxygen atoms. The proteinase-inhibitor complexes are described in detail and are compared with other known structures. An adamalysin-based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors. ; Financial support of NIH grant HL22996 (L.F.K.), the Robert Welsh Foundation (A-328 to E.F.M.), Schering-Plough and Zeneca (E.F.M.), the Human Capital and Mobility Programme of the European Union (ERBCHRXCT9400535), and the Ministerio de Educación y Ciencia (grant EX9446121 143). ; Peer reviewed

El pdf es el manuscrito de autor (PMCID: PMC3077575).-- et al. ; Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52 kDa multidomain protein comprising a central 18 kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analysed the 3D structure of Kly18 in the absence and presence of Mg2+ or Ca2+, which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side-chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg2+ or Ca2+ binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally characterized non-mammalian MMP is a xenologue co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs. ; This study was in part supported by grants from European, Spanish, Polish and Catalan public agencies (FP7-HEALTH-F3-2009-223101 'AntiPathoGN', FP7- HEALTH-2010-261460 'Gums&Joints', BIO2008-04080-E, BIO2009-10334, CSD2006-00015, PSE-010000-2009-8, 2009SGR1036 and MNiSzW 1642/B/P01/2008/35) and by grant DE 09761 from the National Institutes of Health, USA. The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University is a beneficent of the structural funds from the European Union (Grant No.: POIG.02.01.00-12-064/08 – 'Molecular biotechnology for health'). ; Peer Reviewed

Trypanosoma cruzi is the aetiological agent of Chagas' disease, a chronic infection that affects millions in Central and South America. Proteolytic enzymes are involved in the development and progression of this disease and two metallocarboxypeptidases, isolated from T. cruzi CL Brener clone, have recently been characterized: TcMCP-1 and TcMCP-2. Although both are cytosolic and closely related in sequence, they display different temporary expression patterns and substrate preferences. TcMCP-1 removes basic C-terminal residues, whereas TcMCP-2 prefers hydrophobic/aromatic residues. Here we report the three-dimensional structure of TcMCP-1. It resembles an elongated cowry, with a long, deep, narrow active-site cleft mimicking the aperture. It has an N-terminal dimerization subdomain, involved in a homodimeric catalytically active quaternary structure arrangement, and a proteolytic subdomain partitioned by the cleft into an upper and a lower moiety. The cleft accommodates a catalytic metal ion, most likely a cobalt, which is co-ordinated by residues included in a characteristic zinc-binding sequence, HEXXH and a downstream glutamate. The structure of TcMCP-1 shows strong topological similarity with archaeal, bacterial and mammalian metallopeptidases including angiotensin-converting enzyme, neurolysin and thimet oligopeptidase. A crucial residue for shaping the S1′ pocket in TcMCP-1, Met-304, was mutated to the respective residue in TcMCP-2, an arginine, leading to a TcMCP-1 variant with TcMCP-2 specificity. The present studies pave the way for a better understanding of a potential target in Chagas' disease at the molecular level and provide a template for the design of novel therapeutic approaches. © 2008 The Authors. ; This study was supported by the following grants: BIO2006-02668, BFU2006-09593, BIO2007-68046, PSE-010000-2007-1 and the CONSOLIDER-INGENIO 2010 Project 'La Factoría de Cristalización' (CSD2006-00015) from Spanish public agencies; EU FP6 Strep Project LSHG- 2006–018830 'CAMP'; EU FP7 Collaborative Project 223101 'AntiPathoGN'; 2005SGR00280 and 2005SGR01027 from the National Catalan Government; and PICT2003 01–15042 from the Agencia Nacional de Promoción Científica y Tecnológica, Ministerio de Ciencia, Tecnología e Innovación Productiva, Argentina. We acknowledge the help provided by European Synchrotron Radiation Facility local contacts. Funding for data collection was provided by the facility ; Peer Reviewed

Xylanases hydrolyze the β-1,4-linked xylose backbone of xylans. They are of increasing interest in the paper and food industries for their pre-bleaching and bio-pulping applications. Such industries demand new xylanases to cover a wider range of cleavage specificity, activity and stability. The catalytic domain of xylanase Xys1 from Streptomyces halstedii JM8 was expressed, purified and crystallized and native data were collected to 1.78 Å resolution with an Rmerge of 4.4%. The crystals belong to space group P212121, with unit-cell parameters a = 34.05, b = 79.60, c = 87.80 Å. The structure was solved by the molecular-replacement method using the structure of the homologue Xyl10A from Streptomyces lividans. In a similar manner to other members of its family, Xys1 folds to form a standard (β/α)8 barrel with the two catalytic functions, the acid/base and the nucleophile, at its C-terminal side. The overall structure is described and compared with those of related xylanases. ; This study was supported by the Ministerio de Educación y Ciencia (grants PB98-1631, BIO08-0898, BIO2000-1659 and BIO2002-03964) and by the Generalitat de Catalunya (grants 1999SGR188, 2001SGR346 and CERBA). Data collection was supported by the European Union (grants HPRI-CT-1999-00017 and ERBFMGCECT980134 to EMBL Outstation Hamburg) ; Peer Reviewed

5 páginas, 1 figura, 1 tabla.-- et al. ; Karilysin is the only metallopeptidase identified as a virulence factor in the odontopathogen Tannerella forsythia owing to its deleterious effect on the host immune response during bacterial infection. The very close structural and sequence-based similarity of its catalytic domain (Kly18) to matrix metalloproteinases suggests that karilysin was acquired by horizontal gene transfer from an animal host. Previous studies by phage display identified peptides with the consensus sequence XWFPXXXGGG (single-letter amino-acid codes; X represents any residue) as karilysin inhibitors with low-micromolar binding affinities. Subsequent refinement revealed that inhibition comparable to that of longer peptides could be achieved using the tetrapeptide SWFP. To analyze its binding, the high-resolution crystal structure of the complex between Kly18 and SWFP was determined and it was found that the peptide binds to the primed side of the active-site cleft in a substrate-like manner. The catalytic zinc ion is clamped by the α-amino group and the carbonyl O atom of the serine, thus distantly mimicking the general manner of binding of hydroxamate inhibitors to metallopeptidases and contributing, together with three zinc-binding histidines from the protein scaffold, to an octahedral-minus-one metal-coordination sphere. The tryptophan side chain penetrates the deep partially water-filled specificity pocket of Kly18. Together with previous serendipitous product complexes of Kly18, the present results provide the structural determinants of inhibition of karilysin and open the field for the design of novel inhibitory strategies aimed at the treatment of human periodontal disease based on a peptidic hit molecule. © 2013. ; This study was supported in part by grants from European, American, Polish, Spanish, Danish and Catalan agencies (2012/04/A/NZ1/00051, 2011/03/N/NZ1/00586, 2137/7.PR-EU/2011/2, DE09761, FP7-HEALTH-F3-2009-223101 'AntiPathoGN', FP7-HEALTH-2010-261460 'Gums&Joints', FP7-PEOPLE-2011-ITN-290246 'RAPID', BIO2009-10334, BFU2012-32862, CSD2006-00015, Lundbeck Foundation grant R54-A5291 and Fundació 'La Marató de TV3' grants 2009-100732 and 2009SGR1036). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University in Kraków (Poland) is a beneficiary of structural funds from the European Union (grant No POIG.02.01.00-12-064/08 'Molecular Biotechnology for Health'). ; Peer Reviewed

The only endogenous protein inhibitor known for metallocarboxypeptidases (MCPs) is latexin, a 25-kDa protein discovered in the rat brain. Latexin, alias endogenous carboxypeptidase inhibitor, inhibits human CPA4 (hCPA4), whose expression is induced in prostate cancer cells after treatment with histone deacetylase inhibitors. hCPA4 is a member of the A/B subfamily of MCPs and displays the characteristic α/β-hydrolase fold. Human latexin consists of two topologically equivalent subdomains, reminiscent of cystatins, consisting of an α-helix enveloped by a curved β-sheet. These subdomains are packed against each other through the helices and linked by a connecting segment encompassing a third α-helix. The enzyme is bound at the interface of these subdomains. The complex occludes a large contact surface but makes rather few contacts, despite a nanomolar inhibition constant. This low specificity explains the flexibility of latexin in inhibiting all vertebrate A/B MCPs tested, even across species barriers. In contrast, modeling studies reveal why the N/E subfamily of MCPs and invertebrate A/B MCPs are not inhibited. Major differences in the loop segments shaping the border of the funnel-like access to the protease active site impede complex formation with latexin. Several sequences ascribable to diverse tissues and organs have been identified in vertebrate genomes as being highly similar to latexin. They are proposed to constitute the latexin family of potential inhibitors. Because they are ubiquitous, latexins could represent for vertebrate A/B MCPs the counterparts of tissue inhibitors of metalloproteases for matrix metalloproteinases. © 2005 by The National Academy of Sciences of the USA. ; This work was supported by Spanish Ministry for Science and Technology Grants BIO2003-00132, GEN2003-20642, and BIO2001-2046 and >Fundación La Caixa> Grant ON03-7-0. Further support was provided by Commission of the European Communities Grant LSHC-CT-2003-503297 >CANCERDEGRADOME>. For data collections, funding was provided by the European Synchroton Radiation Facility and by the European Union Grant RII3-CT-2004-506008 ; Peer Reviewed

Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of hemebound HmuY reveals a new all-b fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection. © 2009 Wójtowicz et al. ; This study was supported by grants from Spanish ministries (BIO2006-02668, BFU2006-09593, BIO2009-XXXXX, PSE-010000-2007-1, and CONSOLIDER-INGENIO 2010 Project "La Factoría de Cristalización" (CSD2006-00015)) and by grant 2005SGR00280 from the Generalitat de Catalunya. Additional funding was obtained from grants N401 029 32/0742 and 1642/B/P01/2008/35 from the Department of Scientific Research, Polish Ministry of Science and Higher Education; from US National Institutes of Health grant DE 09761; and from the European Union through EU FP6 Strep Project LSHG-2006-018830 "CAMP" and EU FP7 Collaborative Project 223101 "AntiPathoGN". Further funding was provided by ESRF (http://www.esrf.eu/) for data collection ; Peer Reviewed

© 2014 by The American Society for Biochemistry and Molecular Biology Inc. Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosisdriven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys477-His444-Asp388, rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solventfilled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates. ; This work was supported, in whole or in part, by National Institutes of Health Grant DE09761/DE/NIDCR. This work was also supported by Polish National Science Center Grant UMO-2012/04/A/NZ1/00051; Ministry of Science and Higher Education of Poland Grant 2137/7.PR-EU/2011/2; European Union Grants FP7-HEALTH-2010-261460 "Gums&Joints," FP7-PEOPLE-2011-ITN-290246 "RAPID," FP7-HEALTH-2012-306029-2 "TRIGGER," and POIG.02.01.00-12-064/08; Spanish Ministry of Economy and Competitiveness Grants BFU2012-32862 and BIO2013-49320-EXP; Spanish Ministry of Science and Education Grant CSD2006-00015; and National Government of Catalonia Grant 2014SGR9. Funding for data collection was provided in part by the European Synchrotron Radiation Facility (Grenoble, France) ; Peer Reviewed

Transforming growth factor β-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported. © 2009 International Union of Crystallography All rights reserved. ; This work was supported by National Eye Institute Grant R01 EY 12712, the Danish National Research Foundation, the Danish Natural Science Research Council, the Danish Association for Prevention of Eye Diseases and Blindness, the Synoptik Foundation, Aarhus University Research Foundation and the Danish Medical Research Council. Further support was provided by BIO2006-02668, PSE-010000-2007-1 and the CONSOLIDER-INGENIO 2010 Project 'La Factoría de Cristalización' (CSD2006-00015) from Spanish public agencies, FP6 Strep Project LSHG-2006-018830 'CAMP' and FPT Collaborative Project 223101 'AntiPathoGN' from the European Union and 2005SGR00280 from the National Catalan Government ; Peer Reviewed

To improve proteome coverage and protein C-terminal identification, we characterized the Methanosarcina acetivorans thermophilic proteinase LysargiNase, which cleaves before lysine and arginine up to 55 °C. Unlike trypsin, LysargiNase-generated peptides had N-terminal lysine or arginine residues and fragmented with b ion-dominated spectra. This improved protein C terminal-peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurred at methylated or dimethylated lysine and arginine, facilitating detection of these epigenetic modifications. ; The German Academic Exchange Service (DAAD) and the Michael Smith Foundation for Health Research (MSFHR) supported P.F.H. P.F.L. was supported by the Alexander von Humboldt Foundation, Breast Cancer Society of Canada and MSFHR; L.D.R. was supported by the Canadian Institutes for Health Research (CIHR) and MSFHR; and U.E. was supported by the MSFHR. This work was supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology (C.M.O.), a grant from the CIHR (grant number M.O.P. 111055) as well as by an Infrastructure Grant from MSFHR and the Canada Foundations for Innovation (C.M.O.). Further support was provided by the European Union FP7 program; Consolider Program of the Spanish Ministry of Science and Technology; and State Plan for Research in Science, Technology and Innovation of the Spanish Ministry of Economy and Competitiveness (F.X.G.-R.) ; Peer Reviewed

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a 'fuzzy complex' that follows a 'dynamic zipper' mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis. ; This study was supported in part by grants from Spanish, French, Danish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00, BES-2015-074583, ANR-10-LABX-12-01, 6108-00031B, 8022-00385B, LF18039, NNF18OC0032724, Novo Nordisk Foundation "Bio-MS", 2017SGR3 and Fundació "La Marató de TV3" 201815). This work was also supported by EPICS-XS, project 823839, funded by the Horizon 2020 programme of the European Union. The CBS is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), which are national infrastructures supported by the French National Research Agency (grants ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively). Finally, we acknowledge the Structural Mass Spectrometry Unit of CIISB, an Instruct-CZ Centre, which was supported by MEYS CR (LM2018127).

20 pages, 5 figures, 2 tables.-- PMID: 17993455 [PubMed].-- PMCID: PMC2772895.-- NIHMSID: NIHMS65935.-- Available online Nov 7, 2007. ; Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defences and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and its self-processed mature form. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin and a latency flap in the zymogen. Dramatic rearrangement of up to 20Å of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain. ; This study was supported by the following grants: BIO2004-20369-E and BIO2003-06653 from the former Spanish Ministry for Science and Technology; BIO2006-02668, BIO2006-14139, BFU2006-09593 and CONSOLIDER-INGENIO 2010 Project "La Factoría de Cristalización" (CSD2006-00015) from the Spanish Ministry for Education and Science; EU FP6 Integrated Project LSHC-CT-2003-503297 "CANCERDEGRADOME"; EU FP6 Strep Project 18830 "CAMP"; and by "AVON-Project" 2005X0648 from the Spanish Association Against Cancer. Additional funding was obtained by J.J.E. from the Danish National Science Research Council and by J.P. from MNiSW (Warsaw, Poland) and an NIH grant DE 09761. Funding for synchrotron diffraction data collection was provided by the European Synchrotron Radiation Facility and the European Union. ; Peer reviewed