METHOD OF CELL SURFACE ACTIVATION AND INHIBITION

Abstract: Disclosed are methods of cell surface activation and inhibition that involve the interaction of an inhibitor of matrix metalloprotease known as TIMP-2, with the enzyme, gelatinase-A (GelA). Critical to the methods of the invention is the discovery of a unique TIMP-2 binding site on the surface of the C-terminal domain (GelA-CTD) of the enzyme, which has been determined to be Asp<656>, but which can also include other residues in the Ge1A-CTD domain with which Asp<656> forms a contiguous surface, namely Gly<651>, Phe<650>, and Tyr<636>. Identification of this binding site provides a useful target for the screening of MMP inhibitors and for prognosis and treatment of diseases in which MMPs are implicated. Compounds which are candidate MMP inhibitors can be structured to competitively inhibit cell surface activation.

Description: METHOD OF CELL SURFACE ACTIVATION AND INHIBITION Field and Objective of the Invention This invention relates to methods for cell surface activation and inhibition. More particularly, the invention relates to methods of cell surface activation and inhibition that involve the interaction of an inhibitor of matrix metalloprotease known as TIMP-2, with the enzyme, gelatinase-A. Matrix etalloproteases (MMPs) are ubiquitous in human disease and development. Most processes that involve a certain amount of tissue repair and damage are believed to be influenced by MMPs such as, for example, in degradation of type IV collagen that might occur in rheumatoid or osteoarthritis and remodeling of endothelial walls in restenosis. MMPs are also implicated in various aspects of cancer such as primary tumor formation, metastasis, and the vascularization of larger tumors (angiogenesis) . It is also known that MMPs are involved in the conversion of inactive tumor necrosis factor (TNF) precursor into active TNF, which in turn is implicated in rheumatoid arthritis, Crohn's disease, multiple sclerosis, cachexia and sepsis. Consequently, the screening for MMP inhibitors as potential drugs is of significant use in the medical and pharmaceutical fields. MMPs are secreted by mammalian cells as zy ogens and upon activation initiate tissue remodeling by proteolytic degradation of collagens and proteoglycans. Activation of the secreted proenzymes and interaction with their specific inhibitors, TIMP-l and TIMP-2, determine the net enzymatic activity in the extracellular space. TIMP-2 forms a specific complex with the proform of gelatinase-λ (GelA) which is mediated by interaction with the C- ter inal domain (GelA-CTD) of the enzyme. The amino acid sequence of the 72 kDa GelA is disclosed in Goldberg, U.S. Patent 4,923,818, and its complex with TIMP-2 is disclosed in Goldberg published European Patent Application, EP 404,750. GelA is a multi-domain protein containing a catalytic domain, a domain with three type II fibronectin-like repeats, and a C-terminal domain. Soluble GelA proenzyme is recruited to the cell surface where it is specifically activated by MTl-MMP, a membrane bound etalloprotease. The binding of GelA to cell surface and its subsequent activation is also mediated by GelA-CTD. Consequently, cell surface activation is inhibited in the presence of exogenously added excess of TIMP-2 or recombinant GelA-CTD. It has not been known previously how the MTl-MMP that is inhibited by complex with TIMP-2 is able to cleave the GelA propeptide to initiate activation of the pro-enzyme. Resolution of this question is critical to an understanding of the mechanism by which GelA-CTD interacts with TIMP-2 and MTl-MMP on the cell surface. Background of the Invention (Note : Literature references on the following background information and on conventional test method and laboratory procedures well known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated in parentheses, and appended at the end of the specification. ) Secreted metalloprotcascs (MMPs) initiate tissue remodeling by degradation of extracellular matrix (ECM) maσomolcculcs (reviewed in 1 -3). Normal physiological processes such as morphogenesis, tissue repair, and angiogenesis, arc dependent upon spatial and temporal regulation of the activity of these enzymes, while malignant cells exploit these same proteases to promote invasion and metastasis (4-7). A clear understanding of the mechanisms governing regulation of MMP activity in extracellular space has remained an elusive goal. The MTl-MMP/GelA system (8-16) provides a first glimpse at a mechanism by which an activity of a soluble MMP, GelA (17), can be spatially regulated via its recruitment to the cell surface where the GelA proenzyme is converted into its active form. Transfcction of Cosl cells with MTl-MMP is sufficient to cause GelA binding to the cell surface and its activation (8,19). The cell surface activation of GelA involves a two step protcolytic processing of its propeptide. The first cleavage of the Asn 37 -Leu peptide bond is dependant on MTl-MMP (9), a membrane bound mctalloproteasc. This cleavage is also dependent on GelA having an intact C-tcrminal domain since a truncated form of the GelA proenzyme lacking a C-terminal domain can not be activated by membrane bound MTl-MMP (13). Consequently the exogenously added recombinant GelA-CTD is a competitive inhibitor of Asn 37 -Lcu cleavage (9,10). Finally this reaction is inhibited in the presence of an excess of inhibitor, TIMP-2, while T1MP-1 has no effect. The consequent cleavage of propeptide is accomplished via an autoprotcolylic, MTl-MMP independent mechanism (9, 10, 18, 19) to generate a 62 kDa active GelA with an amino-tcrminal residue Tyr 81 . These data demonstrate that binding of GelA to the cell surface via its CTD is a prerequisite for enzyme activation. We have previously shown that two closely related proenzymes GelA and B form specific complexes with TIMP-2 and TIMP-1 respectively (20). These complexes are also formed via inhibitor interaction with the carboxyl-end domain of proenzyme (21 ,22). Thus TIMP-2 and cell surface binding activities of GelA-CTD appear to be interrelated. We have purified activated form of MTl-MMP using affinity chromatography approach (9) and demonstrated that it acts as cell surface T1MP-2 receptor with Kd=l.65xl0 9 M. T e MT1-MMP-TIMP-2 complex in rum acts as a receptor for GelA-CTD (Kd =0.42xlO * M). The data we have presented support the hypothesis that the cell surface binding of GelA-CTD occurs via formation of a tri-molcculax complex of activated MTl-MMP πMP-2/pro-GelA that promotes pro-GelA activation. This model, however, docs not satisfactory resolve the GelA activation mechanism for the following reasons. The inhibitor TIMP-2 consists of two domains. The amino-tcrminal, inhibitory domain interacts with the active center of MMPs to form an inhibitory complex (23,24 ). The C-tcrminal domain binds to GelA-CTD. Thus the inhibitory complex of TIMP-2 with activated MTl-MMP can leave the C-tcrminal domain of the inhibitor exposed and available for interaction with GelA-CTD. In fact we have reported an analogous tri-molecular complex between GcIB, TIMP-1 and activated interstitial collagcnasc (22) where the collagcnasc component of the complex was inhibited. Moreover the specific inhibition of soluble form of MTl-MMP by TIMP-2 has been recently demonstrated (25,26). Thus, the model of cell surface GelA activation that requires assembly of the MTl-MMP/TlMP-2/pro-GelA complex leaves unanswered the question of how the MTl-MMP inhibited by TIMP-2 is able to cleave the Asn 37 -Leu peptide bond to initiate activation of the pro-enzyme. An answer to this question demands a better understanding of the mechanism by which GelA-CTD interacts with TIMP-2 and MTl-MMP on the eel! surface. We have recently reported the high resolution crystal structure of Gel A-CTD (27). Here we report the results of extensive alanine scanning mutagenesis of solvent exposed GelA-CTD amino-acid residues and, using the coordinates of the GelA-CTD structure, define a TIMP-2 binding site on the surface of this domain. By comparison of the TIMP-2 binding site to the same regions in related MMP structures, we characterize structural features required for general ΗMP binding and the specificity of T1MP-2 - GelA-CTD interaction. We also report analysis of GelA activation inhibition activity of GelA-CTD mutants relative to that of wild type. Brief Description of the Invention In accordance with the present invention, methods of cell surface activation and inhibition are provided which are useful for the screening of MMP inhibitors that are potentially useful for the treatment of diseases that involve tissue repair and damage and other diseases in which MMPs are implicated. Critical to the methods of the invention is the discovery of a unique portion of the TIMP-2 binding site on the surface of the GelA-CTD domain, which has been determined herein to be the very strongly binding residue Asp 656 . This critical TIMP-2 binding site can also include other residues in the GelA-CTD domain with which Asp 656 forms a contiguous surface, namely the less strongly binding residues Gly 651 , Phe" 0 , and Tyr 636 . In accordance with another embodiment of the invention, the TIMP-2 binding site includes the foregoing four residues and additionally the very strongly binding residues Asp 815 , Lys S€ , Lys 576 , Trp" 4 , and Arg 5 ' 0 , and the less strongly binding residues Lys 575 , Lys $0< and Asn 611 . The effect of these residues on the TIMP-2 binding of GelA-CTD has been confirmed by utagenesis. Point mutations can be made at these residues in the TIMP-2 binding site to impact the TIMP-2 binding to GelA-CAD, e.g., to inhibit or retard the binding, and thereby provide a unique screening method. Identification of this TIMP-2 binding site provides a useful target for the screening of MMP inhibitors and for the prognosis and treatment of diseases in which MMPs are implicated. Compounds which are structured to competitively inhibit cell surface activation can be candidate MMP inhibitors. Detailed Description of the Invention The following description of the invention taken in conjunction with the accompanying drawings is provided to further illustrate the invention and preferred embodiments in greater detail. Brief Description of the Drawings Figure legends. Fig 1. Space-filling model (A, 34) and ribbon diagram (B, 35) of Gel A-Ctd showing residues which interact with TIMP-2. A) The residues which arc thought to directly interact with TlMP-2 arc shown colored and labeled. Residues which showed between a 2- to 100-fold loss in T1MP-2 binding when mutated to alaninc arc colored cyan, while those which showed over 100-fold loss in TIMP-2 binding when mutated to alaninc arc colored dark blue. TBS-1 and TBS-2 regions arc indicated by the dashed magenta boxes which cover their respective regions. B) A ribbon diagram of Gel A-Ctd shows the canonical β-propeller fold. Each blade of Gel A-Ctd is labeled with a roman numeral. The Ca2+ ion is shown in red along the central axis of symmetry. The disulfidc bond connecting blades I and IV is shown as arc the N- and C- termini. Residues which are thought to directly interact with TIMP-2 are shown in magenta and arc labeled. All the residues lie on blade III, blade IV or the loop connecting the two blades. Fig. 2. Competition Assay of GelA-Ctd Binding to TIMP-2. One hundred μL of solution containing l.7xl0 9 M of ,25 l-labclcd WT GelA-Ctd (10 8 cpm/μg) and unlabelcd purified recombinant WT or mutant of the GelA-Ctd at the indicated concentrations were incubated in T1MP-2 (50ng) coated wells of microtitcr plates and washed as described in Methods. Bound radioactivity was determined by counting individual wells in a gamma counter. After background substraction the CPM retained in the wells was normalized to 1.00 and plotted versus the concentration of unlabeled, competing Gel A-Qd for wild type (0, i/Kd=l) or mutants Gly 65 i (x, i/Kd=3); Lys 579 (Q Ki/Kd=6); Lys 6 °« (Δ, Ki/Kd=25); and Asp*" 5 (o, Ki/Kd=300) as indicated in the figure. The values for wild type self competition are the mean of 8 separate experiments and the error bars represent the standard error. The mutant values are the average of two determinations. Computer generated theoretical curves were fitted to the data and the apparent Kd for WT and Ki for mutants were determined from the fit as shown in the figure by each solid curve. Fig. 3. Molecular surface of Gel A-Qd showing the TIMP-2 binding site. A molecular surface of Gel A-Qd was calculated and displayed using GRASP. The TIMP-2 binding site is colored magenta. The yellow labels denote some of the T1MP-2 binding residues and their position with respect to the surface. The boundary residues described in the paper arc shown in green and arc seen to suπound the TIMP-2 binding site. Fig. 4. Comparison of molecular surface and electrostatic potential at surface of Gel A-Ctd and Cll-Ctd. The molecular surface of both (4λ) Gel λ-Ctd and (4B) Cll-Ctd are displayed. Electrostatic potential for each was calculated and displayed with positive potential shown in blue and negative potential in red. The dashed magenta line conforms to the approximate TIMP-2 binding site described in the text. TBS-1 and TBS-2 are shown and conform to the same regions described in Fig. 1. The TBS-1 region of Gel A-Ctd displays a lot of positive potential, whereas Cll-Ctd has much less positive potential and has a significant amount of negative potential across the TBS-1 binding interface. The TBS-2- regions of both molecules show differing potentials as well. The molecular surfaces of both molecules suggests that they would present significantly different van der Waal contact surfaces. Fig. 5. Sequence alignment of β-propeller blades III and IV from C-terminal domains of MMP family members. The amino acid sequence of GelA containing all the residues defining the TIMP-2 binding site was aligned with other MMPs. Sequences which are found in blade III or blade IV are beneath the underlined regions. GelA residues which are part of the TIMP-2 binding site and corresponding residues from other enzymes are bolded. Those residues constituting the TBS-1 region (see text) are bolded and underlined, while the remaining residues are part of TBS-2 are merely bolded. An ' *' marks residues whose effect on TIMP-2 binding of GelA-CTD were confirmed by mutagenesis. Fig. 6. Inhibition of membrane dependent activation of GelA by GelA-CTD mutants. The 15 ng of purified GelA were incubated in 25 mM HEPES-KOH buffer, pH 7.5, containing 0.1 mM CaCl 2 with, 20 μg of plasma membrane protein from HT1080 cells for 2h at 37 β C in the presence of increasing concentration (1-6) of recombinant Gel-A-CTD WT or mutants #28 (Asp"'), #31 (Lys"') , #39 (Lys 604 ), #41 (Asp 615 ), #229 (Asp 516 ), #234 (Arg 5 ' 0 ), #247 (Lys 646 ), #250 (Trp 514 ), #252 (Tyr 6J6 ), #255 (Phe 650 ), #257 (Gly 651 ), #258 (Asp 65 ), #259 (Asn 611 ), as indicated in each panel. The results of activation reaction were analyzed on zymogram as described previously (9,10). The images of resulting zymograms were acquired using flat bed scanner and concerted to a negative. The colored areas in FIG. 1A and FIG. 3 are shown in black and white copies as follows: FIG. 1A - Residues shown in dark blue are Asp" 6 , Asp 615 , Lys'", Lys 516 , Trp 574 and Arg 5 ' 0 . Residues shown in cyan are Gly" 1 , Phe" 0 , Tyr" s , Asn 611 , Lys 579 and Lys βtH . FIG. 3 - Boundary residues shown in the green colored area are Lys' 4 ', Gin 641 , Lys" 8 , Lys 6 ", Asp" 8 and Asp 618 . The red colored TIMP-2 binding site shows residues Asp" 6 , Phe" 0 , Tyr" 6 , Asp 615 , Asn 611 , Lys 646 , Lys 576 , τrp 574 and Lys 604 . In order to further illustrate the invention, the following detailed examples were carried out although it will be understood that the invention is not limited to these examples or the details described therein. Examples Materials and Methods Cell Culture. HT1080 fibrosarcoma cells were grown in monolayer culture in RPMI 1640 media supplemented with 4% fetal calf scrum and 2 mM glutamine in the presence of 5% CO 2 and treated with 12-O-tetradccanoyl-phorbol acetate (TPA) (50ng/ml for 16 h). Isolation of plasma membranes from HT1080 cells was performed using discontinuous sucrose gradient as described (9,10). Enzyme Purification. The GelA expression plasmid p6R72hyg was transfcctcd into ElA-cxprcssing p2AHT2a cells and GelA was purified from conditioned medium of stably transfcctcd ceil line p2AHT7212A as described (9,10) Expression and Purification of TIMP-2. Recombinant T1MP-2 was expressed in p2AHT2a cells transfcctcd with TIMP-2 cDNA in the pδRhyg expression vector and purified from serum free conditioned media of p2AHT2aT2 cells as described earlier (9,10) using Reactive Red- 120- Agarose (Sigma, R-0503), Q-Sepharose (Pharmacia #17-0510-01), CM- Sepharose CL-6B (Sigma #CCL-6B-100) and RP-HPLC column chromatography. Expression and purification of the FLAG GelA-CTD fusion protein. Expression vector pFLAG72CT was constructed by cloning a fragment from GelA cDNA (17) coding for Leu 444 - Cys 660 into E.Coli secretion vector pFlagl (IBI Inc.). The resulting vector coding for the fusion protein FLAG-CT was transfectcd into an E.coli TOPP5 host (Stratagene). Protein was purified from a periplasmic fraction by chromatography on Reactive Rcd-120-Agarose (Sigma, R-0503) and Ml anti-flag antibody affinity column as described previously (9, 10,27 ). Each of the 50 mutants and wild type GelA CT were purified using this procedure. Mutageoesis of the FLAG GelA-CTD fusion protein. Expression vector pFLAG72CT was mutagenized directly using PCR mediated site directed mutagencsis. A pair of anti-parallel 33 base pair long primers was synthesized for each mutant. These primers containing a desired mutation were used in a pair of PCR reactions with cither of two primers flanking the coding sequence. Both resulting PCR products contained mutation. They were mixed, melted and annealed to generate a partial hetcroduplex encompassing the whole coding sequence. The latter served as a template in a third PCR reaction primed by both of the flanking primers. Each of the resulting PCR products was cloned back into the pFLAG72CT expression vector and subjected to a sequence analysis to confirm the presence of mutation. All resulting mutant proteins were purified and assayed for TIMP-2 binding as described below. The sequence of mutants that had negative effect on TIMP-2 binding was verified by sequencing of the entire coding region to exclude the appearance of secondary, PCR generated, mutations. Secondary mutations, when present, were separated from the desired mutant by cither a second round of PCR or using restriction enzyme mediated subcloning. TIMP-2 binding of the FLAG-GelA-CTD fusion protein. The TTMP-2 binding and competition assays were performed in 96 well modular plates (Costar). TIMP-2 coated plates were prepared by addition of 100 μ\ of loading buffer (20 mM Tris HCI, pH 9) containing 50ng of purified TIMP-2 to each well and incubated for lh at RT. This solution was replaced with 200 μ\ of blocking buffer (0.5 % BSA and 0.02 % Brij in PBS, pH 7.2) and incubated ON at 4°C. For binding experiments increasing concentrations of competing cold ligand in lOO l of binding buffer (1 mg/ml BSA and 0.01% Brij in PBS) were added to TIMP-2 or BSA (control) coated wells and incubated for 30min prior to addition of 10 9 M of 125 I-GelA-CTD (between 6,5xl0 7 and lxlO dpm/μg). Incubation continued for lh, after which plates were washed 5 times with Binding Buffer and each well was counlcd to determine retained radioactivity. Activation of the GelA proenzyme. Between 15-50 ng of the GelA proenzyme was used for activation with plasma membranes (1-4 μg of plasma membrane protein) in 10 μl final volume of 25 mM HEPES-KOH buffer, pH 7.5, containing 0.1 mM CaCl2. T e reaction was incubated at 37°C for 120 rπin, terminated by addition of the sample buffer and subjected to gelatin zymogram analysis as described (9, 10). Protein Structure Analysis. Residues whose mutation to alaninc caused a loss in TIMP-2 binding were divided into those that most likely directly interact with TIMP-2 and those whose effect on TIMP-2 binding arc most likely a result of indirect structural perturbations based on a detailed examination of the environment of each of the mutant. The set of residues which interact with TIMP-2 arc all confined to a single, contiguous surface of GelA-CTD which is divided into two adjacent regions, TBS1 and TBS2. Using boundary residues which arc near the TIMP-2 binding residues but whose mutation to alaninc had no effect on TIMP-2 binding permitted us to defined the TlMP-2 binding site as a molecular surface that includes residues not mutated in the analysis. GelA-CTD and the C-tcrminal domain of interstitial collagcnasc (Cll-Ctd) were aligned along their respective C α atoms. The two structures aligned with an average root mean square difference in C 0 position of 3.7 A and were visualized using the graphics program O (28). The model of GelB-CTD was constructed using the modeling software, Sybyl (version 6.2, Tripos Associates, St. Louis, Missouri). The GelA-CTD structure provided the basic template for the structure and the coordinates of the C α atoms were preserved in regions of sequence identity. In these regions, the conformation of the sidechains were preserved as well. In regions with no sequence identity, the C α positions were held constant but the side chain conformation was chosen from a rotamer library set. Stcric clashes due to the insertion of GelB residues were relieved by moving either the neighboring atoms (whether they be sidechain or backbone atoms) or by moving the C α position of the substituted residue. Regions requiring the insertion or deletion of residues in the sequence only occurred along loops or turns and were modeled by choosing a turn or loop from the Brookhavcn protein data bank that had a similar sequence and made the fewest van dcr Waal contacts with nearby atoms. Finally, the model was completed by minimizing van der Waal contacts over the entire structure. T e final GelB-CTD model was aligned with GelA-CTD along their respective C α atoms. Results. Description of GelA-CTD structure The GelA-CTD coordinates arc from a high resolution crystal structure (resolution = 2.15 A) with a low R-factor (18.8%) and low average coordinate error (< 0.25 A) (27), so the positions of the backbone and sidcchain atoms are well determined. The structure includes all residues between Leu 461 and Cys 660 where the only residues with poorly defined positions arc Glu 529 and Glu 530 . The overall structure of GelA-CTD is best described as a four-bladcd β-propclleτ (Fig. 1). The four "blades' arc each composed of four strands of anti-parallel β-shect. The fished domains arc twisted making the fourth, outer most strand form nearly an 80 * angle with the inner most strand. Each blade is arrayed about a central pscudo four-fold axis so that a 90 * rotation about the axis positions one blade on top of another. A channel formed by the four blades, parallel to the rotation axis, contains a CaJ« ion, a Na«Cl ion pair and a number of stably bound water molecules. The inner most strands of each blade are all parallel and the Ca*- ion protrudes from the N-tcrminal end of the channel. The regions between the four blades are composed of hydrophobic residues (primarily Phe, Tyr and Trp) which arc large enough to contact one another across such a wide interface. Connecting loops lay across the hydrophobic interface and connect adjacent blades. Blade IV is connected covalcntly to blade I via a disulfide bond between Cys 469 and Cys 660 . Identification of the TIMP-2 Binding Site in the GelA-CTD by Alanine Scanning Mutagenesis. Alanine scanning mutagenesis of solvent exposed amino-acid residues of GelA-CTD was used to define its molecular surface that interacts with TIMP-2. The results were interpreted by examination of the location and environment of each point mutant in the crystal structure of GelA-CTD (Fig. 1, 27), so that only residues of GelA-CTD which can directly interact with TIMP-2 are identified. Expression vector pFLAG72CT was mutagenized directly using PCR mediated site directed mutagenesis as described in Methods. All fifty resulting mutant proteins were purified as described previously (9, 10) and assayed for TIMP-2 binding as described in Methods. The sequence of mutants that had negative effect on TIMP-2 binding was verified by sequencing of the entire coding region to exclude the appearance of secondary, PCR generated, mutations. To quantitatc the TlMP-2 binding affinity of the different GelA-CTD mutants relative to wild type (WT) GelA-CTD we developed TIMP-2 binding and competition assay in 96 well modular plates. For binding experiments a solutions of 10 9 M of , 25 I-GelA-CTD containing increasing concentrations of competing cold ligand were added to TIMP-2 or BSA (control) coated wells and retained radioactivity was determined by counting individual wells as described in Methods. The apparent Ki for each mutant was determined by a fit of computer generated series of curves to the data from the competition assay. A 25% variation in apparent Ki thus determined produced curves which were clearly less representative of the data. An example of the results of this analysis for WT GelA-CTD and four mutants are shown in Fig. 2. The mutants presented in Fig. 2 were chosen to illustrate the range of variation encountered. All the mutants that had an effect on TIMP-2 binding (Ki / Kd > 1) arc summarized in Table 1. Substitution of Ala for one of the following amino acid residues Lys 470 , Arg 482 , Arg 491 , Arg 495 , Asp 501 , Glu 515, Glu 518, Lys i9, Glu 529, Lys 53i, Glu 539, Glu 5 4 9, Arg 550, Asp 564, Arg 567, Lys 578, Asp 586, Lys 596, Asp«>8, Asp «i8, Hys«8, Lys«3, Lys " , Glu «!, Lys "9, Leu "8, Gin * 43 , and Leu 5 « did not affect mc binding affinity of GelA- CTD to T1MP-2 (Kd=Ki) in this assay. Single replacement of Lys 5 Ϊ 9 with Arg, Ala 479 with Thr, or Leu 54β with Arg also had no effect. Localization of TIMP-2 binding residues on GelA-CTD. Among all the point mutants of GelA-CTD which show a loss in binding, only Asp 569 j s not considered part of the TIMP-2 binding surface. T e remaining mutants all lie within two adjacent areas of the GelA-CTD shown as TIMP-2 Binding Surfacc-1 (TBS-1) and TIMP-2 Binding Surfacc-2 (TBS-2) in Fig. 1. The TIMP-2 binding site of GcLA-CTD is divided into two regions in order to facilitate discussion of the different features seen in this broad binding site and to simplify comparison of these regions on related proteins. There is no physical basis for dividing the binding site into two regions, but we do so in order to discuss different features seen in the TIMP-2 binding site. TBS-1 is formed between blades III and IV and includes a non- polar interface composed of large aromatic residues (contacting Trp 574 ) which pack between the two adjacent blades and form a small, hydrophobic cavity. Suπrounding this non-polar part of TBS-1 are a number of positively charged residues which are contributed mostly from the second (Lys 576 , Lys 579 ), third (Arg590), an d fourth (Lys 604 ) strands of blade III as well as Lys 646 which is on a large turn made between the third and fourth strands of blade IV. The non- polar cavity is bounded by a looping strand which lies across the cavity and connects blades III and IV. This loop region, which contains Asn 6* ' , is considered part of TBS-1 but is adjacent to TBS-2 and forms part of the putative TIMP-2 binding surface of GelA-CTD. TBS-2 contains residues required for TIMP-2 binding that arc mostly located on blade IV. Phe 650 and Gly 651 arc located on the fourth strand of blade IV. Tyr 636 comes from the third strand of blade rV but forms an adjacent surface with Phe 650 and Gly 651 . Asp 656 is located on a single α-hclical rum at the end of blade IV. Asp 615 is part of the loop section connecting blades III and IV, but is positioned adjacent to Tyr 636 . Together, TBS-1 and TBS-2 make up the entire putative TIMP-2 binding surface of GelA-CTD. From Fig. 1 , it can be seen that residues whose mutation caused at least 100-fold loss in TIMP-2 binding are predominantly found in TBS-1 in and about the cavity. Asp 6i 5 is the only residue from TBS-2 which showed more 100 fold loss in TIMP-2 binding when mutated to alanine. In modeling a TIMP-2 binding surface of GelA-CTD, it is possible to also make use of point mutations which had no effect on TIMP-2 binding. Some residues on GelA-CTD near or adjacent to the putative binding region did not impact T1MP-2 binding when mutated to alaninc . These mutants are considered boundary residues because they help define the outer limits of the TIMP-2 binding surface. They include Lys 5 ™, Asp 586 , Asp 608 , Asp 6 !", Lys 633 , Lys 639 , Glu 641 , Gin 643 , and Lys 649 . While the list is not an exhaustive one and docs not completely sunound the site, it is a considerable number, and as seen in Fig. 1 , they contribute greatly to determining the shape of the TlMP-2 binding surface on GelA-CTD. The effects of point mutations on GelA-CTD binding of TIMP-2 can be characterized as 'direct' or 'indirect'. Point mutations with direct effect presumably show a loss in binding due to direct interaction with TIMP-2 since in the crystal structure these residues arc almost entirely solvent exposed making no significant van der Waals contact, salt bridges or hydrogen bonds with nearby sidechain or backbone atoms. Those classed as 'indirect' are point mutants of residues which are involved in such interactions with neighboring atoms. The effect of these mutants on TIMP-2 binding may be either a result of loss of direct interaction with TIMP-2 or due to a perturbation of the local structure as a result of the point mutation which 'indirectly' causes a loss in TIMP-2 binding. Most of the point mutants which have an effect on TIMP-2 binding are classed as 'direct' including mutants of Lys 576 , Lys 579 , Arg 590 , Lys 604 , Asn 61 1 , Asp 615 , Lys 646 , and Phe 6J 0 (see Table 1). Tyr 636 may also be considered direct in that most of the ring including the hydroxyl group is solvent exposed and the van der Waals interactions of its Cjl and C t l atoms are not likely to significantly perturb local geometry. The residues classed as 'indirect' are Asp 569 , Trp 574 , Gly 65 ι, and Asp 656 . The residues classed as 'indirect' are Asp 569 , Trp 574 , Gly 65 ', and Asp 656 . The entire TIMP-2 binding site shown in Fig. 3 represents a surface area of 1027 A 2 . The inte-rior of the surface is defined by residues whose point mutants show a loss in TIMP-2 binding. The boundary of the surface is defmed by the outermost residues which show an effect on TTMP-2 binding and by the boundary residues described above. In order to create the entire surface other residues for which mutations were not made needed to be included as part of the binding surface. These residues were selected by the criteria that they could have no atoms outside the boundary of the binding surface and must have surface accessible atoms within the interior of the surface. The non-mutated residues included as part of the TIMP-2 binding surface are residues Asn 5 ", Tyr 58 ι, Phe 588 , Ala 609 , Trp 610 , Ala 6 ' 2 , llc 6 ^, Pro 6 ' 4 , Leu 645 , and Vafl 648 as well as the C ζ and C t l ring carbons of Phe 602 . All the aromatic residues of this group as well as Leu 645 contribute to form the non-polar cavity in TBS-1. Ala 612 , He 613 , and Pro 6!4 are on the loop connecting blades III and IV. He 6 ' 3 is unique in that only its backbone atoms arc surface accessible. Val 648 makes up part of the van der Waal contact surface of TBS- 2. The hole in the binding surface is present because Leu 638 (which is in a small depression between Phe 650 and Tyr 636 ) did not show a loss in TIMP-2 binding when mutated to alanine. Tbus, the C γ , l and C&2 atoms of Leu 638 are not considered part of the TIMP-2 binding surface. Rationalizing the effects of these mutations on TIMP-2 binding requires a broader description of the structural environment of these residues. Structural analysis of 'indirect' mutants As discussed above, a some of the residues included in the TIMP-2 binding site of GelA-CTD may be classed as 'indirect' mutants including Asp 569 , Trp 574 , Gly 651 , and Asp 656 . Here, we rationalize the effects of mutating them to alaninc on T1MP-2 binding given the fact that these residues also interact with other portions of the GelA-CTD molecule. The Ojl of Asp 569 forms a hydrogen bond with a backbone amide proton of Gly 585 which is located on a tight turn formed between the second and third strands of blade III. The Asp 569 -> AJa mutation causes only a small reduction in TlMP-2 binding. Strand three of blade III contains a residue, Arg 590 , whose mutation to alanine shows a large loss in binding (> 100-fold) and directly interacts with TIMP- 2. Also, Asp 569 is away from the contiguous binding surface formed by the other point mutants which affect TlMP-2 binding. While it is possible that TIMP-2 directly interacts with Asp 569 , the effect of the Asp 569 -> AJa mutation is most likely mediated by alteration of the position of Arg 59 o as a result of the loss of an important structural H-bond with Gly 585 which constrains the conformation of the turn. Trp 574 is one of a number of hydrophobic residues forming the pocket between blades III and IV. It makes van der Waals contact with a number of atoms from neighboring sidechains including Tyr M1 and Trp 610 . Since only the C ζ 3 and C η 2 atoms of Tip 574 arc surface accessible, the large loss in binding of the Trp 574 -> Ala mutant is most likely not due to a loss in the interaction of these atoms with TIMP-2 but to a rearrangement of neighboring residues as a result of the mutation. The most reasonable interpretation of the effect of the mutation is that TlMP-2 makes van der Waals contact with this pocket upon binding GelA-CTD and that Trp 574 -> Ala disrupts T1MP-2 binding by altering the van der Waal surface presented by GelA-CTD. So while the effect of the Trp 574 -> Ala mutation is 'indirect', it is suggestive of direct binding of TIMP-2 to surface atoms in the pocket. The Gly 651 -> Ala mutation has a moderate effect on TIMP-2 binding. Since alaninc, by virtue of its Cp, is stcrically restricted in its allowable φ, ψ angles, it is possible that the effect on TIMP-2 binding of the Gly 65 1 -> Ala mutation is the result of alteration in the protein backbone. Alanine is more energetically restricted than glycine in the φ, ψ conformations it may adopt. However, since Gly 651 is found in a well formed β-sheet of the outer most β-strand of blade IV and adopts phi psi angles (φ=- 157.9, ψ=-174.8) commensurate with an anti-parallel β-stand conformation, alaninc is likely to adopt the same conformation at the site. Thus, loss in TIMP-2 binding for the alaninc mutant is due to the addition of the C β atom on residue 651 which, by approximation, blocks interaction of TIMP-2 with the C α of 6 1. The double mutant, Glu 64 ! -> Ala/ Gly 65 1 -> Arg shows > 100-fold loss in TIMP-2 binding. Glu 64 ! j s entirely solvent exposed and docs not interact with neighboring atoms, and the single mutant, Glu 64 1 -> Ala shows no effect on TIMP-2 binding. Since Glu 641 is entirely solvent exposed docs not appear to interact with any neighboring atoms, the effects of the double mutant are not considered to be a result of coopcrativity. Thus, the effect of the double mutant is due exclusively to the Gly 651 - > Arg mutation. Presumably, the Gly 65 1 -> Arg mutant covers a nearby surface on GelA-CTD upon which TIMP-2 normally binds. Since argininc is much larger than alaninc and is also charged, it is not surprising that it had a much more dramatic effect on TIMP-2 binding than alanine. The fact that both Gly 651 -> Ala and Gly 651 -> Arg reduce TIMP-2 binding suggests that TIMP-2 contacts GelA-CTD at the C 0 of Gly 65 ) as well as surface residues near Gly 65 ! . Asp 656 is solvent exposed and forms an H-bond with the hydroxyl group of Tyr 637 . The effect on TIMP-2 binding of the Asp 656 -> Ala mutation may be a result of perturbation of the orientation of Tyr 637 by loss of this H-bond. While it is possible that TIMP-2 interacts with only Tyr 637 , the simplest interpretation of the effect of the mutation is that Asp 656 directly interacts with TIMP-2. This conclusion would partially explain the large loss in TIMP-2 binding of the Gly 65 ! .> Arg mutation which puts a positive charge near Asp 656 . Also, Asp 656 forms a contiguous surface with Gly 651 , Phe 650 , and Tyr 636 (other TIMP-2 binding residues). TIMP-2 may interact with Tyr 637 but that residue was not mutated in the study so it cannot explicitly be considered as part of the T1MP-2 binding surface of GelA-CTD. Comparison of GelA-CTD and interstitial collagenase. Having defined a TIMP-2 binding site on the surface of GelA-CTD, it is instructive to compare the known structure of the C-terminal domain of interstitial collagenase (Cll-Ctd) (29) which does not bind TIMP-2 to identify which structural features of the TIMP-2 binding site are shared and which arc divergent. It was surprising to find that many of the positively charged residues arc conserved both in terms of sequence and structure. Lys 579 , Arg 590 , and Lys 604 of GelA- CTD are conserved in Cll-Ctd and adopt similar conformations in the structures (Figs 3 and 4). Furthermore, Lys 646 in GelA-CTD aligns with Arg 453 in Cll-Ctd, so while the sequence is not identical, charge is conserved and the residues overlay well when their C α atoms arc aligned. Point mutants of Arg590 and Lys646 all show at least 100-fold loss in TlMP-2 binding in GelA-CTD. Lys576, which also shows over a 100-fold loss in TIMP-2 binding when mutated to alaninc, is not conserved in Cll-Ctd where it becomes a negatively charged Asp residue. It is interesting to note that some of the charged residues, like Arg590 and Lys646, which seem to make a large contribution to TIMP-2 binding arc also conserved in a Cll-Ctd which docs not bind TIMP-2. Clearly, other features of GelA-Ctd, which are not found in Cll-Ctd, must be identified to account for its TIMP-2 binding properties. Further examination of the aligned structures reveals that the non-polar cavity in GelA-CTD is covered by a number of negatively charged residues in Cll-Ctd. Trp 574 , Lys 576 , and Ala 609 of GelA-CTD align with negatively charged residues of Cll-Ctd. In Cll-Ctd, Asp 385 is on the periphery of the pocket; Glu 383 protrudes from the pocket and Glu 4 ' 8 extends over the pocket. The effect of these negative charges on TIMP-2 binding is still not known but their negative potential could shield the nearby positive charges from TIMP-2. Alternatively, if TIMP-2 does make van der Waals contacts with the non-polar cavity of GelA-CTD, the effect of all the charged groups in the cavity would be to block this interaction and in fact bury the negative charges inside the TIMP-2/GclA-CTD binding interface. The negative potential in the cavity of σi-Ctd is partially reduced by the presence of Lys 452 which is Leu 645 in GelA-CTD. Leu 645 is a non-polar residue which points into the hydrophobic cavity of TBS- 1. Fig. 3 shows the charge potentials of both GelA-CTD and Cll-Ctd as calculated and displayed by GRASP. Comparison of the TBS-1 regions of GelA-CTD and Cll-Ctd suggests qualitatively that the pockets formed present different accessible surfaces. Some residues in the pocket are conserved, notable exceptions are Trp 6 ' 0 and Phe 588 of GelA-CTD. Other differences arc seen in the loop connecting blades III and IV. Here, Asp 615 becomes isosteric, but uncharged Asn 424 in Cll-Ctd, while Asn 6 ' 1 , Ala 6 " 2 , Pro 6 ' 4 of GelA-CTD arc changed to other residues in Cll-Ctd. Only He 623 is conserved but this residue has only backbone atoms which arc surface accessible in the structure. Clearly, GelA-CTD and Cll-Ctd would present a very different charge distribution and contact surface along their connecting loops. Comparison of TBS-2 of the aligned molecules, reveals more subtle effects. Phe 650 , which protrudes out into solvent in the GelA-CTD, as well as Tyr 636 , Gly 65 1 and Asp 656 arc not conserved in Cll-Ctd. Again, these changes create both a different contact surface and different surface potentials which would reduce the possibility of Cll-Ctd binding TIMP-2 at this region. Comparison of related sequences A sequence alignment of other MMP C-tcrminal domains was performed (Fig. 5) to sec if features noted in the comparison of Cll-Ctd and GelA-CTD held true for other MMP family members particularly those not known to bind TlMP-2. One of the most striking features of the alignment is how well conserved some of the residues necessary for full TIMP-2 binding are throughout many members of the MMP family. Just as in the comparison with Cll-Ctd, Lys 579 , Arg 590 , Lys 604 and Lys 645 are well conserved in many members of the family. GelB-CTD shows the least homology among this group of positively charged residues. Also, the negative charges in Cll-Ctd, which occurred at Trp 574 , Lys 576 , and Ala 609 in GelA-CTD arc also seen in many of the members of the MMP family. Only GelA, GclB and MTl-MMP do not place negative charges in the cavity. Further examination of the sequence alignment shows that GelA- CTD has very little homology with other member in the region between Ala 609 - Pro 614 . These residues make up the loop region which connect blades III and IV. Other members show a lot of homology over the region and fit well to a DFPGIX (where X is either G, D, E or P) consensus sequence. It is interesting to note that GelA is only homologous in this region at He 6 ' 3 whose sidechain is buried in the structure and could not interact directly with bound T1MP-2 . The alignment of residues from the TBS-2 region shows that GelA and GclB arc most similar, although not identical, over this stretch. Many of these residues, except for Leu 645 and Lys 646 , make up most of what is considered TBS-2 in GclA-CTD. Asp 615 is also considered part of TBS-2 and is homologous in GclB. MTl-MMP and stromclysin-3 arc the next most similar with residues which arc identical to or make conservative substitutions at Asp* 1 * and Asp* 56 . Comparison of GelA-CTD and GelB-CTD A comparison of aligned structures made between GelA-CTD and the model of GelB-CTD shows they share more homology over the TIMP-2 binding surface than Cll-Ctd. As seen from the sequence alignment, residues in TBS-2 were highly homologous. Tyr 636 , Val 648 , Gly 65 !, Asp 6 ! 5 nd Asp 656 from GelA-CTD arc structurally conserved in GelB-CTD. Only one residue is significantly different, Phe 650 becomes Val 694 in GelB-CTD. The turn connecting the third and fourth strands of blade IV required rebuilding in GelB-CTD due to the insertion of residues. But for the most part, these residues were arranged similarly in both structures. The loop connecting the third and fourth strand of blade IV had to be rebuilt to accommodate the insertion of two residues. This increased the size of the loop, but still placed Leu 688 and Asn 689 of GelB- CTD near Leu 645 and Lys 646 of GclA-CTD. So while no new charges are introduced, the contact surface in this region would be somewhat different in GelB-CTD. In contrast to TBS-2, TBS-1 of the model of GelB-CTD diverges dramatically from GclA-CTD. A great number of changes have been made in the non-polar cavity residues. Trp 574 , Tyr 581 , Phe 588 , Phe 602 , and Trp 610 are not conserved in GelB-CTD. The sequence changes make the cavity much deeper in GelB-CTD with a cavity floor defined by the contribution of non-polar atoms from Leu 688 and Met 653 . Other residues conserved between the two in TBS-1 are some of the positively charged residues which lie about the cavity. Lys 579 and Arg 590 of GelA-CTD arc conserved in GelB-CTD. GelB-CTD makes a conservative substitution at Lys 576 where the positive charge is conserved. Other positive charges, such as Lys 604 and Lys 646 of GelA-CTD, become polar, but uncharged residues in GelB-CTD. Overall, there arc fewer positively charged residues in the TBS-1 region of GelB-CTD than found in either GelA-CTD or Cll-Ctd. The loop region connecting blades III and IV in GelB-CTD, which shows intermediate homology to GclA-CTD, required slight rebuilding due to the insertion of Leu 659 in GelB-CTD. The insertion makes it impossible to model the C α positions of the loop residues identically, so it is modeled to have a different structure than cither GelA-CTD of Cll-Ctd. Pro 6 ! 4 0 f GclA-CTD is conserved in GelB-CTD but docs superimpose due to the rebuilding of the loop. Asn 61 ■ and Ala 6!2 arc different in GelB-CTD, but arc identical to residues seen in the Cll-Ctd structure. Mutants of GelA-CTD That Don't Inhibit Membrane Dependant Activation of GelA Are Clustered Within The TIMP-2 Binding Site. interaction of the GclA-CTD with cell surface is essential for activation of the pro-enzyme. Consequently membrane dependent activation of GelA is competitively inhibited in the presence of the recombinant GelA-CTD (sec introduction and discussion). The results we have reported earlier support the hypothesis that assembly of MMP/ΩMP-2/GelA-CTD complex promotes activation of GelA and inhibition of GelA activation in the presence of excess of GelA-CTD is due to a direct competition with the binding of GelA to the inhibitor TI P-2 in the complex. A direct approach to the question whether the assembly of this complex is indeed a prerequisite for GelA activation is to determine whether activation inhibition and TIMP-2 binding properties of GelA-CTD can be separated. Therefore we investigated the ability of all 50 GelA-CTD mutants described above to inhibit membrane dependent activation of GelA in vitro. Increasing amounts of purified WT or mutant GelA-CTD protein was added to membrane GelA activation reaction and the amount of remaining proenzyme species, a measure of activation inhibition, was analyzed on zymograms. The results arc presented in Fig. 6. Most noticeable, is the fact that point mutations outside of the TIMP-2 binding site have inhibited GelA activation as did WT GelA-CTD (T2 Ai + phenotype). Furthermore, the only point mutations which showed a loss in activation inhibition were those found in the TI.MP-2 binding site described above. However, mutants that exhibited a dramatic loss of TIMP-2 binding activity (Ki/Kd>100) segregated into two groups. Mutants of Lys 5 6, Arg 590 , and Trp S74 completely failed to inhibit GelA activation (T2-Ar phenotype). Mutants of Asp 61 ϊ , and Lys 646 were indistinguishable from WT, while mutant Glu 6 ] +Gly 65 ' → Arg shown only a slight loss of activation inhibition activity. Mutants Asp 656 and Tyr 636 exhibited a significant loss of TlMP-2 binding (Ki/Kd=10) and a comparable loss of activation inhibition activity. Mutant Lys 604 showed a considerable loss in T1MP-2 binding (Ki Kd=25) but had little or no effect on activation inhibition. All other mutants (see table 1 and Fig. 6) characterized by a very moderate loss of TTMP-2 binding (Ki Kd<10) and were indistinguishable from WT in the activation inhibition assay. Thus point mutants of residues in the TIMP-2 binding site do not always show a complete correlation between the degree of loss of T1MP-2 binding and their respective loss of activation inhibition activity. Mutants that do show such correlation arc distributed between TBSl and 2. Those with severe loss of both functions (Trp 574 , Lys 576 , and Arg590) arc clustered together in the TBS-1 region of the TIMP-2 binding site (see Fig. 1). Two mutants with moderate effect on both functions (Asp 656 and Tyr 636 ) arc found in TBS2. Two mutants with the greatest disparity in effect on TiMP-2 binding and activation inhibition (Asp 61 5 and Lys 646 ) are found on the border between TBSl and 2. Finally it is important to note an absence of the mutants with T2b + Ar phenotype. Discussion. Since GclA-CTD disp.iys pscudo four-fold symmetry, it is interesting to consider what structural features distinguish lh: T1MP-2 binding site located roughly at the interface between blades III and IV from similar sites whic would be found at the interfaces between the three other blades. A GRASP representation of the G;IA-CTD structure with electrostatic potentials displayed at the surface of the molecule shows that tic interface between blades III and IV is unique in having a high concentration of positive charge (Fig. 3) located near the interface. Furthermore, the outermost strand of blade IV is unique in the GclA-CTD structure in that it forms a regular anti-parallel P-strand with no β-bulgcs as seen in blades II and III. The fourth strand of blade 1 contains no β-bulgcs, but its backbone H- bonding pattern with tie third strand is significantly distorted by the presence of cis prolinc, Pro 500 . Cis prolines are identified in the fourth strands of all the blades except IV. Thus, the highly localized positive charge and a canonical β-strand conformation of an adjacent blade would, in part, create a unique binding surface which would not be found at related positions of this highly symmetrical molecule. Having defined a T1M7-2 binding site on GelA-CTD, it is possible to look at known structures and sequences of related MMPs and develop an idea of how binding and specificity arc achieved. The two basic assumptions in such an analysis arc that 1) all related MMP sequences adopt the same fold as described for GclA-CTD and Cll-Ctd and 2) TIMP-1 binds Gel B-Ctd in a manner comparable to the T1MP-2 binding of GelA-CTD. If these two assumptions are true than some interesting observations on the narjre of TIMPs binding to MMPs may be credibly made and are discussed below. 1) The positively, charged residues in TBS-1 of GelA-CTD are required but not sufficient for binding TIMP-2. While the mutation studies show that these residues arc clearly required for full TIMP-2 binding activity, the fact that many of these charged residues arc conserved in MMPs which are net known to bind TIMP-2 suggests that the presence of these residues is not sufficient for causing TIMP-2 binding. TTMP-2 has a negatively charged C-terminal tail sequence, EFLDEEDP, which when removed shows a reduced binding kinetics profile similar to that of TIMP-1 (30). TIMP-1 does not have a negatively charged sequence at its C-tcrminus. Since electrostatic forces often effect long range interactions between molecules, the positive charges may serve to draw the TIMP-2 molecule near the binding site of GclA-CTD prior to docking. Once bound, the electrostatic interactions arc maintained, but van der Waal forces predominate in directing full, specific binding. It is possible that the negative charges described in the TBS-1 region of other non-TIMP binding MMPs reduce the effect of the long range interaction and also minimize the electrostatic interaction between the negatively charged TIMP-2 sequence and the conserved positively charged residues of these MMPs. It is also interesting to note that Gel B, which specifically binds TIMP-1, has two fewer positively charged residues than GelA in the TIMP-2 binding surface. Perhaps, these two residues, Lys 604 and Lys 646 , play a role in binding the negatively charge tail of TIMP-2. Also, Lys 595 and Lys 597 , which were not mutated in this study, but are near the binding site, may interact with the TIMP-2 tail. Lys 597 is of particular interest since it is not conserved in any of the other MMPs. 2) Interaction With TBS-1 Is Likely To Contribute More Than TBS-2 to Specificity of TIMP-2 Binding to GelA-CTD. GclA-CTD and Gel B-CTD share considerable homology in the TBS-2 region so specificity will most likely not be determined in that region. Presumably, TTMP-1 and TlMP-2 will bind the TBS-2 region similarly in both molecules. The region of the TIMP-2 binding site that diverges the most between GelA and B arc found in TBS-1. Here, Gel B is missing two positively charged residues. Also, sequence analysis and model comparison show the two would have different non-polar cavities. The Gel B cavity is deeper and broader than that of GelA. Furthermore, the loop Ala 609 - Asp 6 ! 5 connecting blades III and IV of GclA-CTD is different than that of Gel B-Ctd. The loop differs in both sequence and backbone structure by virtue of an insertion of a Leu residue in the Gel B sequence. „„,.„ «« PCT/U O 98/12309 3) Van der Waal forces play a major role in TIMP-2 binding and specificity. The TIMP-2 binding site of GclA-CTD represents a broad surface which is conservatively estimated to cover just over 1000 A ? and is composed mainly of uncharged residues. Of the charged residues in the binding site, many arc found in the C-tcrminal domains of non-TIMP binding MMPs suggesting that the presence of the charged residues alone is not enough to account for binding. Likewise, the fact that GelA-CTD shares so many charged residues in common with Gel B-Ctd suggests that specific binding of TIMP-2 is not a result of simple electrostatic interactions. Most likely, the strength and specificity of the binding comes as much from van der Waal interactions as from electrostatic attraction. Biochemical studies have shown that TIMP-2 binding to GclA-CTD is sensitive to low pH and ionic detergent but resistant to high salt (20,30). These results suggest that there is both a significant ionic and van der Waal component to the TIMP-2 binding of GclA-CTD. The TIMP-2 binding site of GclA-CTD described in this paper represents a broad surface of approximately 1000 A* with a high positively charged region clustered about a hydrophobic cavity and an extended, mostly uncharged, van der Waal contact surface. The charged region of the site accounts for the pH and ionic strength dependence of binding, while the cavity and broad, van der Waal surface of the site accounts for the requirement of detergent to fully disassociate the complex. One of the most prominent sequence characteristics of non-TIMP binding MMPs is their propensity to have negatively charged residues in or near the cavity in TBS-1. These charges were seen as potentially having a detrimental effect on T1MP-2 binding. As noted earlier, besides GelA and B, only MTl-MMP is identified as not having negative charges at residues found in or near the cavity. Furthermore, as seen in Fig. 4, many of the sequence features shared by GelA and B are also found in MTl-MMP. Pro 6 ! 4 , Asp 6 ' 5 , and Asp 656 residues of GelA are conserved in MTl-MMP as well. While there are still many sequence features among the TIMP-2 binding site residues not shared by GelA and MTl-MMP, MTl-MMP is by far the most homologous of the non-Gclatinasc MMPs. Taken together, these observations suggest that MTl-MMP may be able to bind TIMP-2. In fact recent observations support this conclusion (25,26). Interaction of inhibitors with pro-gelatiπases is mediated by its C-terminal domain (20-22). The TIMP-2 C-tcrminal domain is 67 residues long from Cys i28 - Pro !94 . It has six cysteines which, by analogy to TlMP-1, arc assumed to form three disulfidc bonds (31). Thus, the C-tcrminal domain of TIMP-2 is likely to be compact and globular. The C-tcrminal portion is separated from the N- tcrminal-domain by only a single residue, Glu' 27 , so the N- and C-tcrminal domains of T1MP-2 must be located extremely close to one another in space. Given the large surface area of the TIMP-2 binding site of GclA-CTD, it is possible that portions of the N-tcrminal domain of TIMP-2 also participate in binding. Since the N-tcrminal domains of TIMP-1 and TIMP-2 show greater homology (44% identity) than their respective C-tcrminal domains (27% identity) and the TBS-2 sections of GelA and B are far more similar than their TBS-1 regions, it is possible that portions of the N- terminal domain of TIMP-2 binds blade [V residues of Gcl-Ctd. This would mean that TBS-1 of GelA-CTD is bound by the C-tcrminal portion of TIMP-2. As stated above, the C-tcrminal domain of TIMP-2 contains a negatively charged sequence which is required for full binding activity. TBS- has a lot of positively charged residues, particularly in GelA, and based on sequence and model comparison of GelA and B shows far less sequence and structural homology than in TBS-2. For this reason, it is likely that TBS-1 of GelA determines its specificity for TIMP-2 as opposed to T1MP-1. Furthermore, assuming the C-tcrminal domain of TIMP-2 is not elongated, portions of TBS-2 may in fact bind parts of the N-terminal domain of TIMP-2. GelA is a multi-domain protein containing a catalytic domain, a domain with three type II fibroncctin- like repeats, and a C-tcrminal domain. The quaternary arrangement of these domains is still unknown. Biochemical evidence from deletion studies and crosslinking experiments suggest that active GelA is bound simultaneously at its catalytic and C-tcrminal domains by the N-tcrminal and C- ter inal domains of TIMP-2. TIMP-2 is a relatively small, globular protein (MW = 21 kDa) whose N-terminal portion is compact, adopts an OB-fold (32), and competitively inhibits substrate cleavage by binding the catalytic domain of MMPs. Given the TIMP-2 binding site described in the paper, it may be assumed that the active site of the catalytic domain is located relatively near the interface between blades Hi and r of the C-terminal domain when bound to T1MP-2. Whether the domains of GelA adopt a rigid conformation or tumble freely in solution has yet to be determined and is the subject of future study. Mechanism of Cell surface GelA Activation. The soluble MMP, GelA, is recruited to the cell surface where it is activated in a MTl-MMP dependent fashion (reviewed 33). The initial MTl-MMP dependent Asn 37 -Lcu pro-pcptide cleavage is inhibited by excess of TIMP-2 and competitively inhibited by GclA-CTD. Accordingly, truncated GelA that lacks its C-tcrminal domain is not activatablc by this mechanism (13). Thus compelling evidence supports the role of GclA-CTD in recruitment of the proenzyme to the cell surface that is a prerequisite to its activation. The role of TIMP-2 in this mechanism is more controversial. It is clear that the recombinant GelA-CTD can interact with cell surface via binding to the activated MT1- MMP TIMP-2 complex to form a tri-molccular complex of activated MTl-MMPmMP-2 GclA-CTD. It is also possible to demonstrate that carefully titrated amounts of T1MP-2 can increase the efficiency of activation in cell membrane dependent, TIMP-2 depleted system. These results support the hypothesis that assembly of MTl-MMP/TIMP-2/GclA-CTD complex promotes cell surface GelA activation. Conversely, it has become evident that soluble MTl-MMP lacking its transmembranc domain can faithfully cleave GelA propeptide at Asn 37 -Leu (26). In this soluble purified system, TIMP-2 functions solely as a specific MTl-MMP inhibitor.Clcavagc of the GelA propeptide docs not depend on the presence of its C-terminal domain and, contrary to membrane dependent GelA activation, truncated GelA is a substrate for soluble activated MTl-MMP. Thus it is essential to ascertain by other approaches whether the assembly of the MTl-MMP/TIMP-2 GclA complex on the cell membrane is indeed a prerequisite for GelA activation. Since inhibition of GelA activation in the presence of excess of GclA-CTD is due to a direct competition with the cell surface binding of GelA, a powerful approach to the above question is to determine whether activation inhibition and TIMP-2 binding properties of GclA-CTD can be separated. Our previous experiments using chemical and protcolytic modifications of GclA-CTD failed to achieve such an effect (9,10). All manipulations of GelA-CTD abolished both itsTIMP-2 binding and the inhibitory activity in the membrane activation assay. Mutagenesis provides an infinitely better approach to address this question. A complete correlation between loss of activation inhibition function and the ability to bind TIMP-2 can be a conclusive evidence that T1MP-2 serves as a mediator of GelA activation, provided that a sufficient number of the TIMP-2 binding site mutants were analyzed. Here, we investigate the ability of all fifty GelA-CTD mutants described above to inhibit membrane dependent activation of GelA in vitro. All mutants outside of the TlMP-2 binding site inhibit GelA activation as well as WT GelA-CTD (T2b + Ai + phenotype). Mutants that exhibited a dramatic loss of TIMP-2 binding activity (Ki/Kd>100) segregated into classes. Mutants with alanine substitution of Lys 576 , Arg 59 °, and Trp 574 failed to inhibit GelA activation (T2b Ai- phenotype). Asp 6 i 5 , and Lys 646 mutants were indistinguishable from WT, while a double with alanine substituting for Glu 6 ] and Arg for Gly 65 ι shown only a slight loss of activation inhibition activity. Other mutants in the TlMP-2 binding site show moderate to no effect on activation inhibition. Importantly no mutants with T2b + Ar phenotype were found. Thus, although all Ar mutants arc concentrated in the TIMP-2 binding site, and no T2b*Aτ mutants were isolated, the correlation between loss of TIMP-2 binding and activation inhibition properties of GclA-CTD mutants is not absolute. This inconsistency can be explained by differences in the assays used to measure the effects of the point mutations. For example, only a part of the TIMP-2 binding site of GclA-CTD described here actually interacts with TIMP-2 bound to MTl-MMP. This may be due to the nature of TIMP-2 interaction with MTl-MMP that exposes only a portion of TIMP-2 C-terminal domain necessary to engage the entire GelA-CTD binding site. Thus only a fraction of mutants in the GclA-CTD TIMP-2 binding site loses the capacity to competitively inhibit activation (T2b- Ai"). In this case the assembly of MTl-MMP/TIMP-2/GelA complex is still a prerequisite for GelA activation and the question remains how the MTl-MMP occupied and inhibited by TIMP-2 is able to cleave the GelA propeptide. Several explanations can be invoked for the mechanism of this reaction. An activation model can be proposed where MT1-MMP TIMP-2 complex acts as a receptor for soluble GelA and forms a tri- molecular presentation complex. Another molecule of TlMP-2 free MTl -MMP may then perform the Asn 37 -Leu pro-pcptide cleavage. As a result, activation of GelA is sensitive to the ratio of the unoccupied activated MTl-MMP to MT1-MMP/T1MP-2 complex and saturating amounts of TIMP-2 inhibit activation. A second set of GelA activation models can be proposed based on the data presented here, if the existence of T2b- Ai" and T2b- Ai* mutants is interpreted to mean that GclA-CTD binds to another, yet to be identified, cell surface receptor and the resulting complex is activated by TIMP-2 free MTl- MMP. For example, binding of the GclA-CTD can occur through interaction with α VβS intcgrin as recently reported (34). The results of mutagenesis indicate that TIMP-2 and putative receptor binding sites on GclA-CTD overlap since no Ai- mutants were found outside of the TIMP-2 binding site. This overlap can potentially explain why TIMP-2 can inhibit binding of GelA to the cell surface even in the case that it is mediated by a receptor other than MT1-MMP T1MP-2 complex. Earlier we have described an analogous but soluble complex of GelB/CII where the C1I and TIMP-1 binding sites of GelB-CTD overlap (22). TABLE 1. Gel A-CTD mutants that affect its TIMP-2 binding activity (Ki / Kd>l). Wild type Kd and mutant Ki was determined as in Figure 7. D and ID - mark mutants affecting TIMP-2 binding directly and indirectly respectively. References: l . Birkedal-Hansen, H. (1995) Proteolytic remodeling of extracellular matrix. Curr. Opin. Cell Biol. 7:728-735. 2. Kleiner, D.E., and Stctlcr-Stcvcnson, W.G. (1993). Curr. Opin. Cell Biol. 5:891-897. 3.Matrisian LM. (1992 ) (Review] Biocssays. 14(7):455-63. 4.Stetler-Stcvenson WG., Aznavoorian S., Liotta LA. (1993) (Review] Annual Review of Cell Biology. 9:541-73. S.Stetler-Stevcnson WG., Liotta LA., Kleiner DE Jr. (1993) (Review] FASEB Journal. 7(15): 1434- 41. 6. Liotta, L.A., Steeg, P.S., and Stctlcr-Stcvcnson, WG. (1991) Cell 64:327-336. 7.Khokha, R., Waterhousc, P., Yagcl, S., Lala, P.K., Overlal, CM., Norton, G., and Dcnhardt, D.T. (1989) Science 243:947-950. 8. Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Sciki M. Nature. 370(6484):61-5, 1994 9.Strongin AY, Marnier BL, Grant GA, Goldberg GI., (1993) J. Biol. Chem.; 268: 14033-14039 lO.Strongin, A.Y., Collier, I.E., Bannikov, G., Marmcr, B.L., Grant, G.A., Goldberg, G.l. (1995) Journal of Biol. Chem. 270(10):5331-8. 11. Ward RV. Atkinson SJ. Slocombe PM. Docherty AJ. Reynolds JJ. Murphy G. (1991) Biochimica et Biophysica Acta. 1079(2):242-6. 12.Azzam HS. Thompson EW. (1992) Cancer Research. 52(16):4540-4. 13. Murphy G. Willenbrock F. Ward RV. Cockett MI. Eaton D. Docherty A J. (1992) Biochemical Journal. 283 ( Pt 3):637-41. 14. Brown PD. Kleiner DE. Unsworth EJ. Stetler-Stevenson WG. (1993 )Kidney International. 43(1): 163-70. IS. Seltzer J. L., Lee A. Y., Akers K. T.. Sudbeck B., Eileen A. Southon, Wayncr E. A., Eisen A. Z. (1994) Experimental Cell Research 213(2):365-74. 1 . Overall CM, Sodek J. (1990) J. Biol. Chem 265, 21141-51 17. Collier, I.E., Wilhelm, S.M., Eisen, A.Z., Marmer, B.L., Grant, G.A., Seltzer, J.L., Kronberger, A.M., He, C, Bauer, E.A., and Goldberg, G.I. (1988) J Biol Chem 263(14):6579- 6587. 18. Atkinson, S.J., et al.(1995) J. Biol. Chem. 270 : 30479-30484. 19. Hiroshi Sato, Takahiso Takino, Takeshi Kinoshita, Kazushi I ai, Yasunori Okada, William G. Stctler- Stevenson, Motoharu Sciki. (1996) FEBS Letters 385, 238-240,. 20. Goldberg, G.L, Marmer, B.L., Grant, G.A., Eisen, A.Z., Wilhelm, S.M. and He, C. (1989) Proc Natl Acad Sci USA 86:8207-8211. 21. Goldberg, G.L, Strongin, A, Collier, I.E., Genrich, L.T., Marmer, B.L. (1992) J Biol Chem. 267(7):4583-91; 22. Fridman, R., Fuerst, T.R., Bird, R.E., Hoyhtya, M., Oclkuct, M., Kraus, S., Komarck, D., Liotta, L.A., Berman, M.L., Stctlcr-Stcvcnson, W.G. (1992) J. Biol. Chem. 267:15398-15405. 23. Willenbrock, F., Murphy G. (1994) American Journal of Respiratory & Critical Care Medicine. 150(6 Pt 2):S165-70. 24. DeClerck, Y.A., Yean, T.D., Lee, Y., Tomich, J.M., Langley, K.E. (1993) Biochemical J 289(Pt l):65-69. 25. Duaπgqing Pci and Stephen Weiss, (1996)J. Biol. Chem. 271 , 9135-9140. 26.Hiroshi Sato, Takeshi Kinoshita, Takahiso Takino, Kazuo Nakayama, and Motoharu Seiki. (1996) FEBS Letters (in press) 27.Libson, A.M., Gittis, A.G., Collier, I.E., Marmer, B.L. , Goldberg, G.I. , Lattman, E.E. (1995) Nature, Structural Biology. 2(l l):938-942. 28. Jones, T.A., et al., (1991) Ada Crystallogr. 47 : 110. 29.U. J., ct al., (1995) Structure 15 : 541-549. 30. Willenbrock, F., et al., (1993) Biochemistry 32 : 330-4337. 31. Williamson, R.A., et al., (1990) Biochem. J. 268 : 267 - 274. 32. Williamson, R., et al., (1994) Biochemistry 33: 11745-11759. 33. Hiroshi Sato, Motoharu Seiki. (1996) A Review J. Biochem. 119, 2C9-215 34. Brooks, P. C, Stromblad, S., Sanders, L C, von Schalscha, T.L.. Aimes, R.T., Steticr- Stevenson, W.G., Quigley, J.P., Cheresh, D.A. (1996) Cell 85:683-695. 35. QUANTA release 4.1.1, (York, England: Molecular Simulations inc. 1990) . 36. Evans, S.V., (1993) SETOR: J. Molcc. Graphics 11: 134-145. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Goldberg, Gregory I. (ii) TITLE OF INVENTION: Method of Cell Surface Activation and Inhibition (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Scott J. Meyer (B) STREET: 800 North Lindbergh Blvd. (C) CITY: St. Louis (D) STATE: MO (E) COUNTRY: USA (F) ZIP: 63167 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Word Perfect 5.0 (Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (Vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 60/026,226 (B) FILING DATE: 17-SEPT-1996 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Meyer, Scott J. (B) REGISTRATION NUMBER: 25,275 (C) REFERENCE/DOCKET NUMBER: WU-2982 ( ix) TELECOMMUNICATION INFORMATION : ( A) TELEPHONE: 314-694-3117 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Asn Trp Ser Lys Asn Lys Lys Thr Tyr lie Phe Ala Gly Asp Lys 5 10 15 Phe Trp Arg Tyr Asn Glu Val Lys Lys Lys Met Asp Pro Gly Phe 20 25 30 Pro Lys Leu lie Ala Asp Ala Trp Asn Ala lie Pro 35 40 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID: O: 2: Arg Ser Gly Arg Gly Lys Met Leu Leu Phe Ser Gly Arg Arg Leu 5 10 15 Trp Arg Phe Asp Val Lys Ala Gin Met Val Asp Pro Arg Ser Ala 20 25 30 Ser Glu Val Asp Arg Met Phe Pro Gly Val Pro Leu 35 40 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Phe Glu Glu Asp Thr Gly Lys Thr Tyr Phe Phe Val Ala His Glu 5 10 15 Cys Trp Arg Tyr Asp Glu Tyr Lys Gin Ser Met Asp Thr Gly Tyr 20 25 30 Pro Lys Met He Ala Glu Glu Phe Pro Gly He Gly 35 40 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Ser Glu Glu Asn Thr Gly Lys Thr Tyr Phe Phe Val Ala Asn Lys 5 10 15 Tyr Trp Arg Tyr Asp Glu Tyr Lys Arg Ser Met Asp Pro Ser Tyr 20 25 30 Pro Lys Met He Ala His Asp Phe Pro Gly He Gly 35 40 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: His Phe Glu Asp Thr Gly Lys Thr Leu Leu Phe Ser Gly Asn Gin 5 10 15 Val Trp Arg Tyr Asp Asp Thr Asn His He Met Asp Lys Asp Tyr 20 25 30 Pro Arg Leu He Glu Glu Asp Phe Pro Gly He Gly 35 40 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (II) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Ser Asp Lys Glu Lys Asn Lys Thr Tyr Phe Phe Val Glu Asp Lys 5 10 15 Tyr Trp Arg Phe Asp Glu Lys Arg Asn Ser Met Glu Pro Gly Pro 20 25 30 Lys Gin He Ala Glu Asp Phe Pro Gly He Asp 35 40 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ser Asp Lys Glu Lys Lys Lys Thr Tyr Phe Phe Ala Ala Asp Lys 5 10 15 Tyr Trp Arg Phe Asp Glu Asn Ser Gin Ser Met Glu Gin Gly Phe 20 25 30 Pro Arg Leu He Ala Asp Asp Phe Pro Gly Val Glu 35 40 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Trp Gly Pro Glu Lys Asn Lys He Tyr Phe Phe Arg Gly Arg Asp 5 10 15 Tyr Trp Arg Phe His Pro Ser Thr Arg Arg Val Asp Ser Pro Val 20 25 30 Pro Arg Arg Ala Thr Asp Trp Arg Gly Val Pro Ser 35 40 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Trp Met Pro Asn Gly Lys Thr Tyr Phe Phe Arg Gly Asn Lys Tyr 5 10 15 Tyr Arg Phe Asn Glu Glu Leu Arg Ala Val Asp Ser Glu Tyr Pro 20 25 30 Lys Asn He Lys Val Trp Glu Gly He Pro 35 40 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Asp Asn Leu Asp Ala Val Val Asp Leu Gin Gly Gly Gly His Ser 5 10 15 Tyr Phe Phe Lys Glu Ala Tyr Tyr Leu Lys Leu Glu Asn Gin Ser 20 25 30 Leu Lys Ser Val Lys Phe Gly Ser He Lys Ser Asp Trp Leu Gly 35 40 45 Cys (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) TOPLOOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Asp Thr His Asp Val Phe Gin Tyr Arg Glu Lys Ala Tyr Phe Cys 5 10 15 Gin Asp Arg Phe Tyr Trp Arg Val Ser Ser Arg Ser Glu Leu Asn 20 25 30 Gin Val Asp Gin Val Gly Tyr Val Thr Tyr Asp He Leu Gin Cys 35 40 45 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Asn Lys Val Asp Ala Val Phe Gin Lys Asp Gly Phe Leu Tyr Phe 5 10 15 Phe His Gly Thr Arg Gin Tyr Gin Phe Asp Phe Lys Thr Lys Arg 20 25 30 He Leu Thr Leu Gin Lys Ala Asn Ser Trp Phe Asn Cys 35 40 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: His Lys Val Asp Ala Val Phe Met Lys Asp Gly Phe Phe Tyr Phe 5 10 15 Phe His Gly Thr Arg Gin Tyr Lys Phe Asp Pro Lys Thr Lys Arg 20 25 30 He He Thr Leu Gin Lys Ala Asn Ser Trp Phe Asn Cys 35 40 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Asp Lys Val Asp Ala Val Tyr Glu Lys Asn Gly Tyr He Tyr Phe 5 10 15 Phe Asn Gly Pro He Gin Phe Glu Tyr Ser He Trp Ser Asn Arg 10 25 30 He Val Arg Val Met Pro Ala Asn Ser He Leu Trp Cys 35 40 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Ser Lys He Asp Ala Val Phe Glu Glu Phe Gly Phe Phe Tyr Phe 5 10 15 Phe Thr Gly Ser Ser Gin Leu Glu Phe Asp Pro Asn Ala Lys Lys 20 25 30 Val Thr His Thr Leu Lys Ser Asn Ser Trp Leu Asn Cys 35 40 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Pro Lys Val Asp Ala Val Leu Gin Ala Phe Gly Phe Phe Tyr Phe 5 10 15 Phe Ser Gly Ser Ser Gin Phe Glu Phe Asp Pro Asn Ala Arg Met 20 25 30 Val Thr His He Leu Lys Ser Asn Ser Trp Leu His Cys 35 40 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Glu He Asp Ala Ala Phe Gin Asp Ala Asp Gly Tyr Ala Tyr Phe 5 10 15 Leu Arg Gly Arg Leu Tyr Trp Lys Phe Asp Pro Val Lys Val Lys 20 25 30 Ala Leu Glu Gly Phe Pro Arg Leu Val Gly Pro Asp Phe Phe Gly 35 40 45 Cys (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Glu Ser Pro Arg Gly Ser Phe Met Gly Ser Asp Glu Val Phe Thr 5 10 15 Tyr Phe Tyr Lys Glu Asn Lys Tyr Trp Lys Phe Asn Asn Gin Lys 20 25 30 Leu Lys Val Glu Pro Gly Tyr Pro Lys Ser Ala Leu Arg Asp Trp 35 40 45 Met Gly Cys

Claims: WHAT IS CLAIMED IS: 1. A target area for screening MMP inhibitors comprising the following residues of the TIMP-2 binding site on the surface of the GelA-CTD domain: Asp 6 ", Gly" 1 , Phe 650 and Tyr 636 . 2. A target area for screening MMP inhibitors comprising the following residues on the TIMP-2 binding site on the surface of the GelA-CTD domain: Asp" 6 , Gly" 1 , Phe" 0 , Tyr" 6 , Asp 6 , Lys 6 ' 6 , Lys 576 , Trp 51 \ Arg" 0 , Lys 575 , Lys 60 ' and Asn 611 . 3. A method of screening for MMP inhibitors comprising determining the effect of a test compound on cell surface activation and inhibition upon reaction at the TIMP-2 binding site of Claim 1. 4. A method of screening for MMP inhibitors comprising determining the effect of a test compound on cell surface activation and inhibition upon reaction at the TIMP-2 binding site of Claim 2.

Inventors: GOLDBERG GREGORY I, GOLDBERG, GREGORY, I.
Applicants: GOLDBERG GREGORY I, UNIV WASHINGTON, GOLDBERG, GREGORY, I., WASHINGTON UNIVERSITY
Application year: 1997
Main classification: C12N9
IPC codes: C07K14, C12N9, C12Q1, G01N33