Catalytic Domain Architecture of Metzincin Metalloproteases

Abstract

Metalloproteases cleave proteins and peptides, and deregulation of their function leads to pathology. An understanding of their structure and mechanisms of action is necessary to the development of strategies for their regulation. Among metallopeptidases are the metzincins, which are mostly multidomain proteins with ∼130–260-residue globular catalytic domains showing a common core architecture characterized by a long zinc-binding consensus motif, HEXXHXXGXX(H/D), and a methionine-containing Met-turn. Metzincins participate in unspecific protein degradation such as digestion of intake proteins and tissue development, maintenance, and remodeling, but they are also involved in highly specific cleavage events to activate or inactivate themselves or other (pro)enzymes and bioactive peptides. Metzincins are subdivided into families, and seven such families have been analyzed at the structural level: the astacins, ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases, snapalysins, leishmanolysins, and pappalysins. These families are reviewed from a structural point of view. Metalloproteases cleave proteins and peptides, and deregulation of their function leads to pathology. An understanding of their structure and mechanisms of action is necessary to the development of strategies for their regulation. Among metallopeptidases are the metzincins, which are mostly multidomain proteins with ∼130–260-residue globular catalytic domains showing a common core architecture characterized by a long zinc-binding consensus motif, HEXXHXXGXX(H/D), and a methionine-containing Met-turn. Metzincins participate in unspecific protein degradation such as digestion of intake proteins and tissue development, maintenance, and remodeling, but they are also involved in highly specific cleavage events to activate or inactivate themselves or other (pro)enzymes and bioactive peptides. Metzincins are subdivided into families, and seven such families have been analyzed at the structural level: the astacins, ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases, snapalysins, leishmanolysins, and pappalysins. These families are reviewed from a structural point of view. Cleavage of peptide bonds is essential for life, and the factors responsible for peptide cleavage are ubiquitous. Among them are MPs, 2The abbreviations used are: MPmetallopeptidaseMMPmatrix metalloproteinaseNSDN-terminal subdomainCSDC-terminal subdomain. which are mostly zinc-dependent peptide-bond hydrolases. They participate in metabolism through both extensive and unspecific protein degradation and controlled hydrolysis of specific peptide bonds (1.Neurath H. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 73: 3825-3832Crossref Scopus (338) Google Scholar). Deregulation of such vast degrading potential leads to pathologies, and in addition, MPs may also act as virulence factors during poisoning and microbial infection. Such a wide range of biological functions makes structural studies of these proteins indispensable to any understanding of their function and to the design of novel, highly specific therapeutic agents to modulate their activity (2.López-Otín C. Overall C.M. Nat. Rev. Mol. Cell Biol. 2000; 3: 509-519Crossref Scopus (632) Google Scholar). metallopeptidase matrix metalloproteinase N-terminal subdomain C-terminal subdomain. Most MPs are members of a protease tribe, the zincins, and they possess a short consensus amino acid sequence, HEXXH. This motif contains two protein ligands of the catalytic zinc and a glutamate that acts as general base/acid during the catalytic process (3.Bode W. Gomis-Rüth F.X. Stöcker W. FEBS Lett. 1993; 331: 134-140Crossref PubMed Scopus (649) Google Scholar, 4.Hooper N.M. FEBS Lett. 1994; 354: 1-6Crossref PubMed Scopus (672) Google Scholar). A third metal ligand is a solvent molecule, further bound to and polarized by the glutamate. This solvent performs nucleophilic attack on the carbonyl carbon of the scissile bond of a bound substrate, leading to a tetrahedral gem-diolate reaction intermediate, which is stabilized by the positively charged metal ion and neighboring protein residues (5.Matthews B.W. Acc. Chem. Res. 1988; 21: 333-340Crossref Scopus (673) Google Scholar). Subsequent evolution of the intermediate under assistance of the general base/acid eventually leads to bond disruption. Zincins are divided into the gluzincin, aspzincin, and metzincin clans (4.Hooper N.M. FEBS Lett. 1994; 354: 1-6Crossref PubMed Scopus (672) Google Scholar, 7.Gomis-Rüth F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar). The latter contains mostly multidomain proteins with an N-terminal prodomain engaged in latency maintenance, a catalytic protease domain, and farther downstream domains engaged in protein-protein and cell-cell interactions and other regulatory functions. The protease domain is characterized by a C-terminally extended zinc-binding motif, HEXXHXXGXX(H/D), with a hallmark glycine and a third zinc-binding histidine or aspartate. In addition, a methionine is present in a conserved downstream turn, the Met-turn (3.Bode W. Gomis-Rüth F.X. Stöcker W. FEBS Lett. 1993; 331: 134-140Crossref PubMed Scopus (649) Google Scholar, 7.Gomis-Rüth F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar, 8.Stöcker W. Grams F. Baumann U. Reinemer P. Gomis-Rüth F.X. McKay D.B. Bode W. Protein Sci. 1995; 4: 823-840Crossref PubMed Scopus (638) Google Scholar). Metzincins split into families, seven of which have been characterized at the structural level for at least one of their members: astacins, ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases, snapalysins, leishmanolysins, and pappalysins. In addition, a series of sequences reported from genome sequencing projects indicate there are further structurally yet uncharacterized families, tentatively referred to as fragilysins, gametolysins, archaemetzincins, thuringilysins, coelilysins, ascomycolysins, helicolysins, and cholerilysins (for a detailed review, see Ref. 7.Gomis-Rüth F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar). To date, >200 structures of metzincins, comprising at least the catalytic domain, have been deposited with the Protein Data Bank (supplemental Table 1). In this review, seven lead structures of each of the aforementioned families are discussed: Astacus astacus astacin, Crotalus adamanteus adamalysin II, Pseudomonas aeruginosa aeruginolysin, human neutrophil collagenase (MMP-8), Streptomyces caespitosus neutral protease (snapalysin), Leishmania major leishmanolysin, and Methanosarcina acetivorans ulilysin (9.Bode W. Gomis-Rüth F.X. Huber R. Zwilling R. Stöcker W. Nature. 1992; 358: 164-167Crossref PubMed Scopus (296) Google Scholar, 10.Baumann U. Wu S. Flaherty K.M. McKay D.B. EMBO J. 1993; 12: 3357-3364Crossref PubMed Scopus (426) Google Scholar, 11.Gomis-Rüth F.X. Kress L.F. Bode W. EMBO J. 1993; 12: 4151-4157Crossref PubMed Scopus (197) Google Scholar, 12.Kurisu G. Kinoshita T. Sugimoto A. Nagara A. Kai Y. Kasai N. Harada S. J. Biochem. (Tokyo). 1997; 121: 304-308Crossref PubMed Scopus (40) Google Scholar, 13.Bode W. Reinemer P. Huber R. Kleine T. Schnierer S. Tschesche H. EMBO J. 1994; 13: 1263-1269Crossref PubMed Scopus (324) Google Scholar, 14.Schlagenhauf E. Etges R. Metcalf P. Structure (Camb.). 1998; 6: 1035-1046Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 15.Tallant C. García-Castellanos R. Seco J. Baumann U. Gomis-Rüth F.X. J. Biol. Chem. 2006; 281: 17920-17928Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). These prototypes cover distinct kingdoms of life and represent archaea, bacteria, protozoa, crustaceans, reptiles, and mammals. The structures reveal that metzincins share a common scaffold and active-site environment, but each family has distinguishing structural elements. Mature catalytic domains are ∼130–260-residue globular moieties that bifurcate into an upper NSD and a lower CSD with respect to a central active-site cleft. Substrates bind horizontally to this cleft from left to right in an approximately extended conformation (supplemental Fig. 1 A). (All topological indications refer to the standard orientation displayed in this figure.) The NSD displays a five-stranded twisted β-sheet at the top (except leishmanolysin, which has four strands) (supplemental Fig. 1A). All strands (βI–βV) except the fourth are parallel to each other and to any substrate that is bound in the cleft. The antiparallel strand, βIV, forms the lower edge of this subdomain and creates an upper rim or northern wall of the active-site crevice (16.Maskos K. Bode W. Mol. Biotechnol. 2003; 25: 241-266Crossref PubMed Scopus (101) Google Scholar). This strand binds a substrate in an antiparallel manner, mainly on its non-primed side. The loop segment connecting strands βIII and βIV (referred to as LβIIIβIV) leads to the appearance of bulge-like elements, which mainly affect subsites S1′ and S2′ (see Ref. 17.Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4783) Google Scholar for subsite nomenclature in proteases). This gives rise to extensive variations in enzyme-substrate interactions on the primed side of the active-site clefts. The NSD also contains two long α-helices, αA and αB, the backing helix and the active-site helix, respectively. Both are arranged on the concave side of the β-sheet in an identical manner in all metzincin structures (supplemental Fig. 1B). Helix αB superimposes well in all seven structures (Fig. 1A) and encompasses the first half of the zinc-binding motif, which includes the first two zinc-binding histidine residues (Fig. 1A and supplemental Fig. 1). At the end of helix αB, the polypeptide chain takes a sharp downward turn, mediated by the glycine of the consensus sequence. The main-chain angles of this residue in the different lead structures indicate that any other residue would be in a high-energy conformation and thus disfavored, with the exception of ulilysin (see below). The CSD starts after this glycine, and the chain leads to the third zinc ligand, a histidine or an aspartate, which approaches the metal from below (supplemental Fig. 1). This subdomain contains few repetitive secondary structure elements, mainly a C-terminal helix αC at the end of the polypeptide chain. Helices αB and αC are connected by structures that vary both in length and conformation. However, all structures coincide at a conserved 1,4-β-turn containing a methionine at position 3, the Met-turn, which is separated from the third zinc-binding histidine by connecting segments of 6–53 amino acids in the different structures. The Met-turn is superimposable (including the conformation of the methionine side chain) (Fig. 1A) and is positioned underneath the catalytic Zn2+, forming a hydrophobic pillow. However, no direct contact with the metal is observed. Mutation studies suggested a role for this methionine in the folding and stability of the catalytic domains, although the strict conservation of this residue remains to be explained (18.Hege T. Baumann U. J. Mol. Biol. 2001; 314: 181-186Crossref PubMed Scopus (26) Google Scholar, 19.Butler G.S. Tam E.M. Overall C.M. J. Biol. Chem. 2004; 279: 15615-15620Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). 3A. E. Oberholzer, M. Bumann, T. Hege, S. Russo, and U. Baumann, unpublished data. The S1′ pocket of metzincins is shaped at the top by a protruding bulge made by LβIIIβIV and at the bottom by a wall-forming segment made up of residues intercalated between the Met-turn and the C-terminal helix αC (16.Maskos K. Bode W. Mol. Biotechnol. 2003; 25: 241-266Crossref PubMed Scopus (101) Google Scholar). This segment diverges in structure and length (ranging from 11 to 37 residues between the Met-turn methionine and the first residue of helix αC) in all seven of the reference structures. The catalytic zinc ion lies roughly at the center of the bottom of the active-site cleft. It is coordinated by the Nϵ2 atoms of the three consensus histidines (two histidine Nϵ2 atoms and one aspartate Oδ2 atom in snapalysin) and the catalytic solvent molecule, substituted by other ligands in the enzyme/inhibitor-product complexes reported (supplemental Table 1). Some metzincins display an additional protein ligand at a slightly greater distance from the catalytic cation in the form of a tyrosine Oη atom, as seen in unbound and and as in ulilysin C. García-Castellanos R. A. F. Y. M. Baumann U. Gomis-Rüth F.X. Biol. Chem. PubMed Scopus Google Scholar). This tyrosine residue lies two of the Met-turn In a tyrosine lies two downstream of the aspartate, no distance of the zinc These tyrosine residues and during substrate and in a referred to as the tyrosine In they may a role in substrate and catalytic solvent and of the tetrahedral intermediate or the amino F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar, W. Gomis-Rüth F.X. Huber R. Zwilling R. Stöcker W. Nature. 1992; 358: 164-167Crossref PubMed Scopus (296) Google Scholar, I. E. Stöcker W. FEBS Lett. 2000; PubMed Scopus Google Scholar, T. Baumann U. J. Mol. Biol. 2001; 314: PubMed Scopus Google Scholar, F. A. I. S. Zwilling R. Bode W. Stöcker W. Biol. 3: PubMed Scopus Google Scholar). Such a role is by residues in A with the exception of the comprising the zinc-binding consensus sequence, is metzincins structural on this between leads (supplemental Fig. between and and between and the at which two be as structurally P. A. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). The range between and below for Protein 12: PubMed Scopus Google Scholar). The structural are and (supplemental Fig. and supplemental H. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 73: 3825-3832Crossref Scopus (338) Google Scholar and C. Overall C.M. Nat. Rev. Mol. Cell Biol. 2000; 3: 509-519Crossref Scopus (632) Google and adamalysin and ulilysin and adamalysin and The are and adamalysin and ulilysin F.X. Rev. Biochem. Mol. Biol. PubMed Scopus Google and and ulilysin is a from the A. astacus and the first of the family to be structurally analyzed (9.Bode W. Gomis-Rüth F.X. Huber R. Zwilling R. Stöcker W. Nature. 1992; 358: 164-167Crossref PubMed Scopus (296) Google Scholar). protein matrix and degradation participate in biological such as development, and tissue and and and Stöcker W. Mol. PubMed Scopus Google Scholar). The structure of a and two of approximately (supplemental Fig. 1A) (9.Bode W. Gomis-Rüth F.X. Huber R. Zwilling R. Stöcker W. Nature. 1992; 358: 164-167Crossref PubMed Scopus (296) Google Scholar). this contains a and a catalytic domain, other display additional C-terminal and (for see 8.Stöcker W. Grams F. Baumann U. Reinemer P. Gomis-Rüth F.X. McKay D.B. Bode W. Protein Sci. 1995; 4: 823-840Crossref PubMed Scopus (638) Google Scholar, Stöcker W. Mol. PubMed Scopus Google Scholar, and Protein Sci. 1995; 4: PubMed Scopus Google Scholar). The family has the of the CSD secondary structure elements, a helix and a short although this is the conserved connecting segment metzincins (see Table 1 in Ref. 7.Gomis-Rüth F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar). The protein scaffold is by four conserved residues forming two (supplemental Fig. 1). The first two residues of the are in an in the and the N-terminal an with a conserved glutamate to the third zinc-binding The unbound of the catalytic zinc is to the of the fourth although tyrosine zinc ligand downstream of the Met-turn (supplemental Fig. 1A). In to astacin, the structures of human and have been reported A. S. A. A. U. P. C. J. Mol. Biol. Scopus Google Scholar). are virulence factors as by U. of Scopus Google Scholar). These are responsible for human such as tissue and other and They a major role in to their to and to of the virulence potential of these bacteria, are factors and protease and and may an The first to be and structurally characterized P. aeruginosa U. Wu S. Flaherty K.M. McKay D.B. EMBO J. 1993; 12: 3357-3364Crossref PubMed Scopus (426) Google Scholar). catalytic domain and is on its C-terminal end by a in astacin, its two are of The polypeptide chain starts with an in the CSD for the that is to the by a conserved with the C-terminal helix αC (supplemental Fig. 1B). The NSD a made up by an and the of the This reported (supplemental Table substrate The CSD of an the segment the Met-turn with the wall-forming and a shaped by residues of the connecting These also modulate substrate of aeruginolysin, which first in with a bound U. Wu S. Flaherty K.M. McKay D.B. EMBO J. 1993; 12: 3357-3364Crossref PubMed Scopus (426) Google with the structure of unbound that the zinc is to It also includes a tyrosine that a substrate U. J. Mol. Biol. 1994; PubMed Scopus Google Scholar). S. caespitosus is a neutral protease that a catalytic domain by an acid N-terminal a peptide and a sequences have been reported for other Streptomyces and they have been and and They is the metzincin and the family that has been structurally structure a and into two G. Kinoshita T. Sugimoto A. Nagara A. Kai Y. Kasai N. Harada S. J. Biochem. (Tokyo). 1997; 121: 304-308Crossref PubMed Scopus (40) Google Scholar). It displays all the metzincin connected by short are a protruding from the upper the a bulge on top of the primed side of the active-site a short helix in and a (supplemental Fig. 1A). In addition, an aspartate is at the position of the third zinc-binding and two in the sequence, a conserved tyrosine approaches but binds the are or and participate in during They are mainly in although sequences have been in and of matrix are involved in tissue remodeling, and as during and development, and and However, their potential or its may also lead to such as and and and and H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have been to be engaged in events as in and protein but also in human and proteins such as other peptides, and and In common with (see are also involved in of and and and from the Cell Biol. 2001; 13: PubMed Scopus Google Scholar). other metzincin families, are proteins by a series of and These may an an a and catalytic domain, a and a C-terminal may a and a a and an an a a and a of with collagenase as and has with different forms in Overall C.M. C. Nat. Rev. 2003; 4: PubMed Scopus Google Scholar). a rise to the (16.Maskos K. Bode W. Mol. Biotechnol. 2003; 25: 241-266Crossref PubMed Scopus (101) Google Scholar, H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in to a or This the prodomain and from an the atom of a residue a conserved motif, the catalytic solvent in the zinc to the H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). are the structurally metzincin with structures reported (supplemental Table C. A. Gomis-Rüth F.X. Biophys. Cell Scholar). The catalytic domain of human neutrophil collagenase W. Reinemer P. Huber R. Kleine T. Schnierer S. Tschesche H. EMBO J. 1994; 13: 1263-1269Crossref PubMed Scopus (324) Google Scholar, C. A. Gomis-Rüth F.X. Biophys. Cell has a active-site which a NSD from a CSD amino and a hydrophobic S1′ bonds are present in the The N-terminal is to the first of two conserved in helix The NSD displays an loop connecting strands βIII and βIV, which a structural zinc cation and a bound The downstream residues of this segment form a bulge that into the active-site and to a on top of the NSD β-sheet (supplemental Fig. A and In the the chain displays the and conserved connecting segment split into three the MPs, the and the S. M. C. C. PubMed Scopus Google Scholar, Nat. Rev. Mol. Cell Biol. 6: PubMed Scopus Google Scholar, Mol. PubMed Scopus Google Scholar, S. Cell Biol. Google Scholar). The are responsible for through digestion of matrix in tissue In turn, to a role in and function in They are involved in development, of and They are also engaged in human and and They also have a major role in protein as for family members the domain and of a and a domain rise to a distinct of the H. K. Res. PubMed Scopus Google Scholar). These by to They are also involved in development and and as well as in degradation in and All are multidomain proteins containing a catalytic and In addition, they display a prodomain and C-terminal domains, as well as a is by the and is to as in by cleavage of the prodomain to a F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar, H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, F. Huber R. Kress L.F. Bode W. FEBS Lett. 1993; PubMed Scopus Google Scholar). The first catalytic domain structure to be analyzed that of adamalysin from C. adamanteus F.X. Kress L.F. Bode W. EMBO J. 1993; 12: 4151-4157Crossref PubMed Scopus (197) Google Scholar). This is a of at the to a cleft. This cleft a NSD from a CSD (supplemental Fig. 1A). Both the and are the is by a to the C-terminal helix αC F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar). from the metzincin consensus the conserved secondary structure elements, at strand and αA and αC (Fig. 1B). into the common two additional are the In the two bonds the connecting segment and helix αC to the respectively. A ion is on the the and to the (supplemental Fig. 1A). The S1′ characterized by a bulge segment is hydrophobic and of In to adamalysin II, a of MPs and and have been structurally analyzed to (supplemental Table 1). are proteins present in and They the major of the and are polypeptide They cleave at the of human and from by a role as a virulence sequences have been in and F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar, S. P. J. Cell Biol. 2004; PubMed Scopus Google Scholar). The structurally analyzed family is major It is as a in the with a and a which includes a highly conserved residue as a (see a of by an C-terminal A domain is between the glycine and the third zinc-binding histidine of the long consensus motif (supplemental Fig. 1A). The domain is the metzincins, with a NSD and an acid is on the left The NSD is characterized by a which strand (supplemental Fig. A and and by the of two acid which for of the in from the other proteins of the The NSD is by two strand βIV, a slightly bulge segment lies on top of the S1′ which is by the wall-forming segment and the of the active-site helix At the end of the helix αC is by a segment in an extended which from left to right the E. Etges R. Metcalf P. Structure (Camb.). 1998; 6: 1035-1046Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The family to be structurally characterized are the pappalysins. They after human a multidomain protein proteins K. C. Biochem. J. 2001; 358: PubMed Scopus Google Scholar). is a protein from M. acetivorans that with but encompasses the prodomain and the catalytic domain C. García-Castellanos R. Seco J. Baumann U. Gomis-Rüth F.X. J. Biol. Chem. 2006; 281: 17920-17928Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The may as suggested by the of a conserved in the in the of ulilysin is the of all metzincin catalytic distinguishing in the NSD a loop strand into two and and a LβIIIβIV that from the and the on its primed side. The segment connecting helix αA with strand is the metzincins and all of the of the from the NSD to the CSD in a and includes two The glycine of the zinc-binding motif is in ulilysin and a of by an under of the main-chain which to a high-energy conformation but to a the chain this residue is from other metzincins (supplemental Fig. F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar). The CSD two bonds and a This is a for as the be through C. García-Castellanos R. Seco J. Baumann U. Gomis-Rüth F.X. J. Biol. Chem. 2006; 281: 17920-17928Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, R. C. A. M. Baumann U. Gomis-Rüth F.X. Biochem. Biophys. PubMed Scopus Google Scholar, C. García-Castellanos R. A. F. Y. M. Baumann U. Gomis-Rüth F.X. Biol. Chem. PubMed Scopus Google Scholar). in the of an unbound ulilysin may possess a zinc-binding tyrosine ligand by the Met-turn that is substrate The metzincins a of MPs present in all kingdoms of life, which are for and pathology. seven families have been structurally The a of evolution from an which may different from the such a evolution has structural conserved each on the of an extended zinc-binding consensus families have been suggested to the although structural be to their on each of the seven families may to common catalytic and mechanisms and to the function of proteins by