Mechanism of action of a Janus-faced single-domain protein inhibitor simultaneously targeting two peptidase classes

Protein inhibitors provide a physiological mechanism for the regulation of proteolytic enzymes. While most single-domain inhibitors have one reactive site with which they target peptidases of a specific catalytic class, selected specimens inhibit two peptidase molecules simultaneously, thus giving rise to ternary complexes. To study such inhibition, we analyzed the function of one of these proteins, sermetstatin, which strongly binds as a dimer to serine proteinases (SPs) and a metallopeptidase (MP). In addition, we determined the structures of the isolated inhibitor dimer and its heterotetrameric complexes with the SP subtilisin and the MP snapalysin, which reveal that inhibition occurs through two independent distal reactive sites. These structures and the derived model for the heterohexameric complex provide for the first time a detailed view of the molecular mechanism of simultaneous inhibition of proteinases belonging to two distinct mechanistic classes by a single-domain protein. © The Royal Society of Chemistry 2013.Peer Reviewe

Folding of small disulfide-rich proteins : clarifying the puzzle

Premi a l'excel·lència investigadora. Àmbit de les Ciències Experimentals. 2008The process by which small proteins fold to their native conformations has been intensively studied over the last few decades. In this field, the particular chemistry of disulfide bond formation has facilitated the characterization of the oxidative folding of numerous small, disulfide-rich proteins with results that illustrate a high diversity of folding mechanisms, differing in the heterogeneity and disulfide pairing nativeness of their intermediates. In this review, we combine information on the folding of different protein models together with the recent structural determinations of major intermediates to provide new molecular clues in oxidative folding. Also, we turn to analyze the role of disulfide bonds in misfolding and protein aggregation and their implications in amyloidosis and conformational diseases

Multiple stable conformations account for reversible concentration-dependent oligomerization and autoinhibition of a metamorphic metallopeptidase

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Molecular plasticity controls enzymatic activity: the native fold of a protein in a given environment is normally unique and at a global free-energy minimum. Some proteins, however, spontaneously undergo substantial fold switching to reversibly transit between defined conformers, the >metamorphic> proteins. Here, we present a minimal metamorphic, selective, and specific caseinolytic metallopeptidase, selecase, which reversibly transits between several different states of defined three-dimensional structure, which are associated with loss of enzymatic activity due to autoinhibition. The latter is triggered by sequestering the competent conformation in incompetent but structured dimers, tetramers, and octamers. This system, which is compatible with a discrete multifunnel energy landscape, affords a switch that provides a reversible mechanism of control of catalytic activity unique in nature. Shape shifting: A minimal metamorphic, selective, and specific caseinolytic metallopeptidase, selecase, reversibly transits between several different states of defined three-dimensional structure (monomer and tetramer represented in picture). The competent conformation is sequestered in incompetent but structured dimers, tetramers, and octamers, which are associated with loss of enzymatic activity due to autoinhibition.This study was supported in part by grants from European, Spanish, and Catalan agencies (FP7-HEALTH-2010-261460 “Gums&Joints”; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; CSD2006-00015; Fundació “La Marató de TV3” grant 2009-100732; 2009SGR1036; and “Pot d’Idees” FGB301793) and FPI Ph.D. fellowships from the former Spanish Ministry for Science and Technology, currently of Economy and Competitiveness, to M.L.-P. and A.C.-P. P.B. acknowledges funds from ANR-CHEX (project SPIN-HD) and ATIP-AvenirPeer Reviewe

Protease inhibitors as models for the study of oxidative folding

The correct balance between proteases and their natural protein inhibitors is of great importance in living systems. Protease inhibitors usually comprise small folds that are crosslinked by a high number of disulfide bonds, making them perfect models for the study of oxidative folding. To date, the oxidative folding of numerous protease inhibitors has been analyzed, revealing a great diversity of folding pathways that differ mainly in the heterogeneity and native disulfide-bond content of their intermediates. The two extremes of this diversity are represented by bovine pancreatic trypsin inhibitor and hirudin, which fold, respectively, via few native intermediates and heterogeneous scrambled isomers. Other proteins, such as leech carboxypeptidase inhibitor, share characteristics of both models displaying mixed folding pathways. The study of the oxidative folding of two-domain inhibitors, such as secretory leukocyte protease inhibitor, tick carboxypeptidase inhibitor, and Ascaris carboxypeptidase inhibitor, has provided some clues about how two-domain protease inhibitors may fold, that is, either by folding each domain autonomously or with one domain assisting in the folding of the other. Finally, the recent determination of the structures of the major intermediates of protease inhibitors has shed light on the molecular mechanisms guiding the oxidative folding of small disulfide-rich proteins.This work has been supported by the Spanish Ministry of Science and Innovation, grant BIO2007-68046, and by the National Catalan Government, grants 2005-SGR01037 and 2009-SGR760.Peer Reviewe

Zinc metallocarboxypeptidases

Kretsinger, Robert H.; Uversky, Vladimir N.; Permyakov, Eugene A. (eds.). Encyclopedia of Metalloproteins. New York: Springer, 2013, p.2473-2479. ISBN 978-1-4614-1533-6 (Online). ISBN 978-1-4614-1532-9 (Print)Peer Reviewe

Ultratight crystal packing of a 10 kDa protein

7 páginas, 2 figuras, 2 tablas.-- et al.While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved.This study was supported in part by grants from the American, European, Lithuanian, Spanish and Catalan agencies (FP7-HEALTHF3-2009-223101 ‘AntiPathoGN’, FP7-HEALTH-2010-261460 ‘Gums & Joints’, FP7-PEOPLE-2011-ITN-290246 ‘RAPID’, 31V-151 ‘COSMETIZYM’, BIO2009-10334, BFU2009-07134/BMC, BFU2012-32862, BFU2012-33516, CSD2006-00015, a JAE postdoctoral contract from CSIC, an FPU PhD fellowship from the Spanish Ministry for Science and Technology and Fundació ‘La Marató de TV3’ grants 2009-100732 and 2009SGR1036).Peer Reviewe

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an >N-like> chain trace.This study was supported in part by grants from European, German, Swiss, Spanish, and Catalan agencies (FP7-HEALTH-F3-2009-223101 “AntiPathoGN”; FP7-HEALTH-2010-261460 “Gums&Joints”; FP7-PEOPLE-2011-ITN-290246 “RAPID”; Deutsche Forschungsgemeinschaft Grants DFG Sto185/3-3, BE 4086/1-2, and SFB877 (project A9) and German Cluster of Excellence “Inflammation at Interfaces”; Swiss National Science Foundation Grant 31003A; BIO2009-10334; BFU2012-32862; CSD2006-00015; a JAE postdoctoral contract from Consejo Superior de Investigaciones Cientificas; Fundació “La Marató de TV3” ref. 2009-100732; and 2009SGR1036).Peer Reviewe

Expression and purification of integral membrane metallopeptidase HtpX

Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-β-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members. © 2014 Elsevier Inc. All rights reserved.The study was supported in part by grants from European, Spanish, and Catalan agencies (FP7-HEALTH-2010-261460 “Gums&Joints”; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; CSD2006-00015; Fundació “La Marató de TV3” 2009-100732; 2009SGR1036), by a “JAE” research contract from CSIC (co-funded by FSE) and a short-term EMBO fellowship (to J.L.A.), and by a “Juan de la Cierva” research contract from the Spanish Ministry (to R.G-C.)Peer Reviewe

The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenolog of animal matrix metalloproteinases

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 Reviewe

A novel family of soluble minimal scaffolds provides structural insight into the catalytic domains of integral-membrane metallopeptidases

et al.In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called minigluzincins. We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin) which are soluble and, with ~100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments hitherto unseen in MPs: intra-molecular, through an extended tail, in proabylysin, and crosswise inter-molecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons, as well as phylogenetic and bioinformatics analyses, revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Interestingly, gluzincins mostly have a glutamate as third characteristic zinc ligand while minigluzincins have a histidine. Sequence and structure similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral-membrane MPs of MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches.This work was supported in part by European, Spanish, and Catalan Agency Grants FP7-HEALTH-F3-2009-223101 “AntiPathoGN,” FP7-HEALTH-2010-261460 “Gums&Joints,” FP7-PEOPLE-2011-ITN-290246 “RAPID,” FP7-HEALTH-2012-306029-2 “TRIGGER,” BFU2010-19310, BFU2012-32862, CSD2006-00015, Fundació “La Marató de TV3” Grants 2009-100732 and 2009SGR1036, postdoctoral JAE contract from Consejo Superior de Investigaciones Científicas (co-funded by FSE), and two FPI Ph.D. fellowships from the Spanish Ministry for Science and Technology, currently part of the Ministry of Economy and Competitiveness.Peer reviewe