Peptide Ligands for the Fibronectin Type II Modules of Matrix Metalloproteinase 2 (MMP-2)

Abstract

The interaction of matrix metalloproteinase 2 (MMP-2) with gelatin is mediated by three repeats homologous to fibronectin type II (FN2) modules, which are inserted in the catalytic domain in proximity of the active site. We screened a random 15-mer phage display library to identify peptides that interact with the FN2 modules of MMP-2. Interestingly, the selected peptides are not gelatin-like and do not share a common, obvious sequence motif. However, they contain a high proportion of aromatic residues. The interactions of two peptides, WHWRH0RIPLQLAAGR and THSHQWRHHQFPAPT, with constructs comprising the in-tandem first and second and second and third FN2 modules of MMP-2 (Col-12 and Col-23, respectively) were characterized by NMR. Both peptides interact with Col-12 and Col-23 with apparent association constants in the mm−1 range. Peptide binding results in perturbation of signals from residues located in the gelatin-binding pocket and flexible parts of the molecule. Although the former finding suggests that the gelatin-binding site is involved in the contact, the interpretation of the latter is less straightforward and may well reflect both the direct and indirect effects of the interaction. The interaction of matrix metalloproteinase 2 (MMP-2) with gelatin is mediated by three repeats homologous to fibronectin type II (FN2) modules, which are inserted in the catalytic domain in proximity of the active site. We screened a random 15-mer phage display library to identify peptides that interact with the FN2 modules of MMP-2. Interestingly, the selected peptides are not gelatin-like and do not share a common, obvious sequence motif. However, they contain a high proportion of aromatic residues. The interactions of two peptides, WHWRH0RIPLQLAAGR and THSHQWRHHQFPAPT, with constructs comprising the in-tandem first and second and second and third FN2 modules of MMP-2 (Col-12 and Col-23, respectively) were characterized by NMR. Both peptides interact with Col-12 and Col-23 with apparent association constants in the mm−1 range. Peptide binding results in perturbation of signals from residues located in the gelatin-binding pocket and flexible parts of the molecule. Although the former finding suggests that the gelatin-binding site is involved in the contact, the interpretation of the latter is less straightforward and may well reflect both the direct and indirect effects of the interaction. matrix metalloproteinase fibronectin type II peptide ACGYTYHPPCARLTV -2, and -3, first, second, and third FN2 modules, respectively, from human MMP-2 with amino-terminal peptide derived from the ॆ-galactosidase moiety of the expression vector the three in-tandem FN2 modules from human MMP-2 with amino-terminal peptide derived from the ॆ-galactosidase moiety of the expression vector collagen-binding FN2 repeat the first FN2 domain from human MMP-2 the first and second FN2 domains from human MMP-2 the Col-1 repeat in Col-12 the Col-2 repeat in Col-12 the second FN2 domain from human MMP-2 the second and third FN2 domains from human MMP-2 the Col-2 repeat in Col-23 the Col-3 repeat in Col-23 the third FN2 domain from human MMP-2 peptide HASHFRFRHSHVYGV the proenzyme form of MMP-2 peptide THSHQWRHHQFPAPT peptide WFPGPITFIPRPWSS peptide WHVSPRHQRLFHGLF peptide WHWRHRIPLQLAAGR Matrix metalloproteinase 2 (MMP-2,1 gelatinase A), and the closely related MMP-9 (gelatinase B) are unique among the metalloproteinases in that three gelatin-binding fibronectin type II (FN2) modules (Col-1, Col-2, and Col-3) are inserted in their catalytic domain in the vicinity of the active site (1Collier I.E. Wilhelm S.M. Eisen A.Z. Marmer B.L. Grant G.A. Seltzer J.L. Kronberger A. He C.S. Bauer E.A. Goldberg G.I. J. Biol. Chem. 1988; 263: 6579-6587Google Scholar). The solution conformation of each FN2 repeat from human MMP-2 has been characterized via NMR spectroscopy (2Briknarová K. Grishaev A. Bányai L. Tordai H. Patthy L. Llinás M Structure. 1999; 7: 1235-1245Google Scholar, 3Briknarová K. Gehrmann M. Bányai L. Tordai H. Patthy L. Llinás M J. Biol. Chem. 2001; 276: 27613-27621Google Scholar, 4Gehrmann M. Briknarová K. Bányai L., H. Patthy L. Llinás M. Biol. Chem. 2002; 383: 137-148Google Scholar). Moreover, the x-ray crystallographic structure of the intact human pro-MMP-2 has been reported (5Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Google Scholar). In the second FN2 module from each MMP-2 and MMP-9, residues that are important for the interaction with gelatin have been identified via site-directed mutagenesis (6Collier I.E. Krasnov P.A. Strongin A.Y. Birkedal-Hansen H. Goldberg G.I. J. Biol. Chem. 1992; 267: 6776-6781Google Scholar, 7Tordai H. Patthy L. Eur. J. Biochem. 1999; 259: 513-518Google Scholar). Additionally, the ligand binding surfaces of all three modules of MMP-2 have been mapped from1H and 15N NMR perturbations induced by (PPG)6 and the longer chain analog, (PPG)12, synthetic peptide mimics of gelatin (2Briknarová K. Grishaev A. Bányai L. Tordai H. Patthy L. Llinás M Structure. 1999; 7: 1235-1245Google Scholar, 3Briknarová K. Gehrmann M. Bányai L. Tordai H. Patthy L. Llinás M J. Biol. Chem. 2001; 276: 27613-27621Google Scholar, 4Gehrmann M. Briknarová K. Bányai L., H. Patthy L. Llinás M. Biol. Chem. 2002; 383: 137-148Google Scholar). In line with the crystallographic evidence, which shows that the FN2 modules in MMP-2 point away from each other (5Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Google Scholar), our NMR studies of the interaction between Col domains and (PPG)6 and (PPG)12 have shown that consecutive Col modules contain distinct ligand-binding sites in which affinities for these ligands are virtually identical to those of the individual domains (3Briknarová K. Gehrmann M. Bányai L. Tordai H. Patthy L. Llinás M J. Biol. Chem. 2001; 276: 27613-27621Google Scholar, 4Gehrmann M. Briknarová K. Bányai L., H. Patthy L. Llinás M. Biol. Chem. 2002; 383: 137-148Google Scholar, 8Gehrmann, M., Structural and Functional Similarities between FII and Kringle Domains.Doctoral dissertation, 2002, Carnegie Mellon University, Pittsburgh, PA.Google Scholar). Although the affinity of the MMP-2 Col domains for collagenous ligands appears by now to be well established, less is known regarding the specificity of the interaction. In our previous studies we found that the peptide PIIKFPGDVA, which corresponds to segment 33–42 of the pro-MMP-2, interacts with the three Col domains of MMP-2 in a manner that mimics the interaction with the collagen-like (PPG)6and (PPG)12 peptides (3Briknarová K. Gehrmann M. Bányai L. Tordai H. Patthy L. Llinás M J. Biol. Chem. 2001; 276: 27613-27621Google Scholar, 4Gehrmann M. Briknarová K. Bányai L., H. Patthy L. Llinás M. Biol. Chem. 2002; 383: 137-148Google Scholar). Preference for binding to Col-3 was indicated, consistent with the x-ray crystallographic structure of the pro-MMP-2 (5Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Google Scholar). In the proenzyme, the prodomain interacts intramolecularly with the putative gelatin-binding site of Col-3 via contacts that involve propeptide amino acid residues Ile-35, Phe-37, and Asp-40. As these studies indicate, the ligand specificity of the Col domains is not restricted to collagen-like peptides. It would be useful to gain more information as to the range of structural diversity acceptable for peptides to interact with the Col-binding sites. In the context of MMP-2 involvement in tumor invasion, metastasis and other physiopathological processes (reviewed in Ref. 9Yu A.E. Murphy A.N. Stetler-Stevenson W.G. Parks W.C. Mecham R.P. Matrix Metalloproteinases. Academic Press, San Diego1998: 85-113Google Scholar), it is highly desirable to identify agents that could block its activity. The suitability of MMP-2 as an anticancer target is supported by the finding that MMP-2-deficient mice display reduced angiogenesis and tumor progression (10Itoh T. Tanioka M. Yoshida H. Yoshioka T. Nishimoto H. Itohara S. Cancer Res. 1998; 58: 1048-1051Google Scholar). However, very few inhibitors specific for MMP-2 have been described to date (11Koivunen E. Arap W. Valtanen H. Rainisalo A. Penate-Medina O. Heikkilä P. Kantor C. Gahmber C.G. Salo T. Konttinen Y.T. Sorsa T. Rouslahti E. Pasqualine R. Nat. Biotechnology. 1999; 17: 768-774Google Scholar). The most potent inhibitors also inhibit several other MMP family members (12Talbot D.C. Brown P.D. Eur. J. Cancer. 1996; 32: 2528-2533Google Scholar, 13Beckett R.P. Davidson A.H. Drummond A.H. Huxley P. Whittaker M. Drug Discov. Today. 1996; 1: 16-26Google Scholar, 14Santos O. McDermott C.D. Daniels R.G. Appelt K. Clin. Exp. Metastasis. 1997; 15: 499-508Google Scholar). Although generic MMP inhibitors prevent tumor dissemination and formation of metastases in animal models (14Santos O. McDermott C.D. Daniels R.G. Appelt K. Clin. Exp. Metastasis. 1997; 15: 499-508Google Scholar, 15Davies B. Brown P.D. East N. Crimmin M.J. Balkwill F.R. Cancer Res. 1993; 53: 2087-2091Google Scholar, 16Taraboletti G. Garofalo A. Belotti D. Drudis T. Borsotti P Scanziani E. Brown P.D. Giavazzi R. J. Natl. Cancer Inst. 1995; 87: 293-298Google Scholar, 17Volpert O.V. Ward W.F. Lingen M.W. Chesler L. Solt D.B. Johnson M.D. Molteni A. Polverini P.J. Bouck N.P. J. Clin. Invest. 1996; 98: 671-679Google Scholar, 18Anderson I.C. Shipp M.A. Docherty A.J.P. Teicher B.A. Cancer Res. 1996; 56: 715-718Google Scholar, 19Eccles S.A. Box G.M. Court W.J. Bone E.A. Thomas W. Brown P.D. Cancer Res. 1996; 56: 2815-2822Google Scholar), they tend to elicit too broad a spectrum of response and often exhibit side effects. It can be speculated that active site inhibitors that also bind to the unique FN2 domains of MMP-2 may be more MMP-2-specific. As a platform for such studies, we have screened random 6-mer and random 15-mer phage display libraries for peptides that interact with the FN2 domains of MMP-2 and characterized the interaction of two selected peptides with the homologous gelatin-binding repeats via 1H/15N NMR studies on the Col-12 and Col-23 constructs. Microtiter plates (Greiner Labortechnik) were coated with the first (ॆgalCol-1), second (ॆgalCol-2), or third (ॆgalCol-3) FN2 modules from human MMP-2 (20Bányai L. Tordai H. Patthy L. Biochem. J. 1994; 298: 403-407Google Scholar) or with the three domains in tandem (ॆgalCol-123) (21Bányai L. Patthy L. FEBS Lett. 1991; 282: 23-25Google Scholar). The recombinant proteins, consisting of the appropriate FN2 module(s) and an amino-terminal peptide derived from the ॆ-galactosidase moiety of the expression vector, were prepared as described previously (20Bányai L. Tordai H. Patthy L. Biochem. J. 1994; 298: 403-407Google Scholar, 21Bányai L. Patthy L. FEBS Lett. 1991; 282: 23-25Google Scholar). The plates were incubated with the proteins (20 ॖg/ml) in 100 mmNaHCO3 buffer for 2 h at 37 °C, after which they were blocked with 30 mg/ml serum albumin in 100 mmNaHCO3 buffer for 2 h at 37 °C and washed six times with TBS buffer (50 mm Tris-HCl, pH 7.5, containing 150 mm NaCl) and 0.57 Tween 20. ॆgalCol-123-Sepharose was prepared using cyanogen bromide-activated Sepharose 4B (Amersham Biosciences) and ॆgalCol-123 according to instructions from the manufacturer. Two phage fUSE5 libraries, which express a foreign 15- or 6-mer random peptide library at the amino-terminal end of all five copies of its pIII coat protein (22Scott J.K. Smith G.P. Science. 1990; 249: 386-390Google Scholar) and the Escherichia coli strain K91Kan (thi/HfrC), carrying a 舠mini-kan hopper舡 element inserted in the lacZ gene, were obtained from Prof. G. Smith (University of Missouri-Columbia). The number of primary clones in the 15-mer library is 2.5 × 108 (23Nishi T. Budde R.J.A. McMurray J.S. Oberyesekere N.U. Safdar N. Levin V.A. Saya H. FEBS Lett. 1996; 399: 237-240Google Scholar), and in the case of the 6-mer library it is 2 × 108 (22Scott J.K. Smith G.P. Science. 1990; 249: 386-390Google Scholar). Approximately 1010 phage/well in 100 ॖl of TBS buffer were incubated for 60 min at room temperature. Nonspecifically adsorbed phage were washed away with 12 times with 200 ॖl of TBS buffer containing 5 mg/ml serum albumin and 0.57 Tween 20. Unless otherwise indicated, bound phage were eluted with 2 times with 200 ॖl of TBS buffer containing 1 mg/ml gelatin type A from porcine skin type I collagen (Sigma). In some experiments with immobilized ॆgalCol-123, elution was performed with buffer containing 2 mg/ml ॆgalCol-123. Alternatively, ∼1010 phage in 200 ॖl of TBS buffer were incubated with 50 ॖl of ॆgalCol-123-Sepharose for 60 min. The resin was then washed with 10 ml of TBS buffer containing 5 mg/ml serum albumin and 0.57 Tween 20, and the bound phage were eluted with 3 × 200 ॖl of TBS buffer containing 1 mg/ml gelatin. The eluted phages were amplified, and a portion was used in the next biopanning cycle (24Parmley S.F. Smith G.P. Gene. 1988; 73: 305-318Google Scholar). After three rounds, individual phage were isolated, and the DNA sequence of the 5′ end of the gene III was determined using a primer complementary to the positions 1663–1680 of the wild type gene. The peptides ACGYTYHPPCARLTV (ACG), WFPGPITFIPRPWSS (WFP), WHWRHRIPLQLAAGR (WHW), THSHQWRHHQFPAPT (THS), and HASHFRFRHSHVYGV (HAS) were synthesized on an AB-PE 431A peptide synthesizer (Applied Biosystems-PerkinElmer Life Sciences) using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. 15N-labeled Col-12 and Col-23 modules from human MMP-2 (residues 223–337 and 278–394 respectively) (Fig. 1) were expressed in E. coliand purified as described previously (3Briknarová K. Gehrmann M. Bányai L. Tordai H. Patthy L. Llinás M J. Biol. Chem. 2001; 276: 27613-27621Google Scholar, 4Gehrmann M. Briknarová K. Bányai L., H. Patthy L. Llinás M. Biol. Chem. 2002; 383: 137-148Google Scholar). To monitor ligand-induced resonance shifts, small aliquots of WHW or THS stock solutions in 907 H2O, 107 D2O, pH 7.0, were added to samples of 0.35 mm15N-labeled Col-12 and Col-23 in 907 H2O, 107 D2O, pH 7.0, and 1H-15N HSQC experiments (25Müller L. J. Am. Chem. Soc. 1979; 101: 4481-4484Google Scholar, 26Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185-189Google Scholar, 27Mori S. Abeygunawardana C. Johnson M.O. van Zijl P.C.M. J. Magn. Reson. B. 1995; 108: 94-98Google Scholar) were recorded at each step. All of the data were acquired at 25 °C on Bruker Avance DMX-500 spectrometer equipped with a 5-mm triple resonance three-axis gradient probe. The spectra were processed and analyzed with the programs Felix 95 and Felix 98 (Molecular Simulations, Inc., San Diego, CA) on a Silicon Graphics Indy R-5000 work station. Protein and peptide ligand concentrations were determined spectrophotometrically (28Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Google Scholar). Values of the equilibrium association constant (Ka) were determined by a combination of linear and nonlinear least squares fitting of the chemical shift changes, as described previously (29Marti D.N. Hu C.-K. An S.S.A. von Haller P. Schaller J. Llinás M. Biochemistry. 1997; 36: 11591-11604Google Scholar, 30An S.S.A. Marti D.N. Carreño C. Albericio F. Schaller J. Llinás M. Protein Sci. 1998; 7: 1947-1959Google Scholar). To identify peptides that bind to the FN2 modules from MMP-2, phage display 6-mer or 15-mer random peptide libraries were screened with ॆgalCol-1, ॆgalCol-2, ॆgalCol-3 and ॆgalCol-123. Nonspecifically adsorbed phage were washed away with buffer containing 5 mg/ml serum albumin, then-in most experiments-specifically bound phage was eluted with buffer containing gelatin. In the case of the 6-mer library-unlike in the case of the 15-mer library-there was no enrichment of bound phage after three rounds of biopanning. This observation suggested that a six-residue-long peptide may be too short for unique recognition of FN2 domains, therefore we concentrated on the 15-mer library. From the latter library, after three rounds of biopanning, individual clones were sequenced and the following peptides were identified: ACGYTYHPPCARLTV (ACG), WFPGPITFIPRPWSS (WFP), WHWRHRIPLQLAAGR (WHW), THSHQWRHHQFPAPT (THS), WHVSPRHQRLFHGLF (WHV) and HASHFRFRHSHVYGV (HAS). The frequencies of the peptides selected under various conditions are summarized in Table I. Interestingly, the peptides are not collagen-like and do not share a common, obvious sequence motif. However, their sequences exhibit biased amino acid composition. For example, although His accounts for only 27 of residues in protein databases, the selected peptides contain 167 His. This trend is even more pronounced for peptides selected on single FN2 domains, which contain 227 His. There also is bias in the aromatic amino acid content: and for of residues in protein databases, in the selected peptides, their proportion is Interestingly, peptide inhibitors of the that have been selected previously from phage display library (11Koivunen E. Arap W. Valtanen H. Rainisalo A. Penate-Medina O. Heikkilä P. Kantor C. Gahmber C.G. Salo T. Konttinen Y.T. Sorsa T. Rouslahti E. Pasqualine R. Nat. Biotechnology. 1999; 17: 768-774Google Scholar) are in aromatic residues those from G. G. J.L. 1996; Scholar) library contain results that not only interaction with the active site also binding to FN2 modules may to of these It is also that peptide sequence which is found in the (residues from the gelatin-binding pocket of Col-3 in the x-ray structure of pro-MMP-2 (5Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Google of phage display peptides selected under various in a The clones selected on ॆgalCol-1, and ॆgalCol-3 exhibit identical sequences with only in their In that the sequence between the modules, the ligand-binding sites of the three domains are to The which was with the on immobilized ॆgalCol-123, has two that may form a the It is a observation with phage display peptides that those with the affinity tend to be (11Koivunen E. Arap W. Valtanen H. Rainisalo A. Penate-Medina O. Heikkilä P. Kantor C. Gahmber C.G. Salo T. Konttinen Y.T. Sorsa T. Rouslahti E. Pasqualine R. Nat. Biotechnology. 1999; 17: 768-774Google Scholar, S.A. S.A. W.F. 1992; Scholar, M.A. W. Gene. 1993; Scholar, E. B. E. 1995; Scholar). Interestingly, was selected on single Col domains, which suggests that the complementary binding on ॆgalCol-123 There is a between clones eluted from ॆgalCol-123 with gelatin and those eluted with ॆgalCol-123. Although gelatin phage that interacts with the gelatin-binding site of ॆgalCol-123 is to phage bound to of the peptides that elution with ॆgalCol-123 to elution with gelatin to a are to interact with a at least of the gelatin-binding The interaction of the peptides with Col-12 and Col-23 was using NMR of only WHW and THS were found for Col-12 and Col-23 chemical shift induced by WHW and THS binding were HSQC spectra and affinity constants (Ka) were from the ligand (Fig. Table The first and second modules of the Col-12 bind to WHW and mm−1 the latter with the derived for For mm−1 was The affinity of THS for the Col modules is mm−1 for and of WHW and binding to Col-12 and Col-23 by NMR. The resonance of each FN2 to the of are the ligand and the ligand and protein The data for and are data point is an of selected or 15N chemical binding via nonlinear least squares to the of WHW and THS for FN2 in a (2Briknarová K. Grishaev A. Bányai L. Tordai H. Patthy L. Llinás M Structure. 1999; 7: 1235-1245Google Scholar, 3Briknarová K. Gehrmann M. Bányai L. Tordai H. Patthy L. Llinás M J. Biol. Chem. 2001; 276: 27613-27621Google Scholar, 4Gehrmann M. Briknarová K. Bányai L., H. Patthy L. Llinás M. Biol. Chem. 2002; 383: 137-148Google Scholar), we mapped the gelatin binding of the Col modules by perturbations induced by the synthetic gelatin-like peptides (PPG)6 and (PPG)12 on the of the modules This to be less straightforward in the from the and the segment of the constructs are by WHW and THS (Fig. effects were of Col-23 with the synthetic gelatin mimics and However, in the latter the from the peptide were to those the gelatin-binding In the on the other most perturbations were of or those in the and it is to the effects by direct with the ligand from related to conformation or to peptide binding shift perturbations at from ligand sites have been previously in other W. I. L. M. J. J. NMR. 1998; of residues and residues according to Col-12 or Col-23 chemical shift induced by WHW and are The gelatin-binding site comprising and a (residues on its is in the The of the is the as for of residues and residues according to Col-12 or Col-23 chemical shift induced by THS and are The of the is the as for and The gelatin-binding surfaces of Col modules an aromatic and its in the comprising residues at the of the The of and on the of Col modules can be summarized as 5 and In the first FN2 module the Col-12 WHW residues the gelatin-binding most residues and In the second FN2 module both the Col-12 and Col-23 constructs and WHW the the on the which is involved in gelatin is to a In the third FN2 module in Col-23 resonance are to the of the domain and the and which among residues to the side is only it may be that WHW interacts with the gelatin-binding pocket of Col-1 M. Briknarová K. Bányai L., H. Patthy L. Llinás M. Biol. Chem. 2002; 383: 137-148Google Scholar) and In with such an WHW has been selected most by gelatin elution from plates coated with and less from those coated with THS from residues on the of the gelatin-binding pocket in all Col modules (Fig. that THS contacts Col modules via site. from the are also of inhibitors that on MMP-2 not on other is a MMP-2 with is unique in FN2 domains next to its catalytic active site inhibitors that also interact with FN2 domains be more we have screened random and 15-mer phage display libraries and identified several peptides from the latter library that interact with the FN2 modules of MMP-2.