Crystal Structure of an Active Form of Human MMP-1

Iyer, Shalini; Visse, Robert; Nagase, Hideaki; Acharya, K. Ravi

doi:10.1016/j.jmb.2006.06.079

Cited by 104 publications

(129 citation statements)

References 32 publications

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“…Comparison of the crystal structure of proMMP-1 (29) with the structure of the activated form of MMP-1(E200A) (6,7) suggested that the Cat-Hpx linker region confers interdomain flexibility. Such flexing was further demonstrated for MMP-1 by NMR and small angle X-ray scattering studies (40,41) and seems to be a common property of MMPs (42,43).…”

Section: Discussionmentioning

confidence: 99%

“…It consists of an N-terminal catalytic (Cat) domain containing an active-site zinc ion and a C-terminal hemopexin (Hpx) domain comprised of a four-bladed β-propeller, which are connected by a linker region (6,7). Although the Cat domain can cleave a number of noncollagenous proteins including heat-denatured collagen (gelatin), its activity on native triplehelical collagen is negligible.…”

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confidence: 99%

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Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1

Manka

Carafoli

Visse

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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193

310

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Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triplehelical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis. extracellular matrix | X-ray crystallography | protease T he interstitial collagens I, II, and III are the major structural proteins in connective tissues such as skin, bone, cartilage, tendon, and blood vessels (1). They consist of three α chains with repeating Gly-X-Y triplets (X and Y are often proline and hydroxyproline, respectively) that intertwine each other to form a triple helix of ∼300 nm in length (2). Interstitial collagens are resistant to most proteolytic enzymes, but vertebrate collagenases cleave them at a single site approximately three-quarters of the way from the N terminus of the triple helix, thus initiating collagenolysis (3). Owing to this unique activity, collagenases play important roles in embryo development, morphogenesis, tissue remodeling, wound healing, and human diseases, such as arthritis, cancer, and atherosclerosis (4, 5).Matrix metalloproteinase 1 (MMP-1) is a typical vertebrate collagenase (3). It consists of an N-terminal catalytic (Cat) domain containing an active-site zinc ion and a C-terminal hemopexin (Hpx) domain comprised of a four-bladed β-propeller, which are connected by a linker region (6, 7). Although the Cat domain can cleave a number of noncollagenous proteins including heat-denatured collagen (gelatin), its activity on native triplehelical collagen is negligible. The combination of the Cat and Hpx domains is required for MMP-1 to be able to degrade native collagen, and the same is true for all other collagenolytic MMPs, namely MMP-2, MMP-8, MMP-13, and MMP-14 (3). How collagenases interact with collagen and how the Hpx domain endows these enzymes with collagenolytic activity is not clearly understood. Another enigma of collagenolysis bec...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1

Manka

Carafoli

Visse

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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193

310

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“…3 Ribbon diagram representation of the full-length MMP-1 structure. This figure was modeled after the X-ray structure of porcine synovial collagenase [29]. Mutated amino acids Val268, Gly272, and Lys277 of the hinge region are shown.…”

Section: Site-directed Mutants Of Mmp-1mentioning

confidence: 99%

“…Since physiological collagenolysis is crucial for several biological processes, such as tissue repair and remodeling, angiogenesis, and wound healing [3,21], and collagenases are the main MMPs involved in collagen turnover and remodeling [22][23][24], a clarification of the mechanism of processing of the triple helix by collagenases is of the utmost importance. Structural data on fibrillar collagen I [25][26][27] indeed have shown that, on the basis of the available three-dimensional structures of collagenases [28,29], the triple-helical structural assembly does not allow collagen I to come into close contact with the catalytic site of collagenases. Therefore, a more complex mechanism of substrate recognition must be postulated, where an exosite topologically distinct from the active site interacts with the substrate (likely through the hemopexin-like domain in collagenases; see [14]) with a very limited alteration of the triple-helical arrangement of collagen I [30][31][32].…”

Section: Introductionmentioning

confidence: 99%

The collagenolytic action of MMP-1 is regulated by the interaction between the catalytic domain and the hinge region

et al. 2012

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The role of the hinge region in the unwinding and cleavage of type I collagen by interstitial collagenase (MMP-1) has been studied at 37°C and pH 7.3. The collagenolytic processing by MMP-1 displays a very similar overall rate for both chains of collagen I, even though the affinity is higher for the a-1 chain and the cleavage rate is faster for the a-2 chain. MMP-1 binding to collagen I brings about a significant unwinding of the triple-helical arrangement only after the first cleavage step of the a-1 and a-2 chains. The proteolytic processing by wild-type MMP-1 on a synthetic substrate and collagen I has been compared with that observed for site-directed mutants obtained either by truncating the hinge region (D255-272) or by individually replacing the conserved amino acids Val268, Gly272, and Lys277 of the hinge region with residues observed for the corresponding position in stromelysin-1 (MMP-3), a noncollagenolytic metalloproteinase. The D256-272 mutant has no collagenolytic activity, clearly demonstrating the crucial role of this region for the enzymatic processing of collagen I. However, among various mutants investigated, only Gly272Asp shows a dramatically reduced enzymatic activity both on the synthetic substrate and on collagen I. This effect, however, is clearly related to the substituting residue, since substitution of Ala or Asn for Gly272 does not have any effect on the kinetic properties of MMP-1. These data suggest that the substrate specificity of MMP-1 is dictated by the reciprocal structural relationships between the catalytic domain and the carboxyterminal domain through the conformational arrangement of the hinge region.

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“…1) (10,11), and anti-GBD antibodies inhibit collagen organization in fibroblast cultures (12). Similarly, experiments in cultures have identified the collagenase/ MMP-1 (13) cleavage site in type I collagen as important for FN-mediated attachment of fibroblasts to collagen ECM (14). Peptides spanning that region (15) or MMP-1 cleavage at this site (6) inhibit attachment.…”

mentioning

confidence: 98%

Identification and structural analysis of type I collagen sites in complex with fibronectin fragments

Erat¹,

Slatter

Lowe

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

136

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Collagen and fibronectin are major components of vertebrate extracellular matrices. Their association and distribution control the development and properties of diverse tissues, but thus far no structural information has been available for the complex formed. Here, we report binding of a peptide, derived from the ␣1 chain of type I collagen, to the gelatin-binding domain of human fibronectin and present the crystal structure of this peptide in complex with the 8 -9 FnI domain pair. Both gelatin-binding domain subfragments, 6 FnI 1-2 FnII 7 FnI and 8 -9 FnI, bind the same specific sequence on D-period 4 of collagen I ␣1, adjacent to the MMP-1 cleavage site. 8 -9 FnI also binds the equivalent sequence of the ␣2 chain. The collagen peptide adopts an antiparallel ␤-strand conformation, similar to structures of proteins from pathogenic bacteria bound to FnI domains. Analysis of the type I collagen sequence suggests multiple putative fibronectin-binding sites compatible with our structural model. We demonstrate, by kinetic unfolding experiments, that the triple-helical collagen state is destabilized by 8 -9 FnI. This finding suggests a role for fibronectin in collagen proteolysis and tissue remodeling.collagen destabilization ͉ extracellular matrix ͉ protein structure C ollagen is the most abundant protein in mammals, accounting for Ϸ25% of the body-protein content. It is crucial for effects as diverse as cell differentiation, cell migration, and mechanical properties of tissues. Many human diseases have their origin in mutations that affect interactions of collagen with other extracellular matrix (ECM) molecules and cell receptors (1). Type I collagen fibrils form spontaneously in vitro; in vivo, however, formation requires integrin receptors and fibronectin (FN) (2). FN is a large glycosylated protein composed of multiple copies of 3 classes of domains, FnI, FnII, and FnIII, that forms fibrils in the ECM (3). It plays a role in many important physiological processes, such as embryogenesis, wound healing, hemostasis, and thrombosis (4), and disruption of the FN gene is embryonic lethal in mice (5).The interaction of these 2 proteins has long been demonstrated in vitro by using denatured collagen (gelatin) (6, 7) and isolated collagen type I chains or chain fragments (8, 9). The collagen-binding site on FN has been localized to the 42-kDa gelatin-binding domain (GBD; for an overview of FN and collagen fragments see Fig. 1) (10, 11), and anti-GBD antibodies inhibit collagen organization in fibroblast cultures (12). Similarly, experiments in cultures have identified the collagenase/ MMP-1 (13) cleavage site in type I collagen as important for FN-mediated attachment of fibroblasts to collagen ECM (14). Peptides spanning that region (15) or MMP-1 cleavage at this site (6) inhibit attachment. However, attempts to reconstitute the FN-collagen interaction in vitro by using synthetic peptides failed to find strong, specific, interactions (8,9), and the precise sequence determinants for FN-binding to collagen remained unknown...

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Crystal Structure of an Active Form of Human MMP-1

Cited by 104 publications

References 32 publications

Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1

Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1

The collagenolytic action of MMP-1 is regulated by the interaction between the catalytic domain and the hinge region

Identification and structural analysis of type I collagen sites in complex with fibronectin fragments

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