Identification of Structural Elements Important for Matrix Metalloproteinase Type V Collagenolytic Activity as Revealed by Chimeric Enzymes. Role of Fibronectin-like Domain and Active Site. ∗

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis as this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme three to four fold. This observation led to the identification of amino acid residues, Leu 397, Ala 406, Asp 410, and Pro 415, in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu 397 and Ala 406 are important for the general proteolytic activity of the enzyme, while Asp 410 and Pro 415 specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.


Introduction
The pET/FC+Fib+Gel.B/AS-COOH expression vector was designed to produce the FC+Fib+Gel.B/AS chimera without the carboxy-terminal domain of FC (Fig. 1). This construct was produced from the pET28/∆3´NG expression vector. pET28/∆3´NG contains the gelatinase B cDNA corresponding to amino acids 1-444 inserted into pET28C (Novagen). An engineered stop codon immediately following the codon for Gly 444 enables this vector to produce a carboxy-terminal truncated form of gelatinase B. Plasmids encoding the mutant enzymes were introduced into E. coli BL21(DE3) cells that were maintained in 50 µg/ml carbenicillin.
Protein Expression and PurificationTransformed cells were grown to mid-log phase and induced with the addition of 100-120 mg/L isopropyl-β-D-thiogalactopyranoside. Cells were then allowed to grow 4-19 h before being pelleted at 6,370 X g for 10 min. The supernatant was decanted and the pellets stored at -20 °C for further purification. Latent gelatinase B, point mutants, FC+Fib, FC+Fib+Gel.B/AS, and FC+Fib+Gel.B/AS-COOH were purified using a gelatin Agarose affinity column essentially as described previously (34).
Activated FC and FC+Gel.B/AS were purified by a zinc-chelating column. FC containing bacterial pellets were resuspended in buffer containing 50 mM Tris, pH 7.5, 5 mM CaCl 2 , 0.5 M NaCl, 0.05% (w/v) Brij-30, and 2 mM phenylmethylsulfonyl fluoride. The resulting suspension was then sonicated three times, 30 sec each time, at 30% duty cycle and centrifuged at > 20,000 X g for 30 min. To activate the enzymes, p-aminophenylmercuric acetate (APMA) was added to the supernatant at a final concentration of 1 mM and the resulting mixture was incubated 4-5 h at 37 °C. During the incubation period, precipitant formed that was removed by centrifugation at > 20,000 X g for 30 min. The supernatant was then dialyzed against buffer containing 25 mM Na 2 B 4 O 7 (Borax), pH 8.0, 5 mM CaCl 2 , 1 M NaCl, and 10 mM imidazole (dialysis buffer) and then loaded onto a 5 ml iminodiacetic acid Sepharose column (Pharmacia) that had been charged with 50 mM ZnCl 2 and equilibrated with dialysis buffer. The column was then washed with dialysis buffer until the A 280 of the effluent was approximately zero. The protein was then eluted with a 10-33 mM gradient of imidazole in dialysis buffer.
Selected fractions were combined and concentrated. FC+Gel.B/AS was purified in essentially the same manner except that the dialysis buffer contained 0.5 M NaCl and a 10-40 mM imidazole gradient was used to elute the enzyme. All of the purified enzymes were finally dialyzed against buffer containing 50 mM Tris, pH 7.5, 5 mM CaCl 2 , 150 mM NaCl, and 2 µM ZnCl 2 and stored at -80 °C for further use.
Activation of Enzymes-Gelatinase B and point mutants were activated either by 1 mM APMA for 12-15 h at 37 °C or 16 units/µl stromelysin-1 for 2 h at 37 °C. One unit of stromelysin-1 degrades 1 nM/s of (7-methoxycoumarin-4-yl) acetyl (Mca)-Pro-Leu-Gly-Leu-[3- Concentrations of active enzymes, [E] T , were found by active-site titration with the aminoterminal, 14-kDa, inhibitory domain of recombinant tissue inhibitor of metalloproteinase-2 (a kind gift from Dr. Harold Tschesche, Lerstuhl für Biochemie, Universitat Bielefeld, Bielefeld, Germany) as described previously (29). This method was used to determine the concentration of enzyme used in every assay in this report. Concentrations of active enzymes found by titration correlated with those determined by the Bradford dye-binding technique (standard Bio-Rad Laboratories assay) using bovine serum albumin (BSA) as a standard.
Analysis of Gelatinolytic Activity-The gelatinolytic activity of enzymes was determined using 14 C-labeled gelatin essentially as described previously (35). The specific activity was calculated by dividing the amount of gelatin degraded per hour (mg or µg) by the nmoles of enzyme used in the reaction. Each assay was conducted in duplicate and the results averaged.

Results
Type V Collagenolytic Activity of Chimeric Enzymes One of the most distinctive characteristics of the gelatinases is their ability to cleave type V collagen. Our previous experiments indicated that the type V collagenolytic activity of gelatinase B is determined, at least in part, by its fibronectin-like domain (29). which is consistent with the addition of the fibronectin-like domain to the FC molecule. This enzyme was processed by APMA to an active 62-kDa species (Fig. 2, lane 6). The activity of these enzymes toward type V collagen was then assessed using APMA-activated gelatinase B as a standard. As shown in Fig. 3, FC, at concentrations up to 5.4 µM (lanes 2 and 3), was not able to cleave type V collagen as previously observed (30). However, at the same   MMPs, there are several residues that distinguish the gelatinases from FC (Fig. 4). This suggests that these residues contribute to the efficiency of type V collagen degradation by the gelatinases.
It has been shown that the carboxy-terminal domain of FC is able to bind collagen (22).
Therefore, the possible contribution of this domain to the type V collagenolytic activity of these chimeras was examined by deleting it from FC+Fib+Gel.B/AS. The truncated chimera, FC+Fib+Gel.B/AS-COOH (Fig. 1), was expressed in E. coli and recovered as a 45-kDa latent species (Fig. 2, lane 7) that was processed to an active 39-kDa species (Fig. 2, lane 8) (38). To exclude the possibility that the differences in type V collagenolytic activity of the chimeras were due to the generation of different aminotermini upon activation, the amino-termini of the APMA-activated enzymes were determined.
Amino-terminal sequence analyses of the active species of FC and FC+Gel.B/AS, enzymes lacking type V collagenolytic activity, yielded a mixture of V 101 LTEG and L 102 TEGN sequences, identical to that reported for native FC (38). This indicated that the substitution of the Gel.B/AS for that of FC had no effect on autocatalytic cleavage within the pro-domain. All the aminoterminal sequences of the active species of the chimeras containing the fibronectin-like domain were L 84 KVMK, which were not processed further by longer activation periods (data not shown).
These data indicated that the differences in specific activity of the chimeras containing the fibronectin-like domain were not due to the generation of different amino-termini upon activation. Interestingly, in contrast to APMA-activated FC and FC+Gel.B/AS but similar to plasmin-activated FC and APMA-activated gelatinase B, these chimeras retained the PRCGVPD sequence in the pro-domain containing the conserved cysteine residue that is involved in the "cysteine switch" mechanism of activation (40) (41). These results suggest that in addition to providing the enzyme with type V collagenolytic activity, the inclusion of the fibronectin-like domain in FC has influenced the autocatalytic processing of the pro-domain.   (Table   I). These data suggest that amino acid residues in the Gel.B/AS are responsible for the observed increases in catalytic efficiency of the enzymes for this substrate.

Activity of Chimeric Enzymes Towards Gelatin and a Peptide
The addition of the fibronectin-like domain to FC, FC+Fib, increased the specific activity of the enzyme towards gelatin by more than four times (Table I) The gelatinase B mutants were expressed in E. coli and purified as latent enzymes as described previously (34). The wild type and the mutant enzymes were activated and processed by stromelysin-1 to 42-kDa species within 2 h (data not shown, (43)). This indicated that all of the mutant enzymes were properly folded and catalytically competent. As seen in Table II

Discussion
We have recently shown that the fibronectin-like domain of gelatinase B is required for its ability to bind and degrade type V collagen (29).  (Fig. 4). In addition, several of these residues are also conserved in NC and collagenase 3 that also have significantly greater activities towards the peptide substrate and gelatin compared to FC (50) (Fig. 4). Therefore, it is likely that these conserved amino acids function not only to increase the proteolytic activity of the gelatinases but of other MMPs as well.
Although the X-ray structure of gelatinase B is not available, we were able to gain further insight into the roles of these particular amino acids in the proteolytic activity of the enzyme by using molecular modeling of the crystal structure of NC (36) (Fig. 5). NC shares relatively high amino acid sequence identity with gelatinase B in the active site region and also contains Leu and Pro residues at positions 213 and 231 that correspond to Leu 397 and Pro 415 in gelatinase B. In order to make a model of the AS of gelatinase B that contained all four of the amino acids that were found to be important for the activity of the enzyme, a molecular modeling program was used to substitute Ser 222 and Ala 226 in the structure of NC for Ala and Asp residues, respectively (Fig. 5). Leu 397 and Ala 406 were found to be important for the general catalytic activity of gelatinase B. This is based on the observation that replacement of Leu 397 and Ala 406 with Arg and Ser residues, respectively, significantly reduced the ability of the enzyme to cleave all three substrates tested here. Leu 397 is situated at the beginning of the first alpha helix in the AS (Fig. 5). It is generally accepted that the amino acid in this position defines the depth and character of the S 1´ pocket of MMPs. It has been noted that MMPs, such as NC (36,51) and stromelysin-1 (52), that contain a Leu at this position have a deep and hydrophobic S 1´ pocket. However, FC contains a longer Arg in this position that actually points towards the substrate to provide a shallower and less hydrophobic pocket (53). On the basis of these observations, it would be logical to surmise that MMPs with a deeper S 1´ pocket would be able to substrates (54,55) have shown that enzymes (gelatinases and NC) with a Leu in the S 1´ pocket tend to accommodate more bulky hydrophobic amino acids at the P 1´ position than FC.
Consistent with this observation is our finding that the type V collagenolytic activity of the L397R mutant was significantly lower than that of the wild type enzyme. Niyibizi et al. (56) have shown that the cleavage of type V collagen by gelatinase B generates peptide fragments having large hydrophobic amino acids, Val and Leu, at the P 1´ positions. Therefore, it is reasonable to assume that the replacement of Leu 397 with an Arg reduces the ability of the enzyme to accept these amino acids in the S 1´ pocket. Being able to accommodate a wider variety of amino acids at the P 1´ position would also facilitate the degradation of a substrate like gelatin in which all the peptide bonds are accessible to the enzyme and numerous sites are cleaved. This is supported by the observation that the substitution of Leu 397 with an Arg also resulted in lower gelatinolytic activity. Ala 406 is located near the end of the first alpha helix in the AS (Fig. 5). The decrease in general catalytic activity caused by the substitution of this residue for a slightly longer Ser suggests that an Ala in this position enhances proteolysis because of its small size.
Site-directed mutagenesis revealed that Asp 410 is specifically important for the type V collagenolytic activity of gelatinase B. Asp 410 is located very close to the catalytic zinc ion (Fig. 5) and may come in close contact with P 2 and P 3 residues of substrate. It is possible that Asp 410 interacts with positively charged residues in type V collagen to provide better contact between enzyme and substrate. It was also noted that the D410S mutant had an approximately two fold higher catalytic efficiency towards the peptide substrate than the wild type enzyme.
Having an uncharged Ser at this position apparently provides for a better interaction with the Leu at the P 2 position in the peptide than the negatively charged Asp residue.
The substitution of Pro 415 for an Ile exclusively reduced the ability of gelatinase B to cleave gelatin. Pro 415 (Fig. 5) is located in the middle of a tight turn at the bottom of the AS.
Proline residues are often found in flexible regions of proteins, suggesting that Pro 415 may play a role in movement within the AS during the binding and hydrolysis of gelatin that is beneficial to catalysis.
In conclusion, our data has provided a more comprehensive understanding of the structural elements that contribute to the substrate specificity and catalytic efficiency of MMPs.
We have shown that the addition of the fibronectin-like domain to an MMP is sufficient to confer type V collagenolytic activity. In addition, our mutational analysis has given insight on the roles of conserved amino acids in the AS of the gelatinases for their important gelatinolytic and type V collagenolytic activities. modeled after the X-ray structure of NC (36). To make a model that highlighted all four of the amino acids that were found to be important for the activity of gelatinase B, a molecular modeling program (see "Experimental Procedures") was used to substitute Ser 222 and Ala 226 in the structure of NC for Ala and Asp residues, respectively. Leu 397, green; Ala 406, white;