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

Mechanisms of proteolytic activity regulation exerted via a unique propeptide in matrix metalloproteinases and intra/intermolecular interactions in a novel family of minimal gluzincins

Les metal·lopeptidases participen de manera decisiva en la fisiologia i patologia de tots els organismes vius. La seva estricta regulació és, per tant, essencial pel correcte funcionament d’aquestes i per a prevenir una activitat proteolítica inadequada en el temps i/o espai que podria causar malalties. Aquesta regulació s’aconsegueix mitjançant un ampli ventall de mecanismes, incloent la seva biosíntesis com a precursors inactius, també coneguts com a zimògens. En la present tesi s’han estudiat diversos mecanismes de regulació de metal·lopeptidases, tot combinant tècniques bioquímiques, biofísiques i estructurals. En el primer projecte, l’estructura cristal·lina d’un fragment zimogènic de la proteïna “karilysin” de Tannerella forsythia ha revelat el propèptid més curt descrit fins al moment per una metal·lopeptidasa. Assajos bioquímics i biofísics addicionals han permès determinar la importància del propèptid en l’expressió, plegament i estabilitat de la proteïna. En el segon projecte, s’ha descobert una nova família de metal·lopeptidases mínimes d’origen procariota, que s’han anomenat “minigluzincins”, les quals proporcionen un model estructural per a metal·lopeptidases pertanyents al clan de les gluzincines i per a d’altres integrals de membrana. Dues membres d’aquesta família, anomenades “proabylysin” i “projannalysin”, han exhibit formes úniques de manteniment de latència exercides, respectivament, de forma intra- i intermolecular mitjançant llurs segments C-terminals. En el tercer projecte, una altra membre d’aquesta nova família, anomenada “selecase”, ha presentat una activitat proteolítica selectiva i específica sobre caseïna. La conjunció d’estudis duts a terme, tant biofísics com estructurals, ha evidenciat que “selecase” transita de manera reversible entre diverses conformacions d’estructura tridimensional definida, associades amb una disminució d’activitat enzimàtica deguda a autoinhibició (és a dir, monòmers actius i dímers, tetràmers i octàmers inactius). En definitiva, aquesta tesi contribueix de manera substancial a ampliar el coneixement previ sobre metal·lopeptidases a nivell molecular i a entendre els mecanismes que en regulen l’activitat, facilitant així el disseny d’inhibidors específics com a part d’aproximacions terapèutiques.Metallopeptidases are major players in the physiology and pathology of all living organisms. Their exquisite regulation is therefore essential for proper function and to prevent misdirected temporal and/or spatial proteolytic activity, which may lead to disease. This regulation is achieved through a wide variety of mechanisms, including their biosynthesis as inactive precursors, also known as zymogens. In the present thesis, various mechanisms of metallopeptidase regulation were studied by using a combination of biochemical, biophysical, and structural techniques. In the first project, the crystal structure of a zymogen fragment of Tannerella forsythia karilysin revealed the shortest propeptide reported to date for a metallopeptidase. Additional biochemical and biophysical assays allowed ascertaining the importance of the propeptide in protein expression, folding and stability. In the second project, a novel family of minimal prokaryotic metallopeptidases termed “minigluzincins” was discovered, providing a minimal soluble scaffold for gluzincin metallopeptidases and integral-membrane metallopeptidases. Two members of this family, called proabylysin and projannalysin, showed two unique zymogenic forms of latency maintenance exerted, respectively, via intramolecular and intermolecular interactions through their C-terminal segments. In the third project, a further member of this novel family, called selecase, evinced selective and specific proteolytic activity against casein. A set of biophysical and structural studies showed that selecase reversibly commutes between several conformations of defined three- dimensional structure, which are associated with loss of enzymatic activity due to autoinhibition (i.e. active monomers vs. inactive dimers, tetramers and octamers). Overall, the present thesis contributes substantially to the field broadening previous knowledge at the molecular level on metallopeptidases and their regulatory mechanisms, which paves the way for the design of specific inhibitors to modulate their activity as part of therapeutic approaches

Mechanisms of proteolytic activity regulation exerted via a unique propeptide in matrix metalloproteinases and intra/intermolecular interactions in a novel family of minimal gluzincins

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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

A novel mechanism of latency in matrix metalloproteinases

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Background: Animal and plant matrix metalloproteinases (MMPs) are kept zymogenic through large prodomains and a cysteine-switch mechanism.This work was supported in part by European, United States American, Polish, Spanish, and Catalan Grants UMO-2012/04/A/NZ1/00051, UMO-2013/08/T/NZ1/00315, 2137/7.PR-EU/2011/2, 2975/7.PR/13/2014/2, DE09761, DE022597, FP7-HEALTH-2010-261460 “Gums&Joints,” FP7-PEOPLE-2011-ITN-290246 “RAPID,” FP7-HEALTH-2012-306029-2 “TRIGGER,” BFU2012-32862, BIO2013-49320-EXP, CSD2006-00015, and 2014SGR9)Peer Reviewe

In vitro reconstitution of Escherichia coli divisome activation (Nature Communications, (2022), 13, 1, (2635), 10.1038/s41467-022-30301-y)

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Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins

60 p.-11 fig.-3 tab.Most bacteria accomplish cell division with the help of a dynamic protein complex called the divisome, which spans the cell envelope in the plane of division. Assembly and activation of this machinery are coordinated by the tubulin-related GTPase FtsZ, which was found to form treadmilling filaments on supported bilayers in vitro1, as well as in live cells, in which filaments circle around the cell division site2,3. Treadmilling of FtsZ is thought to actively move proteins around the division septum, thereby distributing peptidoglycan synthesis and coordinating the inward growth of the septum to form the new poles of the daughter cells4. However, the molecular mechanisms underlying this function are largely unknown. Here, to study how FtsZ polymerization dynamics are coupled to downstream proteins, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division. We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ–FtsA filaments, but despite their directed collective behaviour, individual peptides showed random motion and transient confinement. Our work suggests that divisome proteins follow treadmilling FtsZ filaments by a diffusion-and-capture mechanism, which can give rise to a moving zone of signalling activity at the division site.This work was supported by the European Research Council through grant ERC-2015-StG-679239 to M.L. and grants HFSP LT 000824/2016-L4 and EMBO ALTF 1163-2015 to N.B., a grant from the Ministry of Economy and Competitiveness of the Spanish Government (BFU2016-75471-C2-1-P) to C.A. and G.R., and a Wellcome Trust Senior Investigator award (101824/Z/13/Z) and a grant from the BBSRC (BB/R017409/1) to W.V.Peer reviewe

A unique network of attack, defence and competence on the outer membrane of the periodontitis pathogen Tannerella forsythia

International audiencePeriodontopathogenic Tannerella forsythia uniquely secretes six peptidases of disparate catalytic classes and families that operate as virulence factors during infection of the gums, the KLIKK-peptidases. Their coding genes are immediately downstream of novel ORFs encoding the 98–132 residue potempins (Pot) A, B1, B2, C, D and E. These are outer-membrane-anchored lipoproteins that specifically and potently inhibit the respective downstream peptidase through stable complexes that protect the outer membrane of T. forsythia, as shown in vivo. Remarkably, PotA also contributes to bacterial fitness in vivo and specifically inhibits matrix metallopeptidase (MMP) 12, a major defence component of oral macrophages, thus featuring a novel and highly-specific physiological MMP inhibitor. Information from 11 structures and high-confidence homology models showed that the potempins are distinct b-barrels with either a five-stranded OB-fold (PotA, PotC and PotD) or an eight-stranded up-and-down fold (PotE, PotB1 and PotB2), which are novel for peptidase inhibitors. Particular loops insert like wedges into the active-site cleft of the genetically-linked peptidases to specifically block them either via a new “bilobal” or the classic “standard” mechanism of inhibition. These results discover a unique, tightly-regulated proteolytic armamentarium for virulence and competence, the KLIKK-peptidase/potempin system

A unique network of attack, defence and competence on the outer membrane of the periodontitis pathogen Tannerella forsythia

Periodontopathogenic Tannerella forsythia uniquely secretes six peptidases of disparate catalytic classes and families that operate as virulence factors during infection of the gums, the KLIKK-peptidases. Their coding genes are immediately downstream of novel ORFs encoding the 98-132 residue potempins (Pot) A, B1, B2, C, D and E. These are outer-membrane-anchored lipoproteins that specifically and potently inhibit the respective downstream peptidase through stable complexes that protect the outer membrane of T. forsythia, as shown in vivo. Remarkably, PotA also contributes to bacterial fitness in vivo and specifically inhibits matrix metallopeptidase (MMP) 12, a major defence component of oral macrophages, thus featuring a novel and highly-specific physiological MMP inhibitor. Information from 11 structures and high-confidence homology models showed that the potempins are distinct $\beta$-barrels with either a five-stranded OB-fold (PotA, PotC and PotD) or an eight-stranded up-and-down fold (PotE, PotB1 and PotB2), which are novel for peptidase inhibitors. Particular loops insert like wedges into the active-site cleft of the genetically-linked peptidases to specifically block them either via a new "bilobal" or the classic "standard" mechanism of inhibition. These results discover a unique, tightly-regulated proteolytic armamentarium for virulence and competence, the KLIKK-peptidase/potempin system