Residue 2 of TIMP-1 Is a Major Determinant of Affinity and Specificity for Matrix Metalloproteinases but Effects of Substitutions Do Not Correlate with Those of the Corresponding P1′ Residue of Substrate
1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês
10.1074/jbc.274.15.10184
ISSN1083-351X
AutoresQi Meng, Vladimir A. Malinovskii, Wen Huang, Yajing Hu, Linda Chung, Hideaki Nagase, Wolfram Bode, K. Maskos, Keith Brew,
Tópico(s)Signaling Pathways in Disease
ResumoThe unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1·MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1′ specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases. The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1·MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1′ specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases. matrix metalloproteinases tissue inhibitors of metalloproteinases N-terminal domain of tissue inhibitor of metalloproteinases-1 The matrix metalloproteinases (MMPs)1 are a family of about twenty Zn2+-dependent endopeptidases that have important roles in connective tissue turnover during physiological processes including development, morphogenesis, and wound healing (1Woessner Jr., J.F. Ann. N. Y. Acad. Sci. 1994; 732: 11-21Crossref PubMed Scopus (436) Google Scholar,2Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis Ltd., London1996: 153-204Google Scholar). Their activities in the extracellular matrix are stringently regulated through transcriptional control, zymogen activation, and the actions of four endogenous inhibitory proteins, tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 (3Docherty A.J.P. Lyons A. Smith B.J. Wright E.M. Stephens P.E. Harris T.J.R. Murphy G. Reynolds J.J. Nature. 1985; 318: 66-69Crossref PubMed Scopus (582) Google Scholar, 4Boone T.C. Johnson M.J. DeClerck Y.A. Langley K.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2800-2804Crossref PubMed Scopus (180) Google Scholar, 5Pavloff N. Staskus P.W. Kishnani N.S. Hawkes S.P. J. Biol. Chem. 1992; 267: 17321-17326Abstract Full Text PDF PubMed Google Scholar, 6Silbiger S.M. Jacobsen V.L. Cupples R.L. Koski R.A. Gene. 1994; 141: 293-297Crossref PubMed Scopus (66) Google Scholar, 7Greene J. Wang M. Liu Y.E. Raymond L.A. Rosen C. Shi Y.E. J. Biol. Chem. 1996; 271: 30375-30380Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Normal matrix homeostasis is associated with an appropriate balance between the levels of TIMPs and active MMPs, whereas an imbalance involving excess MMP activity is linked with disease processes including arthritis, tumor cell metastasis, and tissue invasion and atherosclerosis (1Woessner Jr., J.F. Ann. N. Y. Acad. Sci. 1994; 732: 11-21Crossref PubMed Scopus (436) Google Scholar, 2Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis Ltd., London1996: 153-204Google Scholar).Mammalian TIMPs have an N-terminal domain of about 125 amino acids and a smaller C-terminal domain of about 65 amino acids; each domain is stabilized by three disulfide bonds (8Williamson E.A. Marston F.A.O. Angal S. Koklitis P. Panico M. Morris H.R. Carne A.F. Smith B.J. Harris T.J.R. Freedman R.B. Biochem. J. 1990; 268: 267-274Crossref PubMed Scopus (155) Google Scholar). The N-terminal domains of different TIMPs fold into a correct native structure which carries the inhibitory activity against MMPs (9Murphy G. Houbrechts A. Cockett M.I. Williamson R.O.-A. Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (283) Google Scholar, 10O'Shea M. Willenbrock F. Williamson R.A. Cockett M.I. Freedman R.B. Reynolds J.J. Docherty A.J.P. Murphy G. Biochemistry. 1992; 31: 10146-10152Crossref PubMed Scopus (73) Google Scholar, 11Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar). Although correctly folded and functional C-terminal domains have not been described, truncation experiments indicate that this region is responsible for the interactions of TIMPs with pro-MMPs (12Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (243) Google Scholar, 13Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Crossref PubMed Scopus (143) Google Scholar). There is little specificity in the inhibitory actions of TIMPs on metalloproteinases, with the exception of the ability of TIMP-2 and TIMP-3 to inhibit membrane-type metalloproteinases-1 and -2, whereas TIMP-1 is a poor inhibitor of these enzymes (12Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (243) Google Scholar, 13Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Crossref PubMed Scopus (143) Google Scholar, 14Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). However, the interactions of TIMPs with pro-MMPs are more specific. For example, TIMP-2 and TIMP-4 form specific complexes with pro-MMP-2 (progelatinase A), whereas TIMP-1 can bind to pro-MMP-9. In addition to their activities as MMP inhibitors and in binding to pro-MMPs, TIMPs promote the growth of various types of cells in tissue culture (15Hayakawa T. Yamashita K. Tanzawa K. Uchijima E. Iwata K. FEBS Lett. 1992; 298: 29-31Crossref PubMed Scopus (645) Google Scholar, 16Hayakawa T. Yamashita K. Ohuchi E. Shimagawa A. J. Cell Sci. 1994; 107: 2373-2379Crossref PubMed Google Scholar) and have anti-angiogenic activity (17Anand-Apte B. Pepper M.S. Voest E. Montesano K. Olsen B. Murphy G. Apte S. Zetter B. Invest. Opthalmol. Visual Sci. 1997; 38: 817-823PubMed Google Scholar). However, the structural basis of these activities is unknown.Crystallographic structures have been recently reported for a complex of TIMP-1 with the catalytic domain of stromelysin-1, MMP-3ΔC (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar), and a complex of TIMP-2 with the catalytic domain of membrane-type matrix metalloproteinase 1 (19Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). Together with a solution NMR structure of the N-terminal domain of TIMP-2, N-TIMP-2 (20Williamson R.A. Martorell G. Carr M.D. Murphy G. Docherty A.J.P. Freedman R.B. Feeney J. Biochemistry. 1994; 33: 11745-11759Crossref PubMed Scopus (95) Google Scholar, 21Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), these reveal that the inhibitory domain of TIMP consists of a 5-stranded β-barrel with three associated α-helices, resembling the folds of members of the OB (oligonucleotide/oligosaccharide binding) protein family (22Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (762) Google Scholar). The TIMP-1·MMP-3 structure reveals that the principle interactions between TIMP and the metalloproteinase involve the N-terminal pentapeptide and part of the loop between β-strands C and D; other interactions are through the A-B loop and some residues in the C-terminal domain (Fig. 1 A). Three quarters of all contacts are by residues adjacent to the disulfide bond between Cys1 and Cys70, specifically residues 1–5 and 66–70 (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). The N-terminal Cys1 is a key to the inhibitory strategy of TIMP because it sits on top of the catalytic Zn2+ of the metalloproteinase and coordinates the metal ion through the α-amino group and peptide carbonyl group (Fig. 1 B). Similar contacts are seen in the TIMP-2·MT1-MMP complex, although there are differences in the relative orientations of the two proteins and in the extensiveness of interactions involving other parts of the structure (19Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar).The reactive site of TIMP revealed by this structure is consistent with the results of a study which showed that TIMP-1 activity is lost when the Val69-Cys70 peptide bond is cleaved by human neutrophil elastase, but that this cleavage is prevented by complex formation between TIMP-1 and MMP-3 (23Nagase H. Suzuki K. Cawston T.E. Brew K. Biochem. J. 1997; 325: 163-167Crossref PubMed Scopus (39) Google Scholar). Previous mutational studies of N-TIMP-1 also show that substitutions at a number of sites between residues 18 and 45 have small effects on the affinity for MMP-3, whereas mutations that disrupt the Cys1 to Cys70 disulfide and substitutions for Thr2, Met66, or Val69 had large effects on activity (24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar). Most significantly, whereas other substitutions that perturb N-TIMP-1 activity have approximately equal effects on binding to MMP-1, MMP-2, and MMP-3, the substitution of Ala for Thr2 produces a 17-fold greater loss in binding to MMP-1 relative to MMP-3 (24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar). This is in accord with the crystallographic structures which indicate that Thr2 of TIMP-1 and Ser2 of TIMP-2 interact with the region of the metalloproteinases that correspond to the binding site for the P1′ residue of peptide substrates, the residue that has a dominant role in MMP specificity (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar, 19Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar).As part of a study of the structural basis of TIMP-1 specificity and as a step toward generating variants that are more selective as MMP inhibitors, we have characterized fifteen N-TIMP-1 mutants with substitutions for Thr2. The results show that this residue has a major influence on the specificity of TIMP for different metalloproteinases but also show that there is little correlation between the effect of an amino acid at position 2 in TIMP-1 and the same residue at the P1′ site of a peptide substrate on their respective activities with an MMP as inhibitor or substrate. The structural basis of these observations is discussed.DISCUSSIONThe present results suggest that protein engineering of TIMP is a viable approach for generating more specific protein inhibitors of MMPs for studies of the biological roles of different MMPs and to facilitate the development of therapeutic agents for diseases linked with excess activity of specific MMPs. The development of specifically targeted high affinity variants will require substitutions at multiple sites but the inhibitory properties of the Met66 and Val69 mutants and full-length TIMP-1 (11Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar, 24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar) indicate that residue 2 has a much greater influence on specificity than other components of the reactive site (see Fig. 3). The effects of systematic substitutions for Thr2 provide information about the determinants of MMP binding by N-TIMP-1 and also some TIMP variants with enhanced specificity. Table II shows that the best inhibitors for MMP-1, MMP-2, and MMP-3, based on affinity and selectivity, have different substitutions at position 2: valine, leucine, or serine, respectively. The substitution of Gly for Thr2 produces a protein that is essentially inactive as an inhibitor at concentrations below 100 nm yet retains a native structure as indicated by CD spectroscopy (Fig. 2). Although the isolated C-domain of TIMP may not be capable of folding, the Gly2 substitution in full-length TIMP-1 will generate a protein that can be used to investigate the interactions of the C-terminal domain in a molecule that has little affinity for a metalloproteinase active site and to determine whether the protease-inhibitory action of TIMP is connected with other functions such as cell growth stimulation and anti-angiogenic activity (15Hayakawa T. Yamashita K. Tanzawa K. Uchijima E. Iwata K. FEBS Lett. 1992; 298: 29-31Crossref PubMed Scopus (645) Google Scholar, 16Hayakawa T. Yamashita K. Ohuchi E. Shimagawa A. J. Cell Sci. 1994; 107: 2373-2379Crossref PubMed Google Scholar, 17Anand-Apte B. Pepper M.S. Voest E. Montesano K. Olsen B. Murphy G. Apte S. Zetter B. Invest. Opthalmol. Visual Sci. 1997; 38: 817-823PubMed Google Scholar).Some properties of the side chain of residue 2 correlate with the affinity for different MMPs but, at present, there is insufficient information to explain the structural basis of the effects of mutations on activity. In the TIMP-1·MMP-3 crystal structure, the N-terminal region of MMP-3 has a different structure from that in free MMP-3 (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). Based on the NMR structure of N-TIMP-2, it appears possible that conformational changes in TIMP may also occur during complex formation (21Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Consequently, mutations in the reactive site could affect the interaction with MMPs through effects on local dynamics (as well as structure). Binding to different MMPs may also be affected differentially by regions distant from the Cys1-Cys70 disulfide bond. The higher affinity of MMP-3 for the Gly2 mutant suggests that interactions outside of the S1′ pocket contribute more of the binding energy for the interaction with MMP-3 than with the other MMPs.The structure of the S1′ pocket of MMP-1 is narrower and less deep than those of other MMPs. Arginine 195, which replaces leucine in MMP-2 and MMP-3, projects into the S1′ pocket toward the catalytic Zn2+, resulting in a less deep more cationic pocket (31Stams T. Spurlino J.C. Smith D.L. Wahl R.C. Ho T.F. Qoronfleh M.W. Banks T.M. Rubin B. Nat. Struct. Biol. 1994; 1: 119-123Crossref PubMed Scopus (201) Google Scholar). The reduced depth may account for the preference of MMP-1 for valine and threonine over leucine, for example, and charge repulsion is also a likely factor in the unfavorable binding of positively charged substituents to MMP-1. The specificity pocket of an MMP with no bound substrate or inhibitor will be expected to contain multiple solvent molecules so that the binding of TIMP mutants will be affected by the ability of the residue 2 side chain to interact with or displace solvent molecules.Large areas of molecular surface become buried in the formation of high affinity heterologous protein-protein complexes (32Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2254) Google Scholar). It has been estimated that approximately 1300 Å2 of the accessible surface of each component is buried on formation of the TIMP-1·MMP-3 complex (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). The C-terminal domain of TIMP-1 accounts for few of these contacts and less than 10% of the free energy of interaction (11Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar). The present results show that a single residue of the reactive site of N-TIMP-1, Thr2, exerts a large influence on the strength and specificity of binding to MMPs. The magnitude of this effect is surprising because, even if the environment of Thr2 changed from being totally exposed to 100% buried on complex formation with the protease, it accounts for less than 8% (102/1300 Å (2Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis Ltd., London1996: 153-204Google Scholar) of the area of the protein−protein interaction site (33Miller S. Janin J. Lesk A.M. Chothia C. J. Mol. Biol. 1987; 196: 641-656Crossref PubMed Scopus (826) Google Scholar). The loss of 33–55% of the free energy of binding on removal of the Thr2 side chain by the substitution of glycine indicates that residue 2 of TIMP-1 could be designated a "hot spot" in the TIMP/MMP interaction interface, a residue that has a uniquely large influence on the strength of the protein-protein interaction (34Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1772) Google Scholar, 35Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1-6Crossref PubMed Scopus (359) Google Scholar). Many other residues form contacts with MMP-3 in the crystal structure (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar), but mutagenesis of others, particularly Met66 and Val69, and the removal of all C-domain contacts in N-TIMP-1 indicates that these residues are less important for binding than Thr2 (24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar). Thus, the present results show that the nature of the contacts in the structure of the TIMP-1·MMP-3 complex are an important step toward understanding the structural basis of the inhibitory strategy and specificity in TIMP, but it is important to experimentally evaluate their contributions to the stability of the complex. The matrix metalloproteinases (MMPs)1 are a family of about twenty Zn2+-dependent endopeptidases that have important roles in connective tissue turnover during physiological processes including development, morphogenesis, and wound healing (1Woessner Jr., J.F. Ann. N. Y. Acad. Sci. 1994; 732: 11-21Crossref PubMed Scopus (436) Google Scholar,2Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis Ltd., London1996: 153-204Google Scholar). Their activities in the extracellular matrix are stringently regulated through transcriptional control, zymogen activation, and the actions of four endogenous inhibitory proteins, tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 (3Docherty A.J.P. Lyons A. Smith B.J. Wright E.M. Stephens P.E. Harris T.J.R. Murphy G. Reynolds J.J. Nature. 1985; 318: 66-69Crossref PubMed Scopus (582) Google Scholar, 4Boone T.C. Johnson M.J. DeClerck Y.A. Langley K.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2800-2804Crossref PubMed Scopus (180) Google Scholar, 5Pavloff N. Staskus P.W. Kishnani N.S. Hawkes S.P. J. Biol. Chem. 1992; 267: 17321-17326Abstract Full Text PDF PubMed Google Scholar, 6Silbiger S.M. Jacobsen V.L. Cupples R.L. Koski R.A. Gene. 1994; 141: 293-297Crossref PubMed Scopus (66) Google Scholar, 7Greene J. Wang M. Liu Y.E. Raymond L.A. Rosen C. Shi Y.E. J. Biol. Chem. 1996; 271: 30375-30380Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Normal matrix homeostasis is associated with an appropriate balance between the levels of TIMPs and active MMPs, whereas an imbalance involving excess MMP activity is linked with disease processes including arthritis, tumor cell metastasis, and tissue invasion and atherosclerosis (1Woessner Jr., J.F. Ann. N. Y. Acad. Sci. 1994; 732: 11-21Crossref PubMed Scopus (436) Google Scholar, 2Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis Ltd., London1996: 153-204Google Scholar). Mammalian TIMPs have an N-terminal domain of about 125 amino acids and a smaller C-terminal domain of about 65 amino acids; each domain is stabilized by three disulfide bonds (8Williamson E.A. Marston F.A.O. Angal S. Koklitis P. Panico M. Morris H.R. Carne A.F. Smith B.J. Harris T.J.R. Freedman R.B. Biochem. J. 1990; 268: 267-274Crossref PubMed Scopus (155) Google Scholar). The N-terminal domains of different TIMPs fold into a correct native structure which carries the inhibitory activity against MMPs (9Murphy G. Houbrechts A. Cockett M.I. Williamson R.O.-A. Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (283) Google Scholar, 10O'Shea M. Willenbrock F. Williamson R.A. Cockett M.I. Freedman R.B. Reynolds J.J. Docherty A.J.P. Murphy G. Biochemistry. 1992; 31: 10146-10152Crossref PubMed Scopus (73) Google Scholar, 11Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar). Although correctly folded and functional C-terminal domains have not been described, truncation experiments indicate that this region is responsible for the interactions of TIMPs with pro-MMPs (12Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (243) Google Scholar, 13Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Crossref PubMed Scopus (143) Google Scholar). There is little specificity in the inhibitory actions of TIMPs on metalloproteinases, with the exception of the ability of TIMP-2 and TIMP-3 to inhibit membrane-type metalloproteinases-1 and -2, whereas TIMP-1 is a poor inhibitor of these enzymes (12Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (243) Google Scholar, 13Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Crossref PubMed Scopus (143) Google Scholar, 14Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). However, the interactions of TIMPs with pro-MMPs are more specific. For example, TIMP-2 and TIMP-4 form specific complexes with pro-MMP-2 (progelatinase A), whereas TIMP-1 can bind to pro-MMP-9. In addition to their activities as MMP inhibitors and in binding to pro-MMPs, TIMPs promote the growth of various types of cells in tissue culture (15Hayakawa T. Yamashita K. Tanzawa K. Uchijima E. Iwata K. FEBS Lett. 1992; 298: 29-31Crossref PubMed Scopus (645) Google Scholar, 16Hayakawa T. Yamashita K. Ohuchi E. Shimagawa A. J. Cell Sci. 1994; 107: 2373-2379Crossref PubMed Google Scholar) and have anti-angiogenic activity (17Anand-Apte B. Pepper M.S. Voest E. Montesano K. Olsen B. Murphy G. Apte S. Zetter B. Invest. Opthalmol. Visual Sci. 1997; 38: 817-823PubMed Google Scholar). However, the structural basis of these activities is unknown. Crystallographic structures have been recently reported for a complex of TIMP-1 with the catalytic domain of stromelysin-1, MMP-3ΔC (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar), and a complex of TIMP-2 with the catalytic domain of membrane-type matrix metalloproteinase 1 (19Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). Together with a solution NMR structure of the N-terminal domain of TIMP-2, N-TIMP-2 (20Williamson R.A. Martorell G. Carr M.D. Murphy G. Docherty A.J.P. Freedman R.B. Feeney J. Biochemistry. 1994; 33: 11745-11759Crossref PubMed Scopus (95) Google Scholar, 21Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), these reveal that the inhibitory domain of TIMP consists of a 5-stranded β-barrel with three associated α-helices, resembling the folds of members of the OB (oligonucleotide/oligosaccharide binding) protein family (22Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (762) Google Scholar). The TIMP-1·MMP-3 structure reveals that the principle interactions between TIMP and the metalloproteinase involve the N-terminal pentapeptide and part of the loop between β-strands C and D; other interactions are through the A-B loop and some residues in the C-terminal domain (Fig. 1 A). Three quarters of all contacts are by residues adjacent to the disulfide bond between Cys1 and Cys70, specifically residues 1–5 and 66–70 (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). The N-terminal Cys1 is a key to the inhibitory strategy of TIMP because it sits on top of the catalytic Zn2+ of the metalloproteinase and coordinates the metal ion through the α-amino group and peptide carbonyl group (Fig. 1 B). Similar contacts are seen in the TIMP-2·MT1-MMP complex, although there are differences in the relative orientations of the two proteins and in the extensiveness of interactions involving other parts of the structure (19Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). The reactive site of TIMP revealed by this structure is consistent with the results of a study which showed that TIMP-1 activity is lost when the Val69-Cys70 peptide bond is cleaved by human neutrophil elastase, but that this cleavage is prevented by complex formation between TIMP-1 and MMP-3 (23Nagase H. Suzuki K. Cawston T.E. Brew K. Biochem. J. 1997; 325: 163-167Crossref PubMed Scopus (39) Google Scholar). Previous mutational studies of N-TIMP-1 also show that substitutions at a number of sites between residues 18 and 45 have small effects on the affinity for MMP-3, whereas mutations that disrupt the Cys1 to Cys70 disulfide and substitutions for Thr2, Met66, or Val69 had large effects on activity (24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar). Most significantly, whereas other substitutions that perturb N-TIMP-1 activity have approximately equal effects on binding to MMP-1, MMP-2, and MMP-3, the substitution of Ala for Thr2 produces a 17-fold greater loss in binding to MMP-1 relative to MMP-3 (24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar). This is in accord with the crystallographic structures which indicate that Thr2 of TIMP-1 and Ser2 of TIMP-2 interact with the region of the metalloproteinases that correspond to the binding site for the P1′ residue of peptide substrates, the residue that has a dominant role in MMP specificity (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar, 19Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). As part of a study of the structural basis of TIMP-1 specificity and as a step toward generating variants that are more selective as MMP inhibitors, we have characterized fifteen N-TIMP-1 mutants with substitutions for Thr2. The results show that this residue has a major influence on the specificity of TIMP for different metalloproteinases but also show that there is little correlation between the effect of an amino acid at position 2 in TIMP-1 and the same residue at the P1′ site of a peptide substrate on their respective activities with an MMP as inhibitor or substrate. The structural basis of these observations is discussed. DISCUSSIONThe present results suggest that protein engineering of TIMP is a viable approach for generating more specific protein inhibitors of MMPs for studies of the biological roles of different MMPs and to facilitate the development of therapeutic agents for diseases linked with excess activity of specific MMPs. The development of specifically targeted high affinity variants will require substitutions at multiple sites but the inhibitory properties of the Met66 and Val69 mutants and full-length TIMP-1 (11Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar, 24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar) indicate that residue 2 has a much greater influence on specificity than other components of the reactive site (see Fig. 3). The effects of systematic substitutions for Thr2 provide information about the determinants of MMP binding by N-TIMP-1 and also some TIMP variants with enhanced specificity. Table II shows that the best inhibitors for MMP-1, MMP-2, and MMP-3, based on affinity and selectivity, have different substitutions at position 2: valine, leucine, or serine, respectively. The substitution of Gly for Thr2 produces a protein that is essentially inactive as an inhibitor at concentrations below 100 nm yet retains a native structure as indicated by CD spectroscopy (Fig. 2). Although the isolated C-domain of TIMP may not be capable of folding, the Gly2 substitution in full-length TIMP-1 will generate a protein that can be used to investigate the interactions of the C-terminal domain in a molecule that has little affinity for a metalloproteinase active site and to determine whether the protease-inhibitory action of TIMP is connected with other functions such as cell growth stimulation and anti-angiogenic activity (15Hayakawa T. Yamashita K. Tanzawa K. Uchijima E. Iwata K. FEBS Lett. 1992; 298: 29-31Crossref PubMed Scopus (645) Google Scholar, 16Hayakawa T. Yamashita K. Ohuchi E. Shimagawa A. J. Cell Sci. 1994; 107: 2373-2379Crossref PubMed Google Scholar, 17Anand-Apte B. Pepper M.S. Voest E. Montesano K. Olsen B. Murphy G. Apte S. Zetter B. Invest. Opthalmol. Visual Sci. 1997; 38: 817-823PubMed Google Scholar).Some properties of the side chain of residue 2 correlate with the affinity for different MMPs but, at present, there is insufficient information to explain the structural basis of the effects of mutations on activity. In the TIMP-1·MMP-3 crystal structure, the N-terminal region of MMP-3 has a different structure from that in free MMP-3 (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). Based on the NMR structure of N-TIMP-2, it appears possible that conformational changes in TIMP may also occur during complex formation (21Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Consequently, mutations in the reactive site could affect the interaction with MMPs through effects on local dynamics (as well as structure). Binding to different MMPs may also be affected differentially by regions distant from the Cys1-Cys70 disulfide bond. The higher affinity of MMP-3 for the Gly2 mutant suggests that interactions outside of the S1′ pocket contribute more of the binding energy for the interaction with MMP-3 than with the other MMPs.The structure of the S1′ pocket of MMP-1 is narrower and less deep than those of other MMPs. Arginine 195, which replaces leucine in MMP-2 and MMP-3, projects into the S1′ pocket toward the catalytic Zn2+, resulting in a less deep more cationic pocket (31Stams T. Spurlino J.C. Smith D.L. Wahl R.C. Ho T.F. Qoronfleh M.W. Banks T.M. Rubin B. Nat. Struct. Biol. 1994; 1: 119-123Crossref PubMed Scopus (201) Google Scholar). The reduced depth may account for the preference of MMP-1 for valine and threonine over leucine, for example, and charge repulsion is also a likely factor in the unfavorable binding of positively charged substituents to MMP-1. The specificity pocket of an MMP with no bound substrate or inhibitor will be expected to contain multiple solvent molecules so that the binding of TIMP mutants will be affected by the ability of the residue 2 side chain to interact with or displace solvent molecules.Large areas of molecular surface become buried in the formation of high affinity heterologous protein-protein complexes (32Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2254) Google Scholar). It has been estimated that approximately 1300 Å2 of the accessible surface of each component is buried on formation of the TIMP-1·MMP-3 complex (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). The C-terminal domain of TIMP-1 accounts for few of these contacts and less than 10% of the free energy of interaction (11Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar). The present results show that a single residue of the reactive site of N-TIMP-1, Thr2, exerts a large influence on the strength and specificity of binding to MMPs. The magnitude of this effect is surprising because, even if the environment of Thr2 changed from being totally exposed to 100% buried on complex formation with the protease, it accounts for less than 8% (102/1300 Å (2Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis Ltd., London1996: 153-204Google Scholar) of the area of the protein−protein interaction site (33Miller S. Janin J. Lesk A.M. Chothia C. J. Mol. Biol. 1987; 196: 641-656Crossref PubMed Scopus (826) Google Scholar). The loss of 33–55% of the free energy of binding on removal of the Thr2 side chain by the substitution of glycine indicates that residue 2 of TIMP-1 could be designated a "hot spot" in the TIMP/MMP interaction interface, a residue that has a uniquely large influence on the strength of the protein-protein interaction (34Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1772) Google Scholar, 35Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1-6Crossref PubMed Scopus (359) Google Scholar). Many other residues form contacts with MMP-3 in the crystal structure (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar), but mutagenesis of others, particularly Met66 and Val69, and the removal of all C-domain contacts in N-TIMP-1 indicates that these residues are less important for binding than Thr2 (24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar). Thus, the present results show that the nature of the contacts in the structure of the TIMP-1·MMP-3 complex are an important step toward understanding the structural basis of the inhibitory strategy and specificity in TIMP, but it is important to experimentally evaluate their contributions to the stability of the complex. The present results suggest that protein engineering of TIMP is a viable approach for generating more specific protein inhibitors of MMPs for studies of the biological roles of different MMPs and to facilitate the development of therapeutic agents for diseases linked with excess activity of specific MMPs. The development of specifically targeted high affinity variants will require substitutions at multiple sites but the inhibitory properties of the Met66 and Val69 mutants and full-length TIMP-1 (11Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar, 24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar) indicate that residue 2 has a much greater influence on specificity than other components of the reactive site (see Fig. 3). The effects of systematic substitutions for Thr2 provide information about the determinants of MMP binding by N-TIMP-1 and also some TIMP variants with enhanced specificity. Table II shows that the best inhibitors for MMP-1, MMP-2, and MMP-3, based on affinity and selectivity, have different substitutions at position 2: valine, leucine, or serine, respectively. The substitution of Gly for Thr2 produces a protein that is essentially inactive as an inhibitor at concentrations below 100 nm yet retains a native structure as indicated by CD spectroscopy (Fig. 2). Although the isolated C-domain of TIMP may not be capable of folding, the Gly2 substitution in full-length TIMP-1 will generate a protein that can be used to investigate the interactions of the C-terminal domain in a molecule that has little affinity for a metalloproteinase active site and to determine whether the protease-inhibitory action of TIMP is connected with other functions such as cell growth stimulation and anti-angiogenic activity (15Hayakawa T. Yamashita K. Tanzawa K. Uchijima E. Iwata K. FEBS Lett. 1992; 298: 29-31Crossref PubMed Scopus (645) Google Scholar, 16Hayakawa T. Yamashita K. Ohuchi E. Shimagawa A. J. Cell Sci. 1994; 107: 2373-2379Crossref PubMed Google Scholar, 17Anand-Apte B. Pepper M.S. Voest E. Montesano K. Olsen B. Murphy G. Apte S. Zetter B. Invest. Opthalmol. Visual Sci. 1997; 38: 817-823PubMed Google Scholar). Some properties of the side chain of residue 2 correlate with the affinity for different MMPs but, at present, there is insufficient information to explain the structural basis of the effects of mutations on activity. In the TIMP-1·MMP-3 crystal structure, the N-terminal region of MMP-3 has a different structure from that in free MMP-3 (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). Based on the NMR structure of N-TIMP-2, it appears possible that conformational changes in TIMP may also occur during complex formation (21Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Consequently, mutations in the reactive site could affect the interaction with MMPs through effects on local dynamics (as well as structure). Binding to different MMPs may also be affected differentially by regions distant from the Cys1-Cys70 disulfide bond. The higher affinity of MMP-3 for the Gly2 mutant suggests that interactions outside of the S1′ pocket contribute more of the binding energy for the interaction with MMP-3 than with the other MMPs. The structure of the S1′ pocket of MMP-1 is narrower and less deep than those of other MMPs. Arginine 195, which replaces leucine in MMP-2 and MMP-3, projects into the S1′ pocket toward the catalytic Zn2+, resulting in a less deep more cationic pocket (31Stams T. Spurlino J.C. Smith D.L. Wahl R.C. Ho T.F. Qoronfleh M.W. Banks T.M. Rubin B. Nat. Struct. Biol. 1994; 1: 119-123Crossref PubMed Scopus (201) Google Scholar). The reduced depth may account for the preference of MMP-1 for valine and threonine over leucine, for example, and charge repulsion is also a likely factor in the unfavorable binding of positively charged substituents to MMP-1. The specificity pocket of an MMP with no bound substrate or inhibitor will be expected to contain multiple solvent molecules so that the binding of TIMP mutants will be affected by the ability of the residue 2 side chain to interact with or displace solvent molecules. Large areas of molecular surface become buried in the formation of high affinity heterologous protein-protein complexes (32Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2254) Google Scholar). It has been estimated that approximately 1300 Å2 of the accessible surface of each component is buried on formation of the TIMP-1·MMP-3 complex (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). The C-terminal domain of TIMP-1 accounts for few of these contacts and less than 10% of the free energy of interaction (11Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar). The present results show that a single residue of the reactive site of N-TIMP-1, Thr2, exerts a large influence on the strength and specificity of binding to MMPs. The magnitude of this effect is surprising because, even if the environment of Thr2 changed from being totally exposed to 100% buried on complex formation with the protease, it accounts for less than 8% (102/1300 Å (2Nagase H. Hooper N.M. Zinc Metalloproteinases in Health and Disease. Taylor & Francis Ltd., London1996: 153-204Google Scholar) of the area of the protein−protein interaction site (33Miller S. Janin J. Lesk A.M. Chothia C. J. Mol. Biol. 1987; 196: 641-656Crossref PubMed Scopus (826) Google Scholar). The loss of 33–55% of the free energy of binding on removal of the Thr2 side chain by the substitution of glycine indicates that residue 2 of TIMP-1 could be designated a "hot spot" in the TIMP/MMP interaction interface, a residue that has a uniquely large influence on the strength of the protein-protein interaction (34Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1772) Google Scholar, 35Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1-6Crossref PubMed Scopus (359) Google Scholar). Many other residues form contacts with MMP-3 in the crystal structure (18Gomis-Rüth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar), but mutagenesis of others, particularly Met66 and Val69, and the removal of all C-domain contacts in N-TIMP-1 indicates that these residues are less important for binding than Thr2 (24Huang W. Meng Q. Suzuki K. Nagase H. Brew K. J. Biol. Chem. 1997; 272: 22086-22091Crossref PubMed Scopus (90) Google Scholar). Thus, the present results show that the nature of the contacts in the structure of the TIMP-1·MMP-3 complex are an important step toward understanding the structural basis of the inhibitory strategy and specificity in TIMP, but it is important to experimentally evaluate their contributions to the stability of the complex. We thank Dr. Per Nissen, Dept. of Biology and Nature Conservation, Agricultural University of Norway, for drawing our attention to the usefulness of log−log plots for identifying mutations that affect TIMP specificity.
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