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Identification of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Binding Site on the Hemopexin Carboxyl Domain of Human Gelatinase A by Site-directed Mutagenesis

1999; Elsevier BV; Volume: 274; Issue: 7 Linguagem: Inglês

10.1074/jbc.274.7.4421

1083-351X

Christopher M. Overall, Angela E. King, Douglas K. Sam, Aldrich Ong, Tim T Y Lau, U. Margaretha Wallon, Yves A. DeClerck, Juliet Atherstone,

Peptidase Inhibition and Analysis

Cell surface activation of progelatinase A occurs in a quaternary complex with the tissue inhibitor of metalloproteinases-2 (TIMP-2) and two membrane-type matrix metalloproteinases. We have mutated the unique cationic clusters found in hemopexin modules III and IV of the carboxyl domain (C domain) of human gelatinase A to determine their role in binding TIMP-2. Twelve single, double, and triple site-directed mutations were produced that exhibited different TIMP-2 binding properties. Notably, single alanine substitutions at Lys547 and Lys617reduced TIMP-2 binding by an order of magnitude from that of the recombinant wild-type C domain. Mutations that completely disrupted the C domain·TIMP-2 interaction were K558A/R561A, K610T/K617A, and K566A/K568A/K617A. A triple mutation, K566A/K568A/K575A, having TIMP-2 binding indistinguishable from the wild-type C domain (K d 3.0 × 10−8m), showed that simple reduction of net positive charge does not reduce TIMP-2 affinity. Because the double mutation K566A/K568A also did not alter TIMP-2 binding, these data do not confirm previously reported chimera studies that indicated the importance of the triple lysine cluster at positions 566/567/568 in TIMP-2 binding. Nonetheless, a subtle role in TIMP-2 interaction for the 566/567/568-lysine triad is indicated from the enhanced reduction in TIMP-2 binding that occurs when mutations here were combined with K617A. Thus, these analyses indicate that the TIMP-2 binding surface lies at the junction of hemopexin modules III and IV on the peripheral rim of the gelatinase A C domain. This location implies that considerable molecular movement of the TIMP-2·C domain complex would be needed for the bound TIMP-2 to inhibit in cis the gelatinase A active site. Cell surface activation of progelatinase A occurs in a quaternary complex with the tissue inhibitor of metalloproteinases-2 (TIMP-2) and two membrane-type matrix metalloproteinases. We have mutated the unique cationic clusters found in hemopexin modules III and IV of the carboxyl domain (C domain) of human gelatinase A to determine their role in binding TIMP-2. Twelve single, double, and triple site-directed mutations were produced that exhibited different TIMP-2 binding properties. Notably, single alanine substitutions at Lys547 and Lys617reduced TIMP-2 binding by an order of magnitude from that of the recombinant wild-type C domain. Mutations that completely disrupted the C domain·TIMP-2 interaction were K558A/R561A, K610T/K617A, and K566A/K568A/K617A. A triple mutation, K566A/K568A/K575A, having TIMP-2 binding indistinguishable from the wild-type C domain (K d 3.0 × 10−8m), showed that simple reduction of net positive charge does not reduce TIMP-2 affinity. Because the double mutation K566A/K568A also did not alter TIMP-2 binding, these data do not confirm previously reported chimera studies that indicated the importance of the triple lysine cluster at positions 566/567/568 in TIMP-2 binding. Nonetheless, a subtle role in TIMP-2 interaction for the 566/567/568-lysine triad is indicated from the enhanced reduction in TIMP-2 binding that occurs when mutations here were combined with K617A. Thus, these analyses indicate that the TIMP-2 binding surface lies at the junction of hemopexin modules III and IV on the peripheral rim of the gelatinase A C domain. This location implies that considerable molecular movement of the TIMP-2·C domain complex would be needed for the bound TIMP-2 to inhibit in cis the gelatinase A active site. Connective tissue remodeling is important for growth, healing, and functional adaptation of tissues. In these processes, activation of matrix metalloproteinase (MMP) 1The abbreviations used are: MMP, matrix metalloproteinase; C domain, carboxyl-terminal domain; MT, membrane-type; PBS, phosphate-buffered saline; TIMP, tissue inhibitor of metalloproteinases.1The abbreviations used are: MMP, matrix metalloproteinase; C domain, carboxyl-terminal domain; MT, membrane-type; PBS, phosphate-buffered saline; TIMP, tissue inhibitor of metalloproteinases. zymogens is a key control step in the degradation of extracellular matrix proteins (reviewed in Refs. 1Overall C.M. Trends Glycosci. Glycotechnol. 1991; 3: 384-399Crossref Scopus (11) Google Scholar and 2Matrisian L.M. BioEssays. 1992; 14: 455-463Crossref PubMed Scopus (1324) Google Scholar). Gelatinase A (EC 3.4.24.24) (MMP-2) is a pivotal MMP in the remodeling of basement membrane, pericellular, and cell attachment proteins. Cellular activation (3Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar, 4Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J.P. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar) and other cell membrane binding properties (5Brooks P.C. Stromblad S. Sanders L.C. vonSchalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Quigley J.P. Cheresh D.A. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1420) Google Scholar, 6Steffensen B. Bigg H.F. Overall C.M. J. Biol. Chem. 1998; 273: 20622-20628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) of gelatinase A are central to the regulation and function of this enzyme (reviewed in Ref. 7Hewitt R.E. Corcoran M.L. Stetler-Stevenson W.G. Trends Glycosci. Glycotechnol. 1996; 8: 23-36Crossref Scopus (8) Google Scholar). The tissue inhibitors of metalloproteinases (TIMPs) form essentially irreversible 1:1 molar inhibitory complexes with active MMPs (8Cawston T.E. Galloway A. Mercer E. Murphy G. Reynolds J.J. Biochem. J. 1981; 195: 159-165Crossref PubMed Scopus (263) Google Scholar, 9Stricklin G.P. Welgus H.G. J. Biol. Chem. 1983; 258: 12252-12258Abstract Full Text PDF PubMed Google Scholar) and so also control MMP activity. Inhibition includes critical interactions between the MMP catalytic Zn2+ ion and Cys1 of the TIMP inhibitory NH2-domain (10Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (111) Google Scholar, 11Gomis-Ruth F.-X. Maskos K. Betz M. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Pourenkov G.P. Bartunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (508) Google Scholar). However, outside the active site TIMP-2 (12Stetler-Stevenson W.G. Krutzsch H.C. Liotta L.A. J. Biol. Chem. 1989; 264: 17374-17378Abstract Full Text PDF PubMed Google Scholar, 13Goldberg G.I. Marmer B.L. Grant G.A. Eisen A.Z. Wilhelm S. He C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8207-8211Crossref PubMed Scopus (543) Google Scholar), TIMP-3, 2C. M. Overall, H. Tschesche, and A. King, unpublished observations. 2C. M. Overall, H. Tschesche, and A. King, unpublished observations. and TIMP-4 (14Bigg 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) also bind progelatinase A in a 1:1 molar complex on the hemopexin carboxyl-terminal domain (C domain) of the enzyme. That is, active gelatinase A can simultaneously bind two molecules of TIMP-2: one at the active site, to inhibit the enzyme, and another on the C domain. Similarly, TIMP-1 binds the progelatinase B C domain (15Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar), but two molecules of TIMP-1 cannot bind gelatinase A (16Howard E.W. Banda M.J. J. Biol. Chem. 1991; 266: 17972-17977Abstract Full Text PDF PubMed Google Scholar).We initially demonstrated that Con A induced the endogenous cellular activation of progelatinase A (3Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar). Paradoxically, the progelatinase A C domain and TIMP-2 carboxyl domain interaction appears essential for enzyme activation on the cell membrane in this process (4Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J.P. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 17Murphy G. Willenbrock F. Ward R.V. Cockett M.I. Eaton D. Docherty A.J.P. Biochem. J. 1992; 283: 637-641Crossref PubMed Scopus (245) Google Scholar, 18Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). Upon binding TIMP-2, a trimolecular complex of progelatinase A first forms with an activator proteinase, membrane type (MT)-MMP (18Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar, 19Sato H. Takino T. Okado Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 20Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). In this interaction, the ihibitory NH2 domain of TIMP-2 binds and inhibits the MT-MMP active site (20Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar, 21Will 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, 22Butler G.S. Butler M.J. Atkinson S.J. Will H. Tamura T. van Westrum S. Crabbe T. Clements J. d'Ortho M.-P. Murphy G. J. Biol. Chem. 1998; 273: 871-880Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 23Zucker S. Drews M. Conner C. Foda H.D. DeClerck Y.A. Langley K.E. Bahou W.F. Docherty A. Cao J. J. Biol. Chem. 1998; 273: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). We (24Overall C.M. Wallon U.M. Steffensen B. DeClerck Y. Tschesche H. Abbey R.S. Edwards D. Hawkes S. Kokha R. Inhibitors of Metalloproteinases in Development and Disease. Harwood Academic Publishing, Lausanne, Switzerland1999Google Scholar) also have proposed that a quaternary activation complex then forms with a second MT-MMP that cleaves the prodomain of progelatinase A at Asn37-Leu38. The domain binding interactions of these proteins and their roles in the activation complex are not yet fully resolved. However, deletions and use of isolated domains have shown that TIMP-2 binds progelatinase A via the C domain of both the enzyme (14Bigg 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, 16Howard E.W. Banda M.J. J. Biol. Chem. 1991; 266: 17972-17977Abstract Full Text PDF PubMed Google Scholar, 17Murphy G. Willenbrock F. Ward R.V. Cockett M.I. Eaton D. Docherty A.J.P. Biochem. J. 1992; 283: 637-641Crossref PubMed Scopus (245) Google Scholar, 20Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar, 25Friedman R. Fuerst T.R. Bird R.E. Hoyhtya M. Oelkuct M. Kraus S. Komarek D. Liotta L.A. Berman M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1992; 267: 15398-15405PubMed Google Scholar, 26Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (216) Google Scholar) and the inhibitor (26Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (216) Google Scholar,27Overall C.M. King A.E. Bigg H.F. McQuibban G.A. Atherstone J. Sam D.K. Ong A.D. Lau T.T.Y. Wallon U.M. DeClerck Y.A. Tam E. Ann. N. Y. Acad. Sci. 1999; (in press)PubMed Google Scholar). 3C. M. Overall, U. M. Wallon, G. A. McQuibban, E. Tam, H. F. Bigg, C. J. Morrison, Y. DeClerck, and A. E. King, manuscript in preparation. 3C. M. Overall, U. M. Wallon, G. A. McQuibban, E. Tam, H. F. Bigg, C. J. Morrison, Y. DeClerck, and A. E. King, manuscript in preparation. Nonetheless, the localization of the respective contact surfaces on these two protein domains and the important molecular determinants of the binding sites have yet to be identified. In this regard, Willenbrock et al. (26Willenbrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (216) Google Scholar) proposed that the highly charged anionic peptide extension (186QEFLDIEDP194) present at the carboxyl terminus of TIMP-2, but not of TIMP-1, mediates binding to the gelatinase A C domain. Of note, TIMP-4 also contains a similar sequence (187KEFVDIVQP195) (28Greene J. Wang M. Liu Y.E. Raymond LA. Rosen C. Shi Y.E. J. Biol. Chem. 1996; 271: 30375-30380Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) and binds to the gelatinase A C domain (14Bigg 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). Thus, these carboxyl-terminal tails may be crucial in forming the gelatinase A binding site on these TIMPs. In addition, an alternate interaction of the gelatinase A C domain with αvβ3 integrin has been described (5Brooks P.C. Stromblad S. Sanders L.C. vonSchalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Quigley J.P. Cheresh D.A. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1420) Google Scholar) but not confirmed (22Butler G.S. Butler M.J. Atkinson S.J. Will H. Tamura T. van Westrum S. Crabbe T. Clements J. d'Ortho M.-P. Murphy G. J. Biol. Chem. 1998; 273: 871-880Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar), and a lower affinity interaction also occurs between the NH2-domain of TIMP-2, but not that of TIMP-1 or TIMP-3, and the gelatinase A C domain (24Overall C.M. Wallon U.M. Steffensen B. DeClerck Y. Tschesche H. Abbey R.S. Edwards D. Hawkes S. Kokha R. Inhibitors of Metalloproteinases in Development and Disease. Harwood Academic Publishing, Lausanne, Switzerland1999Google Scholar, 27Overall C.M. King A.E. Bigg H.F. McQuibban G.A. Atherstone J. Sam D.K. Ong A.D. Lau T.T.Y. Wallon U.M. DeClerck Y.A. Tam E. Ann. N. Y. Acad. Sci. 1999; (in press)PubMed Google Scholar).3 Finally, the MMP-independent growth factor effects of TIMP-2 (29Corcoran M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1995; 270: 13453-13459Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) have been ascribed to the C domain of the inhibitor. These may be masked upon binding the gelatinase A C domain. Thus, locating the TIMP-2 binding site on the gelatinase A C domain and generating mutations that disrupt this interaction will be invaluable in dissecting the mechanistic aspects and relative importance of the different cell membrane binding and activation mechanisms proposed for progelatinase A. These mutant proteins will also be useful in assessing the consequences of TIMP-2 binding to the gelatinase A C domain on the growth factor properties ascribed to TIMP-2.The overall shape of the gelatinase A C domain is a squat cylinder composed of four β-sheets, each representing a hemopexin module (I–IV) and each forming a blade of the four bladed β-propeller structure (30Libson A.M. Gittis A.G. Collier I.E. Marmer B.L. Goldberg G.I. Lattman E.E. Nat. Struct. Biol. 1995; 2: 938-942Crossref PubMed Scopus (64) Google Scholar, 31Gohlke U. Gomis-Rüth F.-Z. Crabbe T. Murphy G. Docherty A.J.P. Bode W. FEBS Lett. 1996; 378: 126-130Crossref PubMed Scopus (81) Google Scholar). Each β-sheet is formed from four antiparallel β-strands. Analysis of these three-dimensional models has revealed cationic clusters in hemopexin module III as a striking feature. These largely result from contiguous stretches of lysine residues, Lys547-Asn-Lys-Lys550 and Lys566-Lys-Lys568, located near the antiparallel β-strand turns at the rim and on the upper surface of the domain. Notably, Lys550, Lys566, and Lys567 from these sequences also contribute to three consecutive BX 7B repeats (B = basic residue, X = undefined): [Lys550–Lys558], [Lys558–Lys566], and [Lys567–Lys575]. A fourth BX 7B motif [Lys617-Lys625] is found in module IV. BX 7B motifs have been implicated in hyaluronan binding in several proteins (32Yang B. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (334) Google Scholar). Although the gelatinase A C domain binds heparin (33Crabbe T. Ioannou C. Docherty A.J. Eur. J. Biochem. 1993; 218: 431-438Crossref PubMed Scopus (89) Google Scholar, 34Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), which enhances progelatinase A activation (22Butler G.S. Butler M.J. Atkinson S.J. Will H. Tamura T. van Westrum S. Crabbe T. Clements J. d'Ortho M.-P. Murphy G. J. Biol. Chem. 1998; 273: 871-880Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar,33Crabbe T. Ioannou C. Docherty A.J. Eur. J. Biochem. 1993; 218: 431-438Crossref PubMed Scopus (89) Google Scholar), it does not bind hyaluronan. 4C. R. Roberts and C. M. Overall, unpublished data. 4C. R. Roberts and C. M. Overall, unpublished data. However, these cationic clusters and BX 7B repeats may be important in binding TIMP-2, possibly interacting through salt bridge formation (24Overall C.M. Wallon U.M. Steffensen B. DeClerck Y. Tschesche H. Abbey R.S. Edwards D. Hawkes S. Kokha R. Inhibitors of Metalloproteinases in Development and Disease. Harwood Academic Publishing, Lausanne, Switzerland1999Google Scholar). This hypothesis has been tested in the present report.In the absence of a three-dimensional structure of the TIMP-2·gelatinase A complex, we adopted a mutagenesis approach to identify the TIMP-2 binding site on the gelatinase A C domain. Rather than individually mutate all 36 basic residues in the C domain, we devised a strategy of using multiple replacements to screen more efficiently for important amino acid sites. Further, these substitutions were only made in the third and fourth hemopexin modules, which contain the unique cationic clusters not found in other MMPs—in particular gelatinase B, which binds TIMP-1 but not TIMP-2. We reasoned that multiple substitutions should also lessen the risk of missing weaker binding residues that as single mutations may have their effects masked by the overall binding energy of the TIMP-2·C domain complex. Reported here are mutations that establish an important role in TIMP-2 binding for several basic residues in the gelatinase A C domain. Analysis of the effects of these mutations and their location on the three-dimensional structure of the C domain has also enabled us to define the approximate boundary of the TIMP-2 binding site. Thus, TIMP-2 binds the upper surface of the human gelatinase A C domain, on its outer rim at the junction of hemopexin modules III and IV. This location has a number of ramifications concerning the proposed actions of the bound TIMP-2.RESULTSTo test the hypothesis that cationic residues in the gelatinase A C domain bind TIMP-2, possibly at the anionic terminal tail, multiple sequence alignment analysis was first used to devise a rational mutagenesis strategy. As shown in Fig. 2, 15 of the 26 basic residues in the C domain are grouped in hemopexin modules III and IV. Comparing the distribution of cationic residues in the C domains of gelatinases A and B, there are more nonconserved basic residues in modules III and IV than in I and II. Therefore, we predicted that the cationic sequences in hemopexin modules III and IV were more likely to be involved in binding TIMP-2. We first focused on the markedly cationic module III, which contains 10 basic residues compared with 5 in module IV, and designed mutations to disrupt the lysine-rich sequences Lys547-Asn-Lys-Lys550 and Lys566-Lys-Lys568. Three point mutations were made to assess the TIMP-2 binding properties of the individual lysines in the Lys547-Asn-Lys-Lys550 sequence. A double mutation (K566A/K568A) was also used to test the effect of charge reduction in the Lys566-Lys-Lys568 triad.Fermenter conditions for each mutant protein were optimized. Expression conditions were mutant protein-specific, but in general, oxygen-limited conditions (5% aeration) proved optimal for the expression of the recombinant gelatinase A C domain and mutants thereof. The mass (Da) measured by electrospray mass spectrometry and the Δ mass from that predicted for the wild-type C domain protein and from the mass of the first round of mutant proteins expressed is shown in TableII. These data confirmed the homogeneity of the protein preparations, that NH2-terminal methionine processing occurred in the recombinant proteins, and that the correct amino acid substitution had been translated.Table IIMolecular mass determination of wild-type and mutant gelatinase A C domain proteins by electrospray mass spectrometryProteinMassaThe masses were measured after sample injection onto a PESCIEX API 300 mass spectrometer from a C18 high pressure liquid chromatography column.Δ Mass from that predicted minus the N-terminal methioninebThe measured mass change from the predicted mass of the protein after N-terminal methionine processing. From the amino acid sequence, the predicted mass of the N-terminal methionine processed recombinant C domain is 25,925 Da. The predicted decrease in mass by a lysine to alanine substitution is 57 Da.DaWild-type C domain25,924−1K547A25,871+3K549A25,869+1K550A25,866−2K566A/K568A25,8110The molecular masses of the wild-type C domain and the four lysine mutants generated in the first round of mutagenesis were measured by electrospray mass spectrometry. The typical error for these mass determinations is 1 in 104 Da. Because of the close agreement between the measured masses with those predicted, this analysis was not carried out on proteins generated in mutagenesis rounds two and three.a The masses were measured after sample injection onto a PESCIEX API 300 mass spectrometer from a C18 high pressure liquid chromatography column.b The measured mass change from the predicted mass of the protein after N-terminal methionine processing. From the amino acid sequence, the predicted mass of the N-terminal methionine processed recombinant C domain is 25,925 Da. The predicted decrease in mass by a lysine to alanine substitution is 57 Da. Open table in a new tab Mutagenic Analysis of the TIMP-2 Binding Properties of the Lys547, Lys549, and Lys550ClusterA differential role in TIMP-2 binding was observed for alanine-substituted proteins at positions 547, 549, and 550 on the upper surface of hemopexin module III (TableIII). An alanine mutation at Lys547 raised the apparent K d of TIMP-2 interaction by approximately 1 order of magnitude (apparentK d 1.7 × 10−7m) and reduced the amount of TIMP-2 bound by ∼50% at saturation (Fig.3 A). Mutation of Lys549 or Lys550 to alanine did not significantly alter the apparent K d values of TIMP-2 interaction (7.8 × 10−8m (Fig.3 A) and 4.8 × 10−8m (Fig.3 B), respectively) from that of the wild-type domain (3.0 × 10−8m). TIMP-2 binding and elution from these mutant proteins on affinity chromatography columns was also no different from the wild-type C domain (not shown). Because a cooperative effect on TIMP-2 binding by residues in this cluster was possible, we then made the triple alanine mutation (K547A/K549A/K550A). However, TIMP-2 binding by the K547A/K549A/K550A protein (apparentK d, 2.6 × 10−7m) was essentially no different from that of the K547A mutant (Fig.3 A). This indicated that cooperative binding by these three residues does not occur and that of these residues, Lys547plays the most important binding role.Table IIITIMP-2 binding constants for gelatinase A C domain mutant proteinsProteinApparentK dmWild-type C domain3.0 × 10−8K547A1.7 × 10−7K549A7.8 × 10−8K550A4.8 × 10−8K547A/K549A/K550A2.6 × 10−7K566A/K568A4.7 × 10−8K550A/K566A/K568A1.8 × 10−7K575A5.2 × 10−8K566A/K568A/K575A7.5 × 10−8K558A/R561AUndetectableK617A1.1 × 10−7K566A/K568A/K617AUndetectableaBinding of TIMP-2 to the mutant protein indicated was not detectable by the solid phase assay, as described under “Experimental Procedures.”K610T/K617AUndetectablea Binding of TIMP-2 to the mutant protein indicated was not detectable by the solid phase assay, as described under “Experimental Procedures.” Open table in a new tab Figure 3Binding of wild-type and mutant C domain proteins to TIMP-2. C domain and mutant proteins (5.0 × 10−6 1.0 × 10−10m) were added to TIMP-2 coated in 96-well plates (0.2 μg/well) and binding quantitated as described under “Experimental Procedures.” Only data from experiments conducted on the same plate are presented in eachpanel, with each panel(A–D) showing different representative plates. Mutant proteins analyzed are as indicated. Data points in each panel are means of replicate samples from the same plate and representative of four separate experiments. Myoglobin served as a negative control protein.View Large Image Figure ViewerDownload (PPT)The enigmatic role of Lys566-Lys567-Lys568 in TIMP-2 BindingA striking feature of the C domain sequence is the unique cationic triad Lys566-Lys-Lys568 on hemopexin module III (see Figs. 1 and 2). This triad also contributes to two BX 7B motifs: [Lys558–Lys566] and [Lys567–Lys575] (see Table I). Rather than initially mutate each of the three residues separately, we first screened for the TIMP-2 binding properties of this cluster as a whole by charge reduction using a double alanine mutation. Sites 566 and 568 were selected, as these were nearer than Lys567 to the other cationic residues in module III (see Fig. 1). The mutant K566A/K568A protein showed TIMP-2 binding properties (apparentK d 4.7 × 10−8m) (Fig. 3 C) that were essentially identical to those of the wild-type C domain. Moreover, an affinity purified antibody raised against a peptide (NH2-RYNEVKKKMDPG-COOH) encompassing the lysine triad did not interfere with TIMP-2 binding to the wild-type C domain immobilized on a Zn2+-chelate affinity column (Fig.4 A). Finally, chromatography of TIMP-2 over a K566A/K568A protein affinity column revealed strong TIMP-2 binding to the mutant domain. Like the wild-type C domain (Fig.4 A), this interaction was not disrupted by 1.0 mNaCl or 10% Me2SO—the bound TIMP-2 and K566A/K568A mutant domain complex were eluted in 50 mm EDTA (not shown). We have previously shown that fibronectin and heparin binding by the gelatinase A C domain is Ca2+ ion-dependent, being disrupted by divalent cation chelators (34Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In microwell plate assays, the C domain was not dissociated from TIMP-2 by 50 mm EDTA (not shown). This indicates that the EDTA elution of C domain with TIMP-2 from the affinity columns was primarily due to chelation and removal of the ligating Zn2+ ion from the metal chelate resin. Thus, the C domain and TIMP-2 in the 50 mm EDTA eluates represent the bound complex. Because the K566A/K568A double mutation did not alter TIMP-2 binding, these two residues were not individually substituted nor was Lys567mutated.Figure 4Elution profiles of TIMP-2 chromatographed over wild-type and mutant C domain protein affinity columns. C domain (A) (260 μg) and the mutant proteins indicated (B and C) (100 μg) were bound to Zn2+- or Ni2+-chelate minicolumns as described under “Experimental Procedures.” U, unbound fraction during loading. After extensive washes with chromatography buffer (W), affinity-purified antipeptide antibodyα72ex12 was loaded and incubated on the column (A). The antibody bands in the 40–60-kDa range and atthe top of the gel (labeled) could be readily seen on elution with the bound complexes in 50 mm EDTA. TIMP-2 was applied to the affinity columns after washes (W). The unbound (TIMP-2) and PBS wash fractions were collected and analyzed on 15% SDS-polyacrylamide gel electrophoresis gels (W in A; PBS wash fractions1–4 in B). Sequential elution with 1.0m NaCl, 10% Me2SO (DMSO), and 50 mm EDTA followed as shown. TIMP-2 did not bind to the zinc chelate resin (not shown). M r, molecular mass marker proteins in Da as indicated; Std, recombinant C domain (rC) and TIMP-2 (T2) before chromatography. For some recombinant proteins, a lower molecular mass form without the His6 NH2-terminal tag is an occasional minor component of the preparation after freeze-thawing (see rC Std in A).View Large Image Figure ViewerDownload (PPT)Of the basic residues in the Lys547-Asn-Lys-Lys550 sequence, Lys550 is topographically closest to the Lys566-Lys-Lys568 triad (see Fig. 1). Therefore, to determine the effects of simultaneous mutations in these two sequences, we made the triple mutation K550A/K566A/K568A. In addition, this selection would also disrupt both the [Lys550–Lys558] and [Lys558–Lys566] BX 7B repeats (Table I). In this regard, Lys550, rather than Lys558, was also the more rational selection because Lys558 is on the underside of the domain. We considered this surface a less likely TIMP-2–binding region candidate given that 7 of the 10 basic residues in hemopexin module III are on the upper surface. As already discussed, the K550A and K566A/K568A mutations alone showed little alteration in apparent K d values for TIMP-2 compared with the wild-type C domain. However, as a triple mutation (K550A/K566A/K568A), a reduction in binding by approximately 1 order of magnitude resulted (apparent K d, 1.8 ×