Portal de Periódicos da CAPES

Unveiling the Surface Epitopes That Render Tissue Inhibitor of Metalloproteinase-1 Inactive against Membrane Type 1-Matrix Metalloproteinase

2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês

10.1074/jbc.m305678200

1083-351X

Meng-Huee Lee, Magdalini Rapti, Gillian Murphy,

Blood Coagulation and Thrombosis Mechanisms

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a zinc-dependent, membrane-associated endoproteinase of the metzincin family. The enzyme regulates extracellular matrix remodeling and is capable of cleaving a wide variety of transmembrane proteins. The enzymatic activity of MT1-MMP is regulated by endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMP). To date, four variants of mammalian TIMP have been identified. Whereas TIMP-2-4 are potent inhibitors against MT1-MMP, TIMP-1 displays negligible inhibitory activity against the enzyme. The rationale for such selectivity is hitherto unknown. Here we identify the surface epitopes that render TIMP-1 inactive against MT1-MMP. We show that TIMP-1 can be transformed into an active inhibitor against MT1-MMP by the mutation of a single residue, namely threonine 98 to leucine (T98L). The resultant mutant displayed inhibitory characteristics of a typical slow, tight binding inhibitor. The potency of the mutant could be further enhanced by the introduction of valine 4 to alanine (V4A) and proline 6 to valine (P6V) mutations. Indeed, the inhibitory profile of the triple mutant (V4A/P6V/T98L) is indistinguishable from those of other TIMPs. Our findings suggest that threonine 98 is critical in initiating MMP binding and complex stabilization. Our findings also provide a potential mechanistic explanation for MMP-TIMP selectivity. Membrane type 1-matrix metalloproteinase (MT1-MMP) is a zinc-dependent, membrane-associated endoproteinase of the metzincin family. The enzyme regulates extracellular matrix remodeling and is capable of cleaving a wide variety of transmembrane proteins. The enzymatic activity of MT1-MMP is regulated by endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMP). To date, four variants of mammalian TIMP have been identified. Whereas TIMP-2-4 are potent inhibitors against MT1-MMP, TIMP-1 displays negligible inhibitory activity against the enzyme. The rationale for such selectivity is hitherto unknown. Here we identify the surface epitopes that render TIMP-1 inactive against MT1-MMP. We show that TIMP-1 can be transformed into an active inhibitor against MT1-MMP by the mutation of a single residue, namely threonine 98 to leucine (T98L). The resultant mutant displayed inhibitory characteristics of a typical slow, tight binding inhibitor. The potency of the mutant could be further enhanced by the introduction of valine 4 to alanine (V4A) and proline 6 to valine (P6V) mutations. Indeed, the inhibitory profile of the triple mutant (V4A/P6V/T98L) is indistinguishable from those of other TIMPs. Our findings suggest that threonine 98 is critical in initiating MMP binding and complex stabilization. Our findings also provide a potential mechanistic explanation for MMP-TIMP selectivity. Membrane type 1-matrix metalloproteinase (MT1-MMP, 1The abbreviations used are: MT1-MMP, membrane type-1 matrix metalloproteinase; ADAM, a disintegrin and metalloproteinase; ADAM-TS, ADAM with thrombospondin-like repeats; MMPs, matrix metalloproteinases; N-TIMP, N-terminal domain form of TIMP; TIMP, tissue inhibitor of metalloproteinases. MMP-14) is a member of the zinc-dependent endopeptidases of the metzincin family. The enzyme is involved in the degradation of extracellular matrix components and tissue remodeling (1Galvez B.G. Matias-Roman S. Albar J.P. Sanchez-Madrid F. Arroyo A.G. J. Biol. Chem. 2001; 276: 37491-37500Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 2Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5134) Google Scholar, 3Brinckerhoff C.E. Matrisian L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 207-214Crossref PubMed Scopus (969) Google Scholar). Among the six membrane-type MMPs identified, MT1-MMP is the most studied not only because of its role in activating other MMPs such as pro-MMP-2 (pro-gelatinase-A) and pro-MMP-13 (4Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2373) Google Scholar, 5Knäuper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar), but also because of its compelling link with cell invasion and tumor malignancy (6Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (363) Google Scholar). As with other members of the matrixin family, MT1-MMP is a multidomain enzyme with clear structural compartmentation. Preceding the catalytic domain is a propeptide that contains a cysteine switch that regulates the catalytic activity of the enzyme. The catalytic domain displays a tertiary fold typical of an enzyme of the metzincin family (7Bode W. Maskos K. Methods Mol. Biol. 2001; 151: 45-77PubMed Google Scholar). Succeeding the catalytic domain are a short hinge, a hemopexin domain, a transmembrane domain, and a cytotail of 2.5 kDa. A crystal structure of the catalytic domain of MT1-MMP was first reported by Fernandez-Catalan et al. in 1998 (8Fernandez-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 (311) Google Scholar). To date, this is the only known variant of membrane-associated MMP to be crystallized and published. The activity of MMPs is regulated by their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMP). There are four mammalian TIMPs, namely TIMP-1-4. TIMP inhibit MT1-MMP by forming a tight, non-covalent 1:1 stoichiometric complex with the catalytic domain of the enzyme. The binding constants (K iapp) of wild type TIMP-2-4 with MT1-MMP are in the low picomolar range (9English W.R. Puente X.S. Freije J.M. Knäuper V. Amour A. Merryweather A. Lopez-Otin C. Murphy G. J. Biol. Chem. 2000; 275: 14046-14055Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Although TIMP-2-4 are superb inhibitors against MT1-MMP, TIMP-1, on the contrary, is not capable of forming a tight binding complex with MT1-MMP. The molecular masses of TIMP vary from 21 to 28 kDa. Crystallography and protein NMR studies carried out on TIMP-1 and -2 revealed that the molecules are folded into two very discrete domains (10Williamson R.A. Martorell G. Carr M.D. Murphy G. Docherty A.J. Freedman R.B. Feeney J. Biochemistry. 1994; 33: 11745-11759Crossref PubMed Scopus (95) Google Scholar, 11Gomis-Rüth F. 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 (513) Google Scholar, 12Tuuttila A. Morgunova E. Bergmann U. Lindqvist Y. Maskos K. Fernandez-Catalan C. Bode W. Tryggvason K. Schneider G. J. Mol. Biol. 1998; 284: 1133-1140Crossref PubMed Scopus (81) Google Scholar). The N-terminal domain of the TIMPs (N-TIMP) is ∼14 kDa and encompasses the first two-thirds of the molecule. The architecture of N-TIMP is typical of a β-barrel scaffold, reminiscent of the oligosaccharide/oligonucleotide-binding (OB) motif. The C-terminal domain, on the other hand, consists mainly of β-sheets and is structurally less well defined. There are three disulfide bonds in each domain of TIMP. Mutagenesis and kinetic analysis on the full-length and N-terminal forms of TIMP-1-3 showed that the MMP-inhibitory function of TIMP resides exclusively at the N-terminal domain (13Huang W. Suzuki K. Nagase H. Arumugam S. Van Doren S.R. Brew K. FEBS Lett. 1996; 384: 155-161Crossref PubMed Scopus (97) Google Scholar, 14Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (285) Google Scholar, 15Nguyen Q. Willenbrock F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar, 16Lee M.H. Knäuper V. Becherer J.D. Murphy G. Biochem. Biophys. Res. Commun. 2001; 280: 945-950Crossref PubMed Scopus (52) Google Scholar). Despite the fact that the co-crystal structure of MT1-MMP/TIMP-2 has been available for some time, hitherto there is no explanation as to why TIMP-1 is not capable of inhibiting MT1-MMP. Here we report a novel strategy adopted by our laboratory in approaching this conundrum. We succeeded in identifying the surface epitopes that render TIMP-1 inactive against MT1-MMP. By exchanging these “obstructive” epitopes with those of other TIMP, we created a N-TIMP-1 mutant that exhibited inhibitory potency indistinguishable from those of other TIMPs. Materials—All chemicals and reagents were purchased from Sigma unless otherwise stated. Restriction enzymes and Vent DNA polymerase for PCRs were obtained from New England Biolabs. The fluorometric substrate for the MT1-MMP assay (QF-24: Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) has been described in our previous papers (16Lee M.H. Knäuper V. Becherer J.D. Murphy G. Biochem. Biophys. Res. Commun. 2001; 280: 945-950Crossref PubMed Scopus (52) Google Scholar, 17Lee M.H. Verma V. Maskos K. Nath D. Knäuper V. Dodds P. Amour A. Murphy G. Biochem. J. 2002; 364: 227-234Crossref PubMed Scopus (40) Google Scholar). Kinetic assays were performed at 27 °C in fluorescence assay buffer (10 mm CaCl2, 50 mm Tris-HCl, pH 7.5, 0.05% Brij-35, 1% Me2SO, 0.02% NaN3) with a PerkinElmer Life Sciences 50B spectrofluorimeter equipped with thermostatic cuvette holders. MT1-MMP enzyme was expressed, refolded, and purified as described in our previous paper (18Will 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). Site-directed Mutagenesis of N-TIMP-1—TIMP-1 mutants in this work were created by PCR using Vent DNA polymerase. Mutations were incorporated into full-length and N-terminal domain forms of human TIMP-1 cDNAs (in pRSET-c Escherichia coli expression vector, Invitrogen) by either forward or reverse primers, depending on the locations. All constructs have been sequenced to confirm that no unwanted mutation had been introduced during the mutagenesis process. Protein Refolding and Assessment of Activity by Titration—N-TIMP-1 mutants were expressed in E. coli and refolded as described previously (19Lee M.H. Maskos K. Knäuper V. Dodds P. Murphy G. Protein Sci. 2002; 11: 2493-2503Crossref PubMed Scopus (28) Google Scholar). Full-length wild type and V4A/P6V/T98L mutant were refolded essentially as with N-TIMP-1 with the exception that 0.5 m arginine was included in the refolding buffer. The concentration and activity of active TIMP in each preparation were assessed and determined by titration against MMP-2 as reported in our previous paper (19Lee M.H. Maskos K. Knäuper V. Dodds P. Murphy G. Protein Sci. 2002; 11: 2493-2503Crossref PubMed Scopus (28) Google Scholar) on N-TIMP-1 engineering. With the exception of T98F, all N-TIMP-1 mutants retained full inhibitory activity against MMP-2, irrespective of their loci. Refolded full-length TIMP-1 and its V4A/P6V/T98L mutant were highly active against MMP-2, their association rates with MMP-2 (k on) being over 10 -8m-1 s-1. The amount of T98F was determined by Bio-Rad protein assay kit. Inhibition Constant Measurement (K iapp )—MT1-MMP (0.15 nm) was pre-incubated with increasing concentrations of N-TIMP-1 mutants, ranging from 0 to 500 nm, depending on the potency of the inhibitors. Incubation was allowed at room temperature for 3 h before steady state (V s) measurement. Quenched fluorescent peptide QF-24 was added to a final concentration of 1 μm to initiate the assays. Measurements of enzyme activities were performed at 27 °C throughout this work. All data were fitted into competitive tight binding equations with the computer program Grafit to obtain an estimation of K iapp values with Equation 1, Vs=(V0/2Et)×(Et-It-Kiapp)+(Kiapp+It-Et)2+4EtKiapp)1/2)(Eq. 1) where V 0 is the rate in the absence of inhibitor; E t is the total enzyme concentration; and It is the total inhibitor concentration. Association Rate Constant Measurement (k on )—k on measurements were performed by adding N-TIMP mutants (3-50 nm) to MT1-MMP enzyme (0.05 nm). The rate of inhibition was followed using a continuous fluorometric assay at 27 °C until steady state was reached. The progress curve was analyzed using Equation 2, P=Vst+(V0-Vs)(1-e-kt)/k(Eq. 2) where p is the product concentration; V 0 is the initial velocity; V s = steady state velocity; and k is the apparent first order rate constant of equilibrium between enzyme and TIMP complex. k on values were calculated by linear regression of k on TIMP concentrations. Strategy for TIMP-1 Mutagenesis—Although members of the TIMP family are well conserved in both primary and tertiary conformation, the inability of TIMP-1 to inhibit MT1-MMP has been a long standing conundrum for protein engineers interested in the mechanism of TIMP-MMPs selectivity (20Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (973) Google Scholar, 21Nagase H. Brew K. Arthritis Res. Ther. 2002; 4: 51-61Crossref Scopus (48) Google Scholar). First and foremost, there is no obvious contrast between the amino acid distribution of TIMP-1 and those of TIMP-2-4 that could account for its inability to inhibit MT1-MMP (Fig. 1). Hypothetical docking of MT1-MMP and TIMP-1 models in our laboratory also failed to reveal significant interfacial clashes that could satisfactorily explain the lack of ability of TIMP-1 to form tight binary complex with MT1-MMP. The most challenging aspect, in our view, is that although the structures of TIMP-1 and -2 and MT1-MMP have been delineated by protein NMR and crystallography for a considerable length of time, all the hypotheses regarding the inactivity of TIMP-1 against MT1-MMP remained hitherto speculative. Taken together, we propose that the lack of TIMP-1 activity against MT1-MMP is not due to simple incompatibility between the surface topology of the two molecules. Instead, we believe that TIMP-1 is not capable of inducing conformational changes in MT1-MMP, an isomerization process that needs to be overcome for tight enzyme-inhibitor (EI) complex formation (22Morrison J.F. Walsh C.T. Methods Enzymol. 1995; 248: 201-301Google Scholar). The reason for this, we hypothesize, is the presence of some “obstructive epitope(s)” that prevent TIMP-1 from initiating conformational changes with the enzyme. These obstructive epitope(s), in our view, must be rather subtle and are not readily identifiable by primary sequence alignment and structural examination. Hence, to approach this problem in a precise manner, we decided to confine our investigation to the N-terminal domain forms of TIMP (N-TIMP) and the catalytic domain form of MT1-MMP. Structural delineation of stromelysin-1·TIMP-1 and MT1-MMP·TIMP-2 complexes by x-ray crystallography showed that the TIMPs inhibit MMPs by inserting an “MMP-binding ridge” into the catalytic site grooves of the enzymes (8Fernandez-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 (311) Google Scholar, 11Gomis-Rüth F. 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 (513) Google Scholar). This MMP-binding ridge, by and large, is composed of the very N terminus, the AB-loop, the CD-loop and the EF-loop of the molecule (Fig. 2). In our opinion, the obstructive epitope(s) are more likely to be located at these loci rather than the OB-core of the molecule. Based on this hypotheses, we divided the MMP-binding ridge of TIMP-1 into five “divisions,” namely the “N terminus” division, the “Pro6” division, the “AB-loop” division, the “CD-loop” division, and finally the “EF-loop” division (Fig. 2). In turn, each of these divisions is sub-divided into independent entities termed “epitopes.” These epitopes could either be a single or multiple amino acids, depending on the locus. Systematically, residues constituting these epitopes were swapped with the corresponding amino acids of TIMP-2-4, and the kinetic profiles of the resultant mutants were monitored throughout the mutagenesis process. Kinetic Analysis of Wild type N-TIMP-1-4 against the Catalytic Domain of MT1-MMP—Before any mutagenesis work was carried out, we started by examining the binding affinities of wild type N-TIMP-1-4 with the catalytic domain of MT1-MMP. The binding constant (K iapp) and association rate (k on) are shown in Table I. Clearly, N-TIMP-2-4 have far superior affinities than N-TIMP-1, their K iapp values being in the range of 0.3 to 1.5 nm. In comparison, N-TIMP-1 is at least 2 orders of magnitude higher in value (K iapp 178 nm). In term of association rate, N-TIMP-2 is slightly superior to N-TIMP-3 and N-TIMP-4 (k on N-TIMP-2, 10 × 10-5m-1 s-1, and N-TIMP-3 and -4, 3-6 × 10-5m-1 s-1) (Table I). N-TIMP-1, on the other hand, is not capable of establishing tight binding complexes with MT1-MMP.Table IApparent inhibition constant (Kiapp) and association rate (kon) of MT1-MMP with wild type N-TIMP-1-4Wild typeKi app (× 10−9m)k on (× 10−5m−1s−1)N-TIMP-1178 ± 20NAN-TIMP-21.30 ± 0.1310.1 ± 3.8N-TIMP-31.38 ± 0.066.04 ± 0.70N-TIMP-40.30 ± 0.023.25 ± 0.35 Open table in a new tab N-terminal Mutants—The side chains of the second and the fourth residues of TIMP (also termed P1′ and P3′ subunits) are critically important in the determination of the selectivity profile of the inhibitor. The residues dock directly into the S1′ and S3′ catalytic pockets of MMPs. The second residue of TIMP-1 and -3 is threonine and that of TIMP-2 and -4 is serine (Fig. 1). Hence, we created only one mutant at the position of Thr2 (T2S). The fourth residue of TIMP-1 is valine. The corresponding residues in TIMP-2-4 are either serine or alanine (Fig. 1). Therefore, two mutants were created at this locus, namely V4S and V4A. The results of the mutagenesis are shown in Table II. Among the three mutants of the N terminus division (T2S, V4A, and V4S), V4A exhibited substantial improvement in binding affinity with MT1-MMP (K iapp 66 nm versus wild type N-TIMP-1 of 178 nm), closely followed by V4S (K iapp 81 nm). Nonetheless, none of the mutants was capable of potentiating tight complex formation with MT1-MMP.Table IIApparent inhibition constant (Kiapp) of MT1-MMP with the first generation N-TIMP-1 mutantsKi app (×10−9 M)N-terminal mutantsT2S220 ± 20V4A66 ± 6V4S81 ± 24Pro6 mutantsP6V78 ± 4P6S95 ± 5P6A165 ± 13AB-loop mutantsT2-AB-loop77 ± 6Long T2-AB-loop117 ± 18T3-AB-loop106 ± 7T4-AB-loop170 ± 13CD loop mutantsM66K462 ± 103M66D>500M66L237 ± 18M66I146 ± 10M66V183 ± 13M66A199 ± 20M66G254 ± 28V69L259 ± 30EF-loop mutantsT98L11.1 ± 1.6 Open table in a new tab Pro6 Mutants—Pro6 is located at the junction between the N terminus and the first α-helix loop of TIMP-1 (Fig. 2). TIMP-1 is unique as it is the only TIMP to have a proline at this locus. In TIMP-2-4, the corresponding residues are valine, serine, and alanine (Fig. 1). Substitution of Pro6 by a smaller residue might arguably release the constraints and bring about more relaxed local dynamics, although none of the crystal or NMR papers on TIMP published so far have emphasized the significance of this residue. Here we mutated Pro6 to valine (P6V), serine (P6S), and alanine (P6A) to mimic TIMP-2-4, respectively. Despite the 2-fold reduction in the K iapp value (K iapp 78-95 nm) for P6V and P6S mutants, there was no manifestation of tight binding inhibition. Surprisingly, replacement of Pro6 by alanine produced no significant changes in binding affinity with MT1-MMP. AB-loop Mutants—The next set of mutants centered around the AB-loop of TIMP-1 (Figs. 1 and 2). MT1-MMP contains a cavity at the far “left” edge of its molecule, a result of the strategic alignment between its MT-loop and the side chains of Asp212, Ser189, and Phe180 (8Fernandez-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 (311) Google Scholar). Crystal structure of MT1-MMP·TIMP-2 complex (Protein Data Bank code 1BUV) shows that this cavity functions as a receptacle for Tyr-36, a conspicuous residue at the tip of the AB-loop of TIMP-2. Although Williamson et al. (23Williamson R.A. Hutton M. Vogt G. Rapti M. Knäuper V. Carr M.D. Murphy G. J. Biol. Chem. 2001; 276: 32966-32970Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) did confirm that the Tyr-36 residue is crucial for TIMP-2 to establish tight binding complex with MT1-MMP, the findings did not explain why TIMP-3 and TIMP-4 are equally good inhibitors against MT1-MMP despite the complete lack of a tyrosine (or an amino acid of similar nature) at the tip of the AB-loop. Hence, the rationale of grafting the entire AB-loops of TIMP-2-4 onto TIMP-1 was not to find out if any of the residues on the AB-loop of TIMP-3 and -4 could interact with the MT1-loop “receptacle” of MT1-MMP. Rather, we were interested in finding out whether the TIMP-1 AB-loop “obstructs” the molecule from initiating conformational changes with MT1-MMP. Our data indicated that TIMP-2 and TIMP-3 AB-loops were slightly beneficial in enhancing the affinity of TIMP-1 mutants against MT1-MMP (K iapp of 77-106 nm) (Table II and Fig. 3). Disappointingly, none of the mutants could be considered tight binding inhibitor in MT1-MMP inhibition. CD-loop Mutants—The fourth group of mutants consisted of the CD-loop mutants. Only two residues were considered important at this site, namely Met-66 and Val-69. We know from the crystal structures of free and TIMP-1-bound forms of stromelysin-1 (Protein Data Bank codes 1QIA and 1UEA) that Met-66 was capable of inducing some degree of conformational change in stromelysin-1 (11Gomis-Rüth F. 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 (513) Google Scholar). Hence, a series of mutants were created to replace Met-66 at this site. The mutations included amino acids of different characteristics, ranging from acidic to basic as well as those hydrophobic in nature (M66K, M66D, M66L, M66I, M66V, M66A, and M66G). On the other hand, given that leucine is the only variant at the Val-69 locus (Fig. 1), only one mutant was created to replace the residue (V69L). Subsequent kinetic analysis showed that the majority of the mutations only impaired the affinity against MT1-MMP (K iapp from 146 nm to an excess of 500 nm) (Table II). EF-loop Mutants—The last mutant in the series was the Thr98 to leucine mutant (T98L) from the EF-loop division. Thr98 is situated right before the second disulfide bond (Cys3-Cys99) of TIMP-1. Interestingly, TIMP-1 is the only member of the TIMP family that has a threonine at this locus (Fig. 1). The corresponding residue in TIMP-2-4 is leucine. Indeed, replacement of threonine by leucine vastly enhanced the affinity of the resultant mutant against MT1-MMP (K iapp 11 nm) (Table II and Fig. 4). Indeed, the K iapp value is 16-fold lower than that of the wild type N-TIMP-1. The most striking effect is that the T98L mutant clearly manifested inhibitory profiles reminiscent of a slow, tight binding inhibitor (Fig. 4). Throughout the series, T98L is the first mutant exhibiting inhibitory profiles akin to those of N-TIMP-2-4. Thr98 Point Mutants—So far, we have identified Thr98 to be the key obstructive residue that renders TIMP-1 inactive against MT1-MMP. It would be interesting to find out the effects of other amino acids on MT1-MMP inhibition. With the exception of cysteine, we mutated Thr98 to all the available amino acids, and the kinetic profiles of the mutants are shown in Table III. Not surprisingly, isoleucine produced the same potentiation effect as leucine, the K iapp value of T98I mutant (12 nm) being indistinguishable from that of T98L. The next two most potent amino acids are valine and methionine (K iapp T98V and T98M ∼30 nm). Glutamine and tyrosine were slightly poorer, the affinity of the T98Q and T98Y mutants (90-120 nm) being marginally better than the wild type protein. Replacement of Thr98 by serine, on the other hand, produced no apparent effect on the activity of N-TIMP-1. The remaining amino acids severely impaired the affinity of N-TIMP-1 against MT1-MMP (Table III).Table IIIThr98 point mutants, apparent inhibition constant (Kiapp) with MT1-MMPKi app (× 10−9m)SmallT98G>1000T98A>1000NucleophilicT98S181 ± 17HydrophobicT98V30.7 ± 1.6T98I12.0 ± 0.8T98M27.1 ± 1.5T98P>1000AromaticT98F>1000T98Y119 ± 13T98W>1000AcidicT98D>1000T98E597 ± 37AmideT98N237 ± 12T98Q94 ± 6BasicT98H530 ± 40T98K>1000T98R>1000 Open table in a new tab Combination of Good Epitopes—Even though T98L was much more active than wild type N-TIMP-1, the mutant was still not as potent as N-TIMP-2, -3, or -4. Hence, in the second phase of this work, we combined four of the major positive epitopes in an attempt to study their effects on MT1-MMP inhibition. Two multiple mutants were made: 1) V4A/P6V/T98L triple mutant, and 2) V4A/P6V/TIMP-2 AB-loop/T98L quadruple mutant. The results of the combination are shown in Table IV. First of all, incorporation of V4A and P6V significantly improved the affinity of the T98L mutant. The affinity of the V4A/P6V/T98L triple mutant (K iapp 1.66 nm) was practically equal to those of N-TIMP-2 (K iapp 1.30 nm) and N-TIMP-3 (K iapp 1.38 nm). The association rate of the mutant (k on 1.48 × 10-5m-1 s-1) exceeded 10-5m-1 s-1, closely resembling N-TIMP-3 and -4 (k on 3-6 × 10-5m-1 s-1). Unexpectedly, the incorporation of the TIMP-2 AB-loop significantly impaired the affinity of V4A/P6V/T98L (K iapp of V4A/P6V/TIMP-2 AB-loop/T98L being 5.2 nm). The association rate, however, did not seem to be affected (Table IV).Table IVApparent inhibition constant (Kiapp) and association rate (kon) of MT1-MMP with N-TIMP-1 mutants of combined epitopesKi app (× 10−9m)k on (× 10−5m−1s−1)Combined mutantsV4A/P6V/T98L1.66 ± 0.121.48 ± 0.35V4A/P6V/T2-AB-loop/T98L5.22 ± 0.201.45 ± 0.14 Open table in a new tab Effects of the C-terminal Domain on V4A/P6V/T98L Activity—So far, we have succeeded in identifying the obstructive epitopes on the N-terminal domain of TIMP-1 that hinder the inhibitor from establishing a tight binding complex with MT1-MMP. What are the effects of the C-terminal domain on MT1-MMP association? To address this issue, we introduced V4A/ P6V/T98L mutations into full-length TIMP-1, and the results are summarized in Table V. At first sight, it appeared that the C-terminal domain might improve the affinity of TIMP-1 against MT1-MMP, because full-length wild type TIMP-1 displayed significantly better affinity than its N-terminal counterpart (K iapp, full-length wild type TIMP-1 91 nm versus N-TIMP-1 of 178 nm). Subsequent comparison of the V4A/P6V/T98L mutant, however, revealed that the C-terminal domain has no beneficial effects on MT1-MMP inhibition (K iapp full-length V4A/P6V/T98L 1.86 nm). Furthermore, the association rate of the full-length mutant (0.9 × 10-5m-1 s-1) was slightly lower than the N-terminal version (Table V and Fig. 5).Table VFull-length wild-type TIMP-1 and V4A/P6V/T98L mutant, apparent inhibition constant (Kiapp) and association rate (kon) against MT1-MMPKi app (× 10−9m)k on (× 10−5m−1s−1)Wild-type TIMP-190.6 ± 5.4NAFull-length V4A/P6V/T98L1.86 ± 0.170.89 ± 0.09 Open table in a new tab Not only are TIMP the endogenous inhibitors of MMPs, they also modulate the enzymatic activities of the ADAM (adisintegrin and metalloproteinase) and ADAM-TS (ADAM with thrombospondin-like repeats) proteinases (reviewed in Ref. 20Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (973) Google Scholar). Membrane-type MMPs, in general, are poorly inhibited by TIMP-1. What is the physiological significance of such inactivity and can protein engineers ever fully understand the ultimate molecular mechanism of TIMP-metalloproteinases selectivity? Contradicting previous perceptions, our study shows that Thr98 is the pivotal obstructive epitope that renders TIMP-1 inactive against MT1-MMP. Thr98 is TIMP-1-specific, and other TIMPs have leucine at the equivalent position. How does leucine potentiate the binding of TIMP-1 to MT1-MMP? Re-examination of the available TIMP/MMP structures failed to provide a satisfactory answer to the question. In the stromelysin-1 (MMP-3)-TIMP-1 complex (Protein Data Bank code 1UEA), Thr98 is situated right before His211 (HEXX- HXXGXX H 211) of the enzyme, the last of the three conserved histidines that forms the catalytic zinc-binding ligands. Distance-wise, the two MMP-3 residues closest to Thr98 are His211 and Pro221, the amino acids being ∼4 Å from the side chain of Thr98 (Fig. 6). This aside, Thr98 does not seem to be in close contact with any particular hydrophobic residue on the surface of stromelysin-1. The TIMP-2 equivalent of Thr98 is Leu100 (Fig. 1). The crystal structure of the MT1-MMP·TIMP-2 complex (Protein Data Bank code 1BUV) again shows that Leu100 is located before the third conserved histidine (His249) of the zinc-binding motif (HEXXHXXGXX H 249) (Fig. 6). The two MT1-MMP residues closest to Leu100 are His249 and Pro259, almost a spitting image of the setting found in TIMP-1·MMP-3 mentioned above (Fig. 6). Hence, could this “His249/Pro259 pair” in MT1-MMP be a deciding factor in its rejection of TIMP-1? As part of our modeling simulation, we replaced the TIMP-2 molecule in MT1-MMP·TIMP-2 (Protein Data Bank code 1BUV) complex with TIMP-1 bearing a Thr98 to leucine mutation. Our study suggests that substitution of Thr98 by leucine does not enhance the interfacial contact between TIMP-1 and MT1-MMP enzyme (not shown). Thr98 is not the only residue we considered that is unique to TIMP-1. As mentioned earlier, Pro6 was also featured prominently on our list of mutagenesis study. Substitution of the residue by valine or serine improved the binding affinities against MT1-MMP significantly. Yet again, examination of the stromelysin-1/TIMP-1 structure suggests that the residue is not directly involved in MMP association. Why should leucine and isoleucine be the best residues? Table III demonstrates that the activity of N-TIMP-1 is critically dependent on the biophysical characteristics of the amino acids occupying the Thr98 position. The best amino acids are those similar in nature to leucine, namely isoleucine, valine, and methionine. Too bulky and hydrophobic (tryptophan and phenylalanine) or minute (glycine and alanine) a side chain resulted in complete abrogation of activity. Strangely, even though phenylalanine is poorly tolerated, tyrosine is beneficial, notwithstanding its similar size with phenylalanine. In general, acidic (aspartate and glutamate) or basic residues (lysine and arginine) are poorly tolerated. Throughout this work, we were intrigued by the fact that some of the individual positive epitopes identified are not always mutually complementary as hoped. Incorporation of V4A and P6V mutations into T98L, for example, enhanced the affinity of N-TIMP-1 against MT1-MMP to a level essentially equal to those of N-TIMP-2-4. The effect is additive. The same could not be said for the TIMP-2 AB-loop; the epitope is clearly incompatible with T98L mutation. The problem is again highlighted by our findings on the full-length forms of the V4A/P6V/T98L mutant. In comparison with wild type N-TIMP-1, full-length TIMP-1 is marginally superior in MT1-MMP binding. Subsequent study on the V4A/P6V/T98L mutant, however, suggests that the C-terminal domain was either inert or slightly detrimental to the affinity and association rate against MT1-MMP. Taken together, it is highly unlikely that the findings in this work could be explained satisfactorily by steric reasons alone. The true answer, we believe, lies in the molecular dynamics of TIMP-1 and MT1-MMP that governs the course of their interaction. Crystallographic studies of MMP·TIMP complexes only portray static representations of the enzymes and their inhibitors. Undeniably, the parts of TIMP with the closest intimacy with MMPs are the N terminus, the AB-loop, and the CD-loop. Here, we show that the key to TIMP/MMPs selectivity might in fact, lie elsewhere. What is most striking is that the pivotal amino acid, Thr98, is a rather “insignificant” residue that has never been emphasized in any of the literature on TIMP analysis and engineering so far (21Nagase H. Brew K. Arthritis Res. Ther. 2002; 4: 51-61Crossref Scopus (48) Google Scholar, 24Douglas D.A. Shi Y.E. Sang Q.A. J. Protein Chem. 1997; 16: 237-255Crossref PubMed Scopus (82) Google Scholar, 25Nagase H. Meng Q. Malinovskii V. Huang W. Chung L. Bode W. Maskos K. Brew K. Ann. N. Y. Acad. Sci. 1999; 878: 1-11Crossref PubMed Scopus (39) Google Scholar, 26Bode W. Fernandez-Catalan C. Grams F. Gomis-Ruth F.X. Nagase H. Tschesche H. Maskos K. Ann. N. Y. Acad. Sci. 1999; 878: 73-91Crossref PubMed Scopus (165) Google Scholar, 27Visse R. Nagase H. Circ. Res. 2003; 92: 827-839Crossref PubMed Scopus (3672) Google Scholar, 28Wei S. Chen Y. Chung L. Nagase H. Brew K. J. Biol. Chem. 2003; 278: 9831-9834Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As mentioned under “Experimental Procedures,” with the exception of T98F, all Thr98 mutants were highly active against MMP-2 regardless of the nature of the amino acids occupying the position. What about other MMPs, ADAM and ADAM-TS proteinases? We are currently evaluating the activity profiles of our TIMP mutants with these proteinases, and the findings will be presented in the near future. This paper is the first in which a variant of inactive TIMP has been successfully transformed into a fully active one against a specific MMP backdrop. We hope the findings in this work will broaden our views on the mechanism of MMP/TIMP selectivity. We thank G. Knight for the provision of QF-24 and V. Knäuper for MT1-MMP constructs.