The interface between catalytic and hemopexin domains in matrix metalloproteinase-1 conceals a collagen binding exosite

Matrix metalloproteinase-1 (MMP-1) is an instigator of collagenolysis, the catabolism of triple helical collagen. Previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilizing the collagen substrate in preparation for hydrolysis of the polypeptide backbone by the catalytic (CAT) domain. Here, we use biophysical methods to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. The two components interact with 1:1 stoichiometry and micromolar affinity via a binding site within blades 1 and 2 of the four-bladed HPX domain propeller. Subsequent site-directed mutagenesis and assay implicates blade 1 residues Phe(301), Val(319), and Asp(338) in collagen binding. Intriguingly, Phe(301) is partially masked by the CAT domain in the crystal structure of full-length MMP-1 implying that transient separation of the domains is important in collagen recognition. However, mutation of this residue in the intact enzyme disrupts the CAT-HPX interface resulting in a drastic decrease in binding activity. Thus, a balanced equilibrium between these compact and dislocated states may be an essential feature of MMP-1 collagenase activity.     

Selective hydrolysis of triple-helical substrates by matrix metalloproteinase-2 and -9

The role of proteases in the tumor cell invasion process is multifaceted. Members of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and degradation of extracellular matrix (ECM) components. Differentiating between the up-regulation of MMP production and the presence of activated MMPs can be difficult but may well dictate which MMPs are critical to invasion. Because the hydrolysis of collagens is one of the committed steps in ECM turnover, we have investigated selective MMP action on collagenous substrates as a means to evaluate active MMPs. Two triple-helical peptide (THP) models of the MMP-9 cleavage site in type V collagen, alpha1(V)436-450 THP and alpha1(V)436-447 fTHP, were hydrolyzed by MMP-2 and MMP-9 at the Gly-Val bond, analogous to the bond cleaved by MMP-9 in the corresponding native collagen. Kinetic analyses showed k(cat)/K(m) values of 14,002 and 5,449 s(-1)m(-1) for MMP-2 and -9 hydrolysis of alpha1(V)436-447 fTHP, respectively. These values, along with individual k(cat) and K(m) values, are comparable with collagen hydrolysis by MMP-2 and -9. Neither THP was hydrolyzed by MMP-1, -3, -13, or -14. alpha1(V)436-447 fTHP and a general fluorogenic THP were used to screen for triple-helical peptidase activity in alpha(2)beta(1) integrin-stimulated melanoma cells. Binding of the alpha(2)beta(1) integrin resulted in the production of substantial triple-helical peptidase activity, the majority (>95%) of which was non-MMP-2/-9. THPs were found to provide highly selective substrates for members of the MMP family and can be used to evaluate active MMP production in cellular systems.     

Pivotal molecular determinants of peptidic and collagen triple helicase activities reside in the S3' subsite of matrix metalloproteinase 8 (MMP-8): the role of hydrogen bonding potential of ASN188 and TYR189 and the connecting cis bond

The mechanism of triple helical collagen unwinding and cleavage by collagenases in the matrix metalloproteinase (MMP) family is complex and remains enigmatic. Recent reports show that triple helicase activity is initiated by the hemopexin C domain of membrane type 1-MMP, whereas catalytically inactive full-length interstitial collagenase (MMP-1) exhibits full triple helicase functionality pointing to active site determinants that are needed to complete the triple helicase mechanism. In MMP-8, the neutrophil collagenase, a conserved Gly at the S(3)' substrate specificity subsite is replaced by Asn(188) that forms a highly unusual cis bond with Tyr(189), a conserved active site residue in the collagenases. Only in MMP-1 is the S(3)' Gly also replaced, and there too a cis configured Glu-Tyr occurs. Thus, this high energy peptide bond coupled to the canonical Tyr may be important in the collagenolytic process. In a systematic mutagenesis investigation of the MMP-8 S(3)' subsite we found that introducing an S(3)' Gly(188) into MMP-8 reduced collagenolytic efficiency by approximately 30% with a corresponding reduction in cleavage of a synthetic peptide fluorescence resonance energy transfer substrate analogue of the alpha2(I) collagen chain cleavage site. The substitution of Asn(188) to Leu, a hydrophobic residue of similar size to the highly polar Asn and designed to retain the cis bond, revealed the importance of hydrogen bonding to bound substrate with both collagenolytic and peptidic activities reduced approximately 3-fold. In contrast, the specificity for type I collagen of the mutant Y189F dropped 3-fold without any significant alteration in general peptidase activity. Therefore, S(3)' and in particular the hydrogen bonding potential of Tyr(189) is a specific molecular determinant for MMP-8 triple helicase activity. The cis bond connection to Asn(188) juxtaposes these two side chains for closely spaced hydrogen bonding with substrate that improves collagenolytic and general catalytic efficiency that could be exploited for new collagenase-specific inhibitor drugs.     

Identification of Specific Hemopexin-like Domain Residues That Facilitate Matrix Metalloproteinase Collagenolytic Activity

Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Several members of the matrix metalloproteinase (MMP) family catalyze the hydrolysis of collagen triple helical structure. This study has utilized triple helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry followed by evaluation of wild type and mutant MMP-1 kinetics led to the identification of three noncatalytic regions in MMP-1 (residues 285–295, 302–316, and 437–457) and two specific residues (Ile-290 and Arg-291) that participate in collagenolysis. Ile-290 and Arg-291 contribute to recognition of triple helical structure and facilitate both the binding and catalysis of the triple helix. Evidence from this study and prior studies indicates that the MMP-1 catalytic and hemopexin-like domains collaborate in collagen catabolism by properly aligning the triple helix and coupling conformational states to facilitate hydrolysis. This study is the first to document the roles of specific residues within the MMP-1 hemopexin-like domain in substrate binding and turnover. Noncatalytic sites, such as those identified here, can ultimately be utilized to create THP inhibitors that target MMPs implicated in disease progression while sparing proteases with host-beneficial functions.The mechanism of collagenolysis, by which proteases catalyze the hydrolysis of amide bonds within triple helical structures, has been investigated for over 30 years. Despite this lengthy period, few inroads have been made in the identification of specific enzyme residues that facilitate collagenolysis. The primary mammalian collagenases have been identified as cathepsin K and several members of the matrix metalloproteinase (MMP)³ family. Most of the early work on MMP collagenolysis focused on analysis of the sites of hydrolysis, and how unique features within these sites may direct collagen catabolism (1). More recent work has evaluated the active sites and domains of MMPs to better understand the dynamic role that the enzyme plays in collagen hydrolysis (2–4).Collagenolytic members of the MMP family possess similar domain organizations, including propeptide, catalytic (CAT), linker, and hemopexin-like (HPX) domains (5). Several of these domains and/or regions within them have been implicated in collagenolysis. For example, MMP-1 residues 183–191, which are on the V-B loop between the fifth β-strand and the second α-helix in the CAT domain, as well as the active site cleft itself, have substantial roles in collagenolysis (6, 7). MMP-1 residue Gly-233 has been implicated as necessary for conformational flexibility of the active site (8). Within the MMP-1 linker domain, residues 262–276 were proposed to form a polyproline type II helix and interact with and destabilize the MMP cleavage site in collagen (9), whereas Gly-272 may allow bending of the linker domain to aid in interaction between the CAT and HPX domains (10).The HPX domain has a critical role in collagenolysis, as removal of the MMP-1, MMP-8, MMP-13, or MMP-14 (MT1-MMP) HPX domain results in a loss of collagenolytic activity (11–16). However, no information has been obtained as to the identity of specific residues within the HPX domain that participate in collagenolysis. Secondary binding sites (exosites) may promote interaction of proteases with large, macromolecular substrates, such as collagen. The identification of exosites involved in collagenolysis may aid in the design of selective MMP inhibitors (17–20). Ultimately, as exosites are identified, the manner in which the CAT, linker, and HPX domains work together to facilitate collagenolysis can be revealed.One approach for the rapid analysis of protein structure and identification of binding sites within proteins involves hydrogen/deuterium exchange (HDX) of protein backbone amide hydrogens with detection by mass spectrometry (MS) (21–23). A protein or protein/ligand pair is incubated for defined intervals in a deuterated environment. After rapid quenching of the HDX reaction, the partially deuterated protein is digested, and the resulting peptide fragments are analyzed by LC-MS. The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. HDX MS has been used previously to monitor the interaction between doxycycline and MMP-7 (24). The interaction sites identified were consistent with other biophysical studies mapping doxycycline binding outside of the catalytic Zn²⁺ (24). This present study has utilized HDX MS with a triple helical peptide (THP) substrate to identify nonactive site MMP-1 regions involved in collagenolysis. Subsequently, site-specific mutagenesis of MMP-1 in combination with THP inhibitors and substrates was utilized to identify, for the first time, specific HPX domain residues that participate in collagenolysis and to provide insight as to how these residues function mechanistically.     

Hydrolysis of triple-helical collagen peptide models by matrix metalloproteinases

The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric triple-helical peptide (THP) models of the collagenase cleavage sites in types I and II collagen. The THPs incorporate either the alpha1(I)772-786 or the alpha1(II)772-783 sequence. The alpha1(I)772-786 and alpha1(II)772-783 THPs were hydrolyzed by MMP-1 at the Gly-Ile and Gly-Leu bonds, respectively, analogous to the bonds cleaved in corresponding native collagens. Thus, the THPs contained all necessary information to direct MMP-1 binding and proteolysis. Subsequent investigations using the alpha1(I)772-786 THP showed hydrolysis by MMP-2, MMP-13, and a COOH-terminal domain-deleted MMP-1 (MMP-1(Delta(243-450))) but not by MMP-3 or a COOH-terminal domain-deleted MMP-3 (MMP-3(Delta(248-460))). Kinetic analyses showed a k(cat)/K(m) value of 1,808 s(-1) m(-1) for MMP-1 hydrolysis of alpha1(I)772-786 THP, approximately 10-fold lower than for type I collagen. The effect is caused primarily by relative K(m) values. MMP-2 and MMP-13 cleaved the THP more rapidly than MMP-1, but MMP-2 cleavage occurred at distinct multiple sites. Comparison of MMP-1 and MMP-1(Delta(243-450)) hydrolysis of alpha1(I)772-786 THP showed that both can cleave a triple-helical substrate with a slightly higher K(m) value for MMP-1(Delta(243-450)). We propose that the COOH-terminal domain of MMPs is necessary for orienting whole, native collagen molecules but may not be necessary for binding to and cleaving a THP. This proposal is consistent with the large distance between the MMP-1 catalytic and COOH-terminal domains observed by three-dimensional structural analysis and supports previous suggestions that the features of the catalytic domain contribute significantly toward enzyme specificity.     

Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of alpha-chain hydrolysis.     

Competitive interactions of collagen and a jararhagin-derived disintegrin peptide with the integrin alpha2-I domain

Integrin alpha2beta1 is a major receptor required for activation and adhesion of platelets, through the specific recognition of collagen by the alpha2-I domain (alpha2-I), which binds fibrillar collagen via Mg(2+)-bridged interactions. The crystal structure of a truncated form of the alpha2-I domain, bound to a triple helical collagen peptide, revealed conformational changes suggestive of a mechanism where the ligand-bound I domain can initiate and propagate conformational change to the full integrin complex. Collagen binding by alpha2-I and fibrinogen-dependent platelet activity can be inhibited by snake venom polypeptides. Here we describe the inhibitory effect of a short cyclic peptide derived from the snake toxin metalloprotease jararhagin, with specific amino acid sequence RKKH, on the ability of alpha2-I to bind triple helical collagen. Isothermal titration calorimetry measurements showed that the interactions of alpha2-I with collagen or RKKH peptide have similar affinities, and NMR chemical shift mapping experiments with (15)N-labeled alpha2-I, and unlabeled RKKH peptide, indicate that the peptide competes for the collagen-binding site of alpha2-I but does not induce a large scale conformational rearrangement of the I domain.     

Selective modulation of matrix metalloproteinase 9 (MMP-9) functions via exosite inhibition

Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly (downward arrow) Val bond selectively by MMP-2 and MMP-9, or the Gly (downward arrow) Leu cleavage site within the consensus interstitial collagen sequence alpha1(I-III)769-783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.     

Identification of the (183)RWTNNFREY(191) region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.     

Evidence of in situ stability of the type IV collagen triple helix in human inflammatory bowel disease using a denaturation specific epitope antibody.

A peptide specific antibody (AH1OW1) was raised against an epitope, AH10 (aa 449-463), of the alpha1(IV) chain adjacent to a cleavage site for matrix metalloproteinases (MMP)-2 and -9 within the triple helix of type IV collagen. The antibody only reacted with denatured and reduced preparations of type IV collagen, or with pepsin isolated type IV collagen digested with MMP-2 and MMP-9. The specificity of this antibody for the denatured triple helix was demonstrated by the lack of staining with pre-immune antibody and by pre-incubation of AH1OW1 antibody with excess AH10 peptide epitope. The AH1OWI antibody was used to detect whether proteolysis of type IV collagen occurs in ulcerative colitis, an inflammatory bowel condition often characterised by a large influx of granulocytes and macrophages and an associated tissue destruction. However, no evidence of in situ proteolysis of the basement membrane type IV collagen was observed. Only in the most actively inflamed mucosa was staining with AH1OW1 antibody observed in the mucosal connective tissue. Digestion of frozen sections of bowel with MMP-1, MMP-2, MMP-3 and MMP-9 did not result in the exposure of the AH10 epitope. These data demonstrate the stability of intact type IV collagen and indicate that susceptibility of alpha1(IV) chain to digestion with MMP-2 and MMP-9 may require other proteolytic/denaturing events in the molecule.     

Molecular dynamics simulation study on the interaction of collagen‐like peptides with gelatinase‐A (MMP‐2)

Although several models have been proposed for the interaction of collagen with gelatinase‐A (matrix metalloproteinases‐2 (MMP‐2)), the extensive role of each domain of gelatinase A in hydrolyzing the collagens with and without interruptions is still elusive. Molecular docking, molecular dynamics (MD) simulation, normal mode analysis (NMA) and framework rigidity optimized dynamics algorithm (FRODAN) based analysis were carried out to understand the function of various domains of MMP‐2 upon interaction with collagen like peptides. The results reveal that the collagen binding domain (CBD) binds to the C‐terminal of collagen like peptide with interruption. CBD helps in unwinding the loosely packed interrupted region of triple helical structure to a greater extent. It can be possible to speculate that the role of hemopexin (HPX) domain is to prevent further unwinding of collagen like peptide by binding to the other end of the collagen like peptide. The catalytic (CAT) domain then reorients itself to interact with the part of the unwound region of collagen like peptide for further hydrolysis. In conclusion the CBD of MMP‐2 recognizes the collagen and aids in unwinding the collagen like peptide with interruptions, and the HPX domain of MMP‐2 binds to the other end of the collagen allowing CAT domain to access the cleavage site. This study provides a comprehensive understanding of the structural basis of collagenolysis by MMP‐2. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 779–794, 2014.     

The roles of substrate thermal stability and P2 and P1' subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity

The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.     

Characterization of the mechanisms by which gelatinase A, neutrophil collagenase, and membrane-type metalloproteinase MMP-14 recognize collagen I and enzymatically process the two alpha-chains

The turnover of native collagen has been ascribed to different members of the matrix metalloproteinase (MMP) family. Here, the mechanisms by which neutrophil collagenase (MMP-8), gelatinase A (MMP-2), and the ectodomain of MT1-MMP (ectMMP-14) degrade fibrillar collagen were examined. In particular, the hydrolysis of type I collagen at 37 degrees C was investigated to identify functional differences in the processing of the two alpha-chain types of fibrillar collagen. Thermodynamic and kinetic parameters were used for a quantitative comparison of the binding, unwinding, and hydrolysis of triple helical collagen. We demonstrate that the MMP family has developed at least two distinct mechanisms for collagen unwinding and cleavage. MMP-8 and ectMMP-14 display a similar mechanism (although with different catalytic parameters), which is characterized by binding (likely through the hemopexin-like domain) and cleavage of alpha-1 and/or alpha-2 chains without distinguishing between them and keeping the gross conformation of the triple helix (at least during the first cleavage step). On the other hand, MMP-2 binds preferentially the alpha-1 chains (likely through the fibronectin-like domain, which is not present in MMP-8 and ectMMP-14), grossly altering the whole triple helical arrangement of the collagen molecule and cleaving preferentially the alpha-2 chain. These distinctive mechanisms underly a drastically different mode of interaction with triple helical fibrillar collagen I, according to which the MMP domain is involved in binding. These findings can be related to the different role exerted by these MMPs on collagen homeostasis in the extracellular matrix.     

Local Conformation and Dynamics of Isoleucine in the Collagenase Cleavage Site Provide a Recognition Signal for Matrix Metalloproteinases

The mechanism by which enzymes recognize the “uniform” collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Gly∼[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P₃ subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.     

Collagen binding properties of the membrane type-1 matrix metalloproteinase (MT1-MMP) hemopexin C domain. The ectodomain of the 44-kDa autocatalytic product of MT1-MMP inhibits cell invasion by disrupting native type I collagen cleavage

Up-regulation of the collagenolytic membrane type-1 matrix metalloproteinase (MT1-MMP) leads to increased MMP2 (gelatinase A) activation and MT1-MMP autolysis. The autocatalytic degradation product is a cell surface 44-kDa fragment of MT1-MMP (Gly(285)-Val(582)) in which the ectodomain consists of only the linker, hemopexin C domain and the stalk segment found before the transmembrane sequence. In the collagenases, hemopexin C domain exosites bind native collagen, which is required for triple helicase activity during collagen cleavage. Here we investigated the collagen binding properties and the role of the hemopexin C domain of MT1-MMP and of the 44-kDa MT1-MMP ectodomain in collagenolysis. Recombinant proteins, MT1-LCD (Gly(285)-Cys(508)), consisting of the linker and the hemopexin C domain, and MT1-CD (Gly(315)-Cys(508)), which consists of the hemopexin C domain only, were found to bind native type I collagen but not gelatin. Functionally, MT1-LCD inhibited collagen-induced MMP2 activation in fibroblasts, suggesting that interactions between collagen and endogenous MT1-MMP directly stimulate the cellular activation of pro-MMP2. MT1-LCD, but not MT1-CD, also blocked the cleavage of native type I collagen by MT1-MMP in vitro, indicating an important role for the MT1-MMP linker region in triple helicase activity. Similarly, soluble MT1-LCD, but not MT1-CD or peptide analogs of the MT1-MMP linker, reduced the invasion of type I collagen matrices by MDA-MB-231 cells as did the expression of recombinant 44-kDa MT1-MMP on the cell surface. Together, these studies demonstrate that generation of the 44-kDa MT1-MMP autolysis product regulates collagenolytic activity and subsequent invasive potential, suggesting a novel feedback mechanism for the control of pericellular proteolysis.     

Activity of matrix metalloproteinase-9 against native collagen types I and III

Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 degrees C and 25 degrees C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters/one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.     

Identification of collagen binding domain residues that govern catalytic activities of matrix metalloproteinase-2 (MMP-2)

An innovative approach to enhance the selectivity of matrix metalloproteinase (MMP) inhibitors comprises targeting these inhibitors to catalytically required substrate binding sites (exosites) that are located outside the catalytic cleft. In MMP-2, positioning of collagen substrate molecules occurs via a unique fibronectin-like domain (CBD) that contains three distinct modular collagen binding sites. To characterize the contributions of these exosites to gelatinolysis by MMP-2, seven MMP-2 variants were generated with single, or concurrent double and triple alanine substitutions in the three fibronectin type II modules of the CBD. Circular dichroism spectroscopy verified that recombinant MMP-2 wild-type (WT) and variants had the same fold. Moreover, the MMP-2 WT and variants had the same activity on a short FRET peptide substrate that is hydrolyzed independently of CBD binding. Among single-point variants, substitution in the module 3 binding site had greatest impact on the affinity of MMP-2 for gelatin. Simultaneous substitutions in two or three CBD modules further reduced gelatin binding. The rates of gelatinolysis of MMP-2 variants were reduced by 20–40% following single-point substitutions, by 60–75% after double-point modifications, and by > 90% for triple-point variants. Intriguingly, the three CBD modules contributed differentially to cleavage of dissociated α-1(I) and α-2(I) collagen chains. Importantly, kinetic analyses (k_cat/K_m) revealed that catalysis of a triple-helical FRET peptide substrate by MMP-2 relied primarily on the module 3 binding site. Thus, we have identified three collagen binding site residues that are essential for gelatinolysis and constitute promising targets for selective inhibition of MMP-2.     

Collagen XXIV,a vertebrate fibrillar collagen with structural features of invertebrate collagens: selective expression in developing cornea and bone

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.     

The Recognition of Collagen and Triple-helical Toolkit Peptides by MMP-13: SEQUENCE SPECIFICITY FOR BINDING AND CLEAVAGE*

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly⁷⁷⁵–Leu⁷⁷⁶ in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.     

Partial covalent structure of the human alpha 2 type V collagen chain

Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).