Identification of the 183RWTNNFREY191Region 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 ¾ and ¼ 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 °C. The results indicate that the nine residues 183RWTNNFREY191 located between the fifth β-strand and the second α-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr191 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.

RWTNNFREY 191 located between the fifth ␤-strand and the second ␣-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.
Interstitial collagen types I, II, and III are the major structural proteins in connective tissues such as tendon, skin, bone, cartilage, and blood vessels. They consist of three ␣ chains with repeating Gly-X-Y triplets where X and Y are frequently Pro and Hyp, respectively. Each chain of the repeating tripeptide adopts a left-handed poly-Pro II helix conformation, and three left-handed chains then intertwine to form a right-handed superhelix (1)(2)(3). This triple helical conformation makes interstitial collagens resistant to most proteinases in vertebrates except for collagenases, cathepsin K (4), and neutrophil elastase (5). The action of cathepsin K is probably important in collagen breakdown in specialized environments such as during bone resorption in an acidic pH environment. Neutrophil elastase may degrade telopeptides of interstitial collagen (6) and the triple-helical region of type I collagen under inflammatory conditions, but the latter activity is much weaker than that of collagenase (5). Vertebrate collagenases, on the other hand, are synthesized by many cell types such as stromal fibroblasts, chondrocytes, keratinocytes, osteoblasts, endothelial cells, and macrophages in response to inflammatory cytokines, growth factors, cellular transformation, and other chemical and physical stimuli (7). These collagenases are members of the matrix metalloproteinase (MMP) 1 family; they include the collagenases (MMP-1, MMP-8, MMP-13, MMP-18) (8), gelatinase A (MMP-2) (9), and MT1-MMP (MMP-14) (10). These enzymes cleave interstitial collagens at a specific site approximately three-fourths of the way from the N terminus of the collagen molecule at neutral pH. Once collagen is cleaved, the resulting triple helical fragments denature at body temperature and become susceptible to nonspecific proteolysis. Thus, the cleavage of interstitial collagens by metallocollagenases is critical for the initiation of collagenolysis under both physiological and pathological conditions. Typical collagenases are secreted from the cell as proenzymes of 55-60 kDa. They consist of an N-terminal propeptide domain of about 80 amino acids, a catalytic domain of about 165 amino acids, and a C-terminal hemopexin-like domain of about 190 amino acids. The catalytic domain and the hemopexin-like domain are linked by an exposed proline-rich linker peptide of 16 amino acids (11), although the linker in MMP-18 (collagenase 4) has 32 amino acids (12). The catalytic domain alone fails to cleave collagen, although it retains activity against other substrates (13)(14)(15). Thus, the expression of collagenolytic activity requires the C-terminal hemopexin domain.
The vertebrate MMPs that have collagenolytic activity ex-hibit overall identities of 44 -55%, exclusive of the fibronectin type II domain in MMP-2. The hemopexin domains of MMP-1 (11), MMP-2 (16 -17), and MMP-13 (18) are similar in threedimensional structure, forming a four-bladed ␤-propeller structure. MMP-3 (stromelysin 1) shares 54% overall identity in amino acid sequence with MMP-1 (58% within the catalytic domain), but it cleaves neither type I nor type II collagen. MMPs have homologous, superimposable substrate binding grooves in the catalytic domains (19). Investigation of the substrate specificity using synthetic substrates shows that all MMPs prefer bulky hydrophobic residues at the P 1 Ј position (20). Both MMP-1 and MMP-3 can cleave all synthetic peptides based on the collagenase cleavage sites of types I, II, and III collagen at the same site, although their rates are different. It was therefore postulated that the C-terminal hemopexin domain plays an important role in the expression of collagenolytic activity. However, when the catalytic domain of MMP-3 was linked with the hemopexin domain of MMP-1, this chimera failed to express collagenolytic activity (21), indicating that additional elements in MMP-1 are required to cleave triple helical collagens. Hirose et al. (22) suggested that the length of the linker region is critical for collagenolysis by MMP-8, but this may not be the only reason because the MMP-3-MMP-1 chimera reported by Murphy et al. (21) had the correct linker peptide of MMP-1. Several other chimeras of collagenases and stromelysins have been constructed and studied (21)(22)(23), but these studies provide only limited information in terms of our understanding of the structural elements essential for collagenolytic activity because these chimeras show little or no collagenolytic activity. We have approached this problem by attempting to transform non-collagenolytic MMP-3 to a collagenolytic enzyme by introducing increasing lengths of MMP-1 into it, starting from the C-terminal end. The present study has determined that the sequence 183 RWTNNFREY 191 located between the fifth ␤-strand and the second ␣-helix in the catalytic domain is one of the key elements in MMP-1 for the expression of triple helicase activity.
Construction of MMP Chimeras-The MMP chimeras shown in Fig.  1 were constructed using wild-type MMP-1 and wild-type MMP-3 cDNAs in the pET3a vector as templates for overlapping polymerase chain reaction. The primers used are listed in Table I. Using the sense T7 promoter primer and an antisense chimeric junction primer containing the MMP-3 sequence at the 5Ј-end and the MMP-1 sequence at the 3Ј-end (see Table I), the N-terminal part of the chimera containing primarily the MMP-3 sequence was made. Using an antisense primer annealing to the vector (pET3a primer) and a sense chimeric junction primer containing the MMP-1 sequence at the 5Ј-end and the MMP-3 sequence at the 3Ј-end (overlapping with the previous primer), the C-terminal part of the chimera containing primarily the MMP-1 sequence was made. A final polymerase chain reaction was performed using these two first round products as the template and the two flanking primers (T7 promoter and pET3a primers). The final reaction resulted in a full-length chimera of MMP-3 and MMP-1 (see Fig. 1). The full-length product was then cut with NdeI and BamHI and ligated into the pET3a vector. The sequence of the final polymerase chain reaction products was confirmed by automated DNA sequencing at the Biotech Facility at the University of Kansas Medical Center.
Amino Acid or Sequence Replacement-Polymerase chain reaction was used to replace a 9-amino acid sequence or a single amino acid of either MMP-1 or MMP-3, using overlapping primers. A sense primer and an antisense primer were constructed in which the 5Ј-ends of the primers contained overlapping portions of the sequence to be inserted and the 3Ј-ends of the primers contained the sequence of the wild-type cDNA (Table I). Using the T7 promoter primer and the antisense mutation primer, the N-terminal portion of the construct was made. Using the sense mutation primer and the pET3a reverse primer, the C-terminal portion of the construct was made. A second reaction was performed using the first round products as a template and the two flanking primers (T7 promoter and pET3a primers) to make the final full-length product. MMP-1(Gln 185 -Thr 193 ) mmp-3 is MMP-1 replaced with 185 QWTKDTTGT 193 of MMP-3, and MMP-3(Arg 183 -Tyr 191 ) mmp-1 is MMP-3 replaced with 183 RWTNNFREY 191 of MMP-1 in their corresponding sequences ( Table I).
Collagenase Assays-All chimeras were tested for their ability to digest pepsin-treated type I collagen at 25°C, and products were analyzed on SDS-PAGE stained with Coomassie Brilliant Blue R250. Various concentrations of activated enzymes were incubated with 30 g of type I collagen for up to 8 h. Reactions were stopped by adding SDS-PAGE loading buffer with 20 mM EDTA, pH 8.0, and the products were subjected to 7.5% SDS-PAGE analysis under reducing conditions. Collagen was then examined for digestion into TC A ( 3 ⁄4) and TC B ( 1 ⁄4) fragments. Activity of each chimera and mutant at 25°C was determined by scanning TC A and TC B fragments on the gel, taking at least 4 earlier time points (less than 10% collagen digestion), and calculating the rate of hydrolysis. Specific activity against a 14 C-labeled type I collagen at 37°C was determined using a diffuse fibril assay according to Cawston and Barrett (29).
Enzyme Activity Assays Using Quenched Fluorescent Substrates-Enzyme assays were performed in TNC buffer containing 0.05% Brij 35 as described previously (24). All enzymes were tested for their ability to digest synthetic fluorogenic substrates: Mca-Pro-Leu-GlyϳLeu-Dpa-Ala-Arg-NH 2 (a general MMP substrate) and Mca-Arg-Pro-Lys-Pro-Val-GluϳNva-Trp-Arg-Lys(Dnp)-NH 2 (specific for MMP-3). Each enzyme was incubated with 1 M substrate at 37°C for 10 -40 min. The reaction was stopped by the addition of 50 M EDTA, pH 8.0. Fluorescence was measured using wavelengths of 325 (excitation) and 393 nm (emission) with a microtiter plate fluorescence reader, Biotek FL600 (Biotek Instruments). The k cat /K m was calculated for each peptide substrate using Equation 1 to measure the initial velocity (v i ) values under first order conditions ( where [S 0 ] and [E 0 ] are the initial concentrations of substrate and enzyme, respectively.

RESULTS
Expression, Folding, and Purification of MMP Chimeras-MMP chimeras designed according to Fig. 1 were expressed in E. coli BL21(DE3) cells as proenzymes. The proteins were extracted from inclusion bodies with 8 M urea and partially purified under denaturing conditions by ion exchange chromatography. Partially purified proteins were folded to the native state as described under "Experimental Procedures." Folded proteins (ϳ85% pure on SDS-PAGE) were further purified by Green A Dyematrex gel chromatography. All chimeras, except LC2, were isolated as the precursor form, and they were activated to the 42-kDa form upon treatment with APMA or chymotrypsin (Fig. 2). The treatment of the activated enzymes with trypsin (10 g/ml) did not degrade these proteins, but they were digested into smaller fragments in the presence of 10 mM EDTA. These results, together with the enzymatic activity detected with the chimeric enzymes (see Table II), support the notion that these proteins were correctly folded in native structure. LC2 was spontaneously activated during purification, and treatment of this species with APMA did not alter the molec-ular mass, indicating that it was a stable, active enzyme.
Digestion of Type I Collagen by MMP-3/MMP-1 Chimeras-To examine collagenolytic activity, wild-type MMP-1 and MMP-3 and their chimeras were activated as described under "Experimental Procedures." Upon activation, some of the 42-45 kDa forms converted to lower molecular mass (22-28 kDa) species. Because only the full-length MMP-1 expresses collagenolytic activity, the amount of each chimeric enzyme corresponding to the 42-45-kDa form generated upon activation was quantified by densitometric analysis after SDS-PAGE and staining of proteins with Coomassie Brilliant Blue R250. As reported previously for natural MMP-3 (30), no collagenolytic activity was detected with recombinant MMP-3 (data not shown). Replacement of the C-terminal segment of MMP-3 with the corresponding sequence of MMP-1 up to Thr 222 (LC1 chimera) (see Fig. 1) did not result in any collagenolytic activity (Fig. 3). The LC2 chimera consisting of MMP-3(Phe 83 -Thr 193 ) Purified zymogens of MMP chimeras were activated as described under "Experimental Procedures." The products were run on a 7.5% SDS-PAGE to check for conversion to a stable full-length product. LC2 was purified as the active form, but it was still activated with 1 mM APMA for 2 h at 37°C before use, as was MMP-1, LC3, and LC4. MMP-3, MMP-3-1-1, and LC1 were activated using 10 g/ml chymotrypsin for 2 h at 37°C. and MMP-1(Asn 192 -Asn 450 ) showed a very low collagenolytic activity (Ͻ 3% of the wild-type MMP-1). A prominent increase of activity was observed with LC3, which contains an additional 9 amino acids of MMP-1 extended toward the N-terminal side (Fig. 3). A further extension of 9 amino acids of the MMP-1 sequence as in LC4 increased the activity only 1.8-fold. A similar pattern of increase in collagenolytic activity with these chimeras was observed at 37°C using pepsin-untreated 14 Clabeled type I collagen (data not shown). The difference between LC2 and LC3 is only a sequence of nine amino acids, 183 RWTNNFREY 191 , indicating that this stretch of residues in MMP-1 is one of the key elements for the expression of collagenolytic activity (Fig. 4).
Replacement of 183 RWTNNFREY 191 -The alignment of the 183 RWTNNFREY 191 region with other collagenolytic MMPs (e.g. MMP-1, MMP-2, MMP-8, MMP-13, and MMP-18) indicates that Tyr 191 is conserved among them, but in MMP-3 the corresponding residue is Thr 193 . We therefore substituted Thr for Tyr 191 in MMP-1, and the proMMP-1(Y191T) mutant was expressed in E. coli, folded from inclusion bodies, and purified. After activation with APMA, collagenolytic activity was measured. This mutation reduced the collagenolytic activity by 4 -5fold. Replacement of the 9 amino acids of MMP-1 with the corresponding sequence QWTKDTTGT of MMP-3 further reduced the collagenolytic activity by 2-fold (Fig. 5). This suggests that the Tyr 191 in this sequence is particularly important in maintaining full collagenolytic activity.
To examine whether the 183 RWTNNFREY 191 sequence is the only part of the catalytic domain that is critical for the unique expression of collagenolytic activity, this sequence was introduced into MMP-3 and MMP-3-1-1 by replacing the 185 QWT-KDTTGT 193 section of MMP-3. Collagen assays were carried out at 25°C, but neither of the variants, MMP-3(Arg 183 -Tyr 191 ) mmp-1 and MMP-3-1-1(Arg 183 -Tyr 191 ) mmp-1 , expressed triple helicase activity (Fig. 5). These results suggest that whereas the RWTNNFREY stretch plays an important role in cleaving triple helices, this segment alone, or even with the linker and the hemopexin domain of MMP-1, is not sufficient to express collagenolytic activity. There must be other additional elements within the catalytic domain that contribute to the collagenolytic activity of MMP-1.
Effects of the RWTNNFREY Region on Substrate Specificity-We used type I gelatin and two synthetic substrates to investigate the substrate specificity of the above chimeras and mutants. In these experiments the active site of each enzyme was titrated with TIMP-1, and the same amount of active enzyme (mixture of 42-kDa and 22-25-kDa forms) was assayed. Using a mixture of catalytic domain and full-length forms is valid, because the enzyme activity on these substrates is not altered by the deletion of the hemopexin domain (data not shown).
Digestion of gelatin by MMP-1 and MMP-3 and the subsequent analyses of the products by SDS-PAGE indicated that MMP-1 and MMP-3 have distinct substrate specificities (Fig.  6A). The chimera LC1 showed substrate specificity similar to the wild-type MMP-3, but it had about 5-fold higher activity on gelatin than did MMP-3. LC2, whose S 1 Ј pocket includes MMP-1-specific Arg 195 , digested gelatin in a pattern similar to that of MMP-1. The activities of LC2 and MMP-1 on gelatin were comparable. LC3 and LC4 also had a similar digestion pattern as LC2, but they were about 5-fold more active on gelatin than was LC2. These studies have demonstrated that the RWTNN-FREY sequence greatly influences the specific activity of MMP-1 on gelatin. When this sequence was introduced into MMP-3 and MMP-3-1-1 by replacement, the chimeric constructs MMP-3(Arg 183 -Tyr 191 ) mmp-1 and MMP-3-1-1(Arg 183 -Tyr 191 ) mmp-1 showed about a 15-fold increase in gelatinolytic activity without significant changes in the cleavage pattern (Fig. 6B). On the other hand, a single mutation of Tyr 191 of MMP-1 to Thr reduced not only collagenolytic activity but also gelatinolytic activity 4 -5-fold (Fig. 6C). Replacement of the QWTKDTTGT sequence with the corresponding sequence of MMP-1 reduced gelatinolytic activity to a level similar to that of MMP-1(Y191T). Gelatin digestion patterns indicate that substrate specificities of these mutants are significantly different from that of the wild-type MMP-1 (Fig. 6C).
Activities of MMP variants were also examined using a general synthetic substrate, Mca-Pro-Leu-GlyϳLeu-Dpa-Ala-Arg-NH 2 (peptide 1), and a substrate specific to stromelysins 1 and 2 (MMP-3 and MMP-10), Mca-Arg-Pro-Lys-Pro-Val-GluϳNva-Trp-Arg-Lys(Dnp)-NH 2 (peptide 2). Table II summarizes the results. MMP-3 cleaves both peptide 1 and peptide 2. When MMP-1 moieties are increased in MMP-3, the k cat /K m values increased against peptide 1 but decreased against peptide 2. About a 10-fold increase of activity against peptide 1 was observed with LC1 compared with the wild-type MMP-3. A second conspicuous increase was seen with LC3 and LC4. Their activities were 55-and 67-fold higher, respectively, than that of MMP-3 and 9-and 12-fold higher, respectively, than that of MMP-1. These results agree with an increase in gelatinolytic activity of these variants. Nonetheless, the increase in these activities did not correlate with an increase in collagenolytic activity.
On the other hand, mutation of Tyr 191 and the replacement of RWTNNFREY in MMP-1 drastically decreased the activity against peptide 1; the k cat /K m values of MMP-1(Y191T) and MMP-1(Gln 185 -Thr 193 ) mmp-3 reduced 33-and 48-fold, respectively, compared with that of MMP-1. In contrast, the MMP-3(T193Y) mutant increased the activity for peptide 1 about 5-fold but decreased the activity for peptide 2 by 26-fold. MMP-3 variants with the RWTNNFREY sequence increased the activity against peptide 1 by 12-fold and reduced it for  (22) also indicated that the correct proline-rich linker is another important region for collagenolysis, because collagenolytic activity was not detected when the linker region of MMP-8 was replaced with that of MMP-3. Those assignments were made based on either reduced or null collagenolytic activity of chimeric or mutated collagenase variants. Replacement of three prolines of the MMP-8 linker region with alanines reduced the collagenase activity to 1.5% (31). Because the chimera consisting of the catalytic domain of MMP-3 and the linker and hemopexin domain of MMP-1 failed to cleave collagen (21), and also because the MMP-3 catalytic domain cleaves the collagen sequence-based synthetic substrates at the same sites as MMP-1 (20), we postulated that there must be additional structural elements in MMP-1 that play an important role in cleaving triple helical collagen. A systematic replacement of the Cterminal portion of non-collagenolytic MMP-3 with the corresponding sequence of MMP-1 allowed us to map the 183 RWT-NNFREY 191 sequence in MMP-1 as an additional region critical for the expression of the collagenolytic activity of this enzyme. Chimera LC2 has Arg 195 of MMP-1, a key constituent of the S 1 Ј pocket of the enzyme. LC2 expressed collagenolytic activity, but it was very low. This chimera is essentially the same as chimera SM:CL4:5 constructed by Sanchez-Lopez et al. (23) using MMP-10 (stromelysin 2) instead of MMP-3. Their studies showed that SM:CL4:5 exhibited MMP-1-like specificity judged by the gelatin cleavage pattern, but it did not have collagenolytic activity. This slight discrepancy may be because of the sensitivity of the assay; in our study very large amounts of chimeras were available for the collagenase assay. However, a striking gain of activity was observed with LC3, which has an additional 9 amino acids toward the N terminus of MMP-1. This 9-amino acid stretch is located in the loop between the fifth ␤-strand (strand V) and the second ␣-helix (helix B) of the catalytic domain of MMP-1 and is exposed to the solvent (Fig.  7A).
The polypeptide folds of the catalytic domains of MMPs are essentially identical (32,33). However, the region corresponding to 183 RWTNNFREY 191 is the most variable in sequence among MMPs, and the molecular surface of MMP-1 and MMP-3 in this region shows significant differences (Fig. 7B), in particular in the region around FREY (188 -191) (Fig. 7C). It is notable that Tyr 191 is conserved among all collagenolytic MMPs except MMP-14, which possesses Leu at this position (34). Thr is found in MMP-3 at this position. Substitution of Thr for Tyr 191 in MMP-1 reduced the activity for collagen and gelatin about 5-fold, and the substitution of 185 QWTKDT-TGT 193 of MMP-3 further decreased the collagenolytic activity 2-fold. Mapping of substrate specificity by gelatin digestion  patterns indicates that alterations in this region significantly influenced the specificity of MMP-1 (see Fig. 6C). MMP-2 and MMP-9 possess three repeats of fibronectin type II domains attached to this loop (35). Without these domains, their gelatinolytic, type IV collagenolytic, and elastinolytic activities are greatly reduced, and their substrate specificities are altered (36 -38). Because the catalytic domain of MMP-1 alone cannot cleave collagen, we propose that this loop of MMP-1 is a part of the components that form a large cleft that accommodates the triple helical collagen molecule. Nonetheless, a surprising result was observed with MMP-3-1-1(Arg 183 -Tyr 191 ) mmp-1 together with the MMP-1 hemopexin domain. This variant has the linker region and hemopexin domain of MMP-1 and the RWTNNFREY element, and it hydrolyzed peptides 1 and 2 well. However, it failed to cleave triple helical collagen. This indicates that additional elements in the catalytic domain are required for the expression of collagenolytic activity.
Studies of MMP variants with synthetic substrates have also emphasized that the loop between strand V and helix B plays a significant role in the substrate specificity of MMPs. Studies with a series of oligopeptide substrates modeled after collagenase cleavage sites of interstitial collagens indicate that MMP-1 and MMP-3 do exhibit some differences in subsite requirements (20). This is partially due to the different geometry of the S 1 Ј pocket between MMP-1 and MMP-3. MMP-3 has a larger S 1 Ј pocket than does MMP-1. The main difference is due to the side chain of Arg 195 in MMP-1 that delimits the S 1 Ј pocket by projecting out toward the catalytic zinc atom (39). Leu 197 is found in the corresponding site of MMP-3, and it is oriented away from the catalytic zinc (19,40). This allows MMP-3 to accommodate a bulky aromatic side chain in the S 1 Ј pocket. Both MMPs, however, will tolerate long aliphatic side chains, and collagen sequence-based peptides are cleaved by them at identical sites, although their rates are different. The crystal structure of MMP-1 shows that Tyr 191 is located close to the S 3 Ј subsite of the substrate binding groove (39). Indeed, MMP-1(Y191T), MMP-1(Gln 185 -Thr 193 ) mmp-3 , and the wild-type MMP-1 all exhibited a different cleavage pattern and efficiency on gelatin (Fig. 6). In the case of MMP-3, mutation of this region influences the catalytic efficiency of gelatin and synthetic substrates (see Table II and Fig. 6B), but there is no significant difference in the digestion patterns of gelatin. Mutation of Thr 193 in MMP-3 to Tyr decreased MMP-3 activity against peptide 2 about 26-fold but increased the activity against a more general MMP substrate, peptide 1, about 10fold. An increase of the catalytic activity toward peptide 1 is even more prominent with MMP-3(Arg 183 -Tyr 191 ) mmp-1 . The single mutation of Tyr 191 of MMP-1 to Thr drastically decreased the k cat /K m value against peptide 1, but it reduced the collagenolytic activity only about 4 -5-fold. This is probably because of a difference in the nature of the synthetic substrate and the native macromolecular substrate. Taken together, these results emphasize that MMPs do have an extended substrate-binding site and that the loop between strand V and helix B is an important element for substrate specificity in MMPs.
Most MMPs digest large extended macromolecular substrates of extracellular matrices. Therefore, an extended substrate recognition site might have developed to selectively cleave a particular substrate, such as collagen. Biological activities of triple helical collagens are often dependent on their conformation (41). Recent studies suggest that cleavage of type I collagen by collagenases is critical for the migration of keratinocytes after wounding (42) and for the parathryroid hormone-induced migration of osteoclasts (43), suggesting that specific cleavage of collagen may express cryptic biological functions of extracellular components.
The three-dimensional structure of collagenases indicates that the cleft in the catalytic domain cannot accommodate the triple helical structure of collagen because it is only about 5 Å wide, whereas the diameter of collagen is about 15 Å. Our molecular docking experiments have shown that the closest peptide bond of collagen is about 7 Å away from the catalytic zinc atom. 2 This suggests that either collagenase itself or the triple helical collagen must undergo considerable changes in conformation to initiate the cleavage of the Gly-Ile bond of the ␣ 1 chain and the Gly-Leu bond of the ␣ 2 chain of type I collagen. Our recent studies 3 suggest that the binding of MMP-1 induces local unwinding of triple helical collagen prior to the cleaving of the peptide bonds. It is, however, not known in which step the RWTNNFREY is involved during collagenolysis: interaction with collagen, reaction of collagen unwinding, or interaction with unwound polypeptide. We are currently investigating the precise role of the residues in this segment.