Thermodynamic Basis of Selectivity in the Interactions of Tissue Inhibitors of Metalloproteinases N-domains with Matrix Metalloproteinases-1, -3, and -14*

The four tissue inhibitors of metalloproteinases (TIMPs) are potent inhibitors of the many matrixins (MMPs), except that TIMP1 weakly inhibits some MMPs, including MMP14. The broad-spectrum inhibition of MMPs by TIMPs and their N-domains (NTIMPs) is consistent with the previous isothermal titration calorimetric finding that their interactions are entropy-driven but differ in contributions from solvent and conformational entropy (ΔSsolv, ΔSconf), estimated using heat capacity changes (ΔCp). Selective engineered NTIMPs have potential applications for treating MMP-related diseases, including cancer and cardiomyopathy. Here we report isothermal titration calorimetric studies of the effects of selectivity-modifying mutations in NTIMP1 and NTIMP2 on the thermodynamics of their interactions with MMP1, MMP3, and MMP14. The weak inhibition of MMP14 by NTIMP1 reflects a large conformational entropy penalty for binding. The T98L mutation, peripheral to the NTIMP1 reactive site, enhances binding by increasing ΔSsolv but also reduces ΔSconf. However, the same mutation increases NTIMP1 binding to MMP3 in an interaction that has an unusual positive ΔCp. This indicates a decrease in solvent entropy compensated by increased conformational entropy, possibly reflecting interactions involving alternative conformers. The NTIMP2 mutant, S2D/S4A is a selective MMP1 inhibitor through electrostatic effects of a unique MMP-1 arginine. Asp-2 increases reactive site polarity, reducing ΔCp, but increases conformational entropy to maintain strong binding to MMP1. There is a strong negative correlation between ΔSsolv and ΔSconf for all characterized interactions, but the data for each MMP have characteristic ranges, reflecting intrinsic differences in the structures and dynamics of their free and inhibitor-bound forms.

The four tissue inhibitors of metalloproteinases (TIMPs) are potent inhibitors of the many matrixins (MMPs), except that TIMP1 weakly inhibits some MMPs, including MMP14. The broad-spectrum inhibition of MMPs by TIMPs and their N-domains (NTIMPs) is consistent with the previous isothermal titration calorimetric finding that their interactions are entropy-driven but differ in contributions from solvent and conformational entropy (⌬S solv , ⌬S conf ), estimated using heat capacity changes (⌬C p ). Selective engineered NTIMPs have potential applications for treating MMP-related diseases, including cancer and cardiomyopathy. Here we report isothermal titration calorimetric studies of the effects of selectivitymodifying mutations in NTIMP1 and NTIMP2 on the thermodynamics of their interactions with MMP1, MMP3, and MMP14. The weak inhibition of MMP14 by NTIMP1 reflects a large conformational entropy penalty for binding. The T98L mutation, peripheral to the NTIMP1 reactive site, enhances binding by increasing ⌬S solv but also reduces ⌬S conf . However, the same mutation increases NTIMP1 binding to MMP3 in an interaction that has an unusual positive ⌬C p . This indicates a decrease in solvent entropy compensated by increased conformational entropy, possibly reflecting interactions involving alternative conformers. The NTIMP2 mutant, S2D/S4A is a selective MMP1 inhibitor through electrostatic effects of a unique MMP-1 arginine. Asp-2 increases reactive site polarity, reducing ⌬C p , but increases conformational entropy to maintain strong binding to MMP1. There is a strong negative correlation between ⌬S solv and ⌬S conf for all characterized interactions, but the data for each MMP have characteristic ranges, reflecting intrinsic differences in the structures and dynamics of their free and inhibitor-bound forms.
Isothermal titration calorimetry can dissect the sources of the free energy changes for protein interactions with other macromolecules and ligands (1). Measurements of the heat released or absorbed during the titration of a protein with a binding partner can quantify the enthalpy of binding (⌬H), association constant K a , and stoichiometry (N) (1), allowing the calculation of the changes in free energy (⌬G) and entropy (⌬S) of binding. Titrations in buffers with different enthalpies of ionization quantify ionization changes linked to binding, whereas the heat capacity change for binding, ⌬C p , measured as the temperature dependence of ⌬H, can be used to estimate the change in solvation entropy on binding (⌬S solv ). These parameters can be correlated with the character of the interaction interface and differences in dynamics and solvation between the free and bound conformer populations (2)(3)(4).
Previously, we used isothermal titration calorimetry to elucidate the thermodynamic profiles of interactions between the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases-1 and -2 (NT1 and NT2) 3 and the catalytic domains of matrix metalloproteinases -1 and -3 (MMP1c and MMP3c) (5,6). TIMP1 and TIMP2 are two of the four human TIMPs, endogenous, broad spectrum, slow binding, high affinity inhibitors of the 23 human MMPs and several disintegrin-metalloproteinases (7). The MMPs catalyze the proteolysis of all polypeptide components of the extracellular matrix and are crucial for tissue remodeling, wound healing, embryo implantation, cell migration, shedding of cell surface proteins, and release of bioactive peptides (8,9); the unregulated activities of various MMPs have been linked to many disease processes including arthritis, heart disease, and tumor metastasis (8).
Here, we have investigated how the mutations in NT1 and NT2 that modify MMP selectivity affect the thermodynamics of N-TIMP⅐MMP interactions, focusing on the T98L mutation in NT1 with MMP3c and MMP14c and the S2D/S4A double substitution in NT2 (with MMP1c). The thermodynamic profiles of the interactions of these MMPs with WT NT1 and NT2 (6) show that all are driven by entropy increases. Entropy-driven binding has been observed in other proteins that bind to multiple targets, including thioredoxin and protein kinase A (33,34). The solvent entropy change for the interaction, ⌬S solv , can be estimated from the heat capacity change, ⌬C p (35). Using ⌬S solv and ⌬S int , the conformation entropy change for the interaction (⌬S conf ) can be determined to provide information about differences in conformational dynamics between the free and bound forms of the two proteins. The results highlight the contributions of changes in conformational dynamics and solvent entropy (the hydrophobic effect) to differences in binding to different MMPs.

Experimental Procedures
Construction, Expression, Purification, and Folding of NT2 Variants, MMP1c, and MMP3c-Reagents and cells were from the same sources as in previous studies (5, 6, 24 -27, 29, 31). The NT1 and NT2 mutants were generated using QuikChange II Site-Directed Mutagenesis kits (Agilent Technologies). Primers were designed using web-based primer design software program (Agilent Technologies). PCR reactions were carried out at 95°C for 30 s, 55°C for 1 min, and 68°C for 5 min for 30 cycles after a 3-min hot start at 95°C. The PCR products were cloned back into pET-42b vector (Novagen) for expression.
NT2 variants were extracted from inclusion bodies and folded as described previously (6). The native proteins were purified by ion exchange followed by gel filtration with columns (2.5 ϫ 35 cm) of Superdex-75 (Amersham Bioscience), equilibrated, and eluted with 20 mM HEPES buffer, pH 7.4, containing 250 mM NaCl and 20 mM CaCl 2 . The eluate was collected in 6-ml fractions at a flow rate of 0.5 ml/min. Fractions containing folded N-TIMPs were identified by polyacrylamide gel electrophoresis, pooled, and concentrated using Centriplus YM-3 centrifugal filter devices (Millipore). MMP1c and MMP3c were expressed as inclusion bodies in Escherichia coli BL21-CodonPlus Competent Cells, folded and purified as in previous studies (6).
Fluorescence Assays for N-TIMP Activity-The inhibition of MMPs by NT2 and NT1 variants was measured by assaying MMP activities for hydrolysis of fluorogenic substrates as described previously (6,13,14). Assays were conducted in HEPES buffer (20 mM), pH 7.4, containing 250 mM NaCl, 10 mM CaCl 2 , and 50 M ZnCl 2 , which was used also for the dilution of MMP and TIMP samples. The K i app of N-TIMP variants for MMP-1c and MMP-3c at 25°C (298 K) were determined as described previously (14,16).
Isothermal Titration Calorimetry of the Interactions of NT1 and NT2 Variants with MMPs-Protein solutions were dialyzed extensively against various buffers at 20 mM concentrations containing 250 mM NaCl, 10 mM CaCl 2 , and 50 M ZnCl 2 at pH 7.4 and degassed before use. N-TIMPs (12-30 M) were titrated with the MMP (120 -300 M) at different temperatures using a MicroCal VP-ITC microcalorimeter as described previously (5, 6). The instrument was programmed to carry out 14 injections of 20 l each over 40 s, spaced at 300-s intervals (see Figs. 2 and 3). The stirring speed was 300 rpm. The data were analyzed by the software package Origin 5.0 from Microcal Inc., which was used to calculate the enthalpy changes (⌬H) and stoichiometry (N) using a single-site binding model. As in previous studies, only ϳ44% of the recombinant NT1 and NT2 variants were active (5, 6) reflecting inactivation by N-acetylation the N terminus by the bacterium (30). The heat capacity change (⌬C p ), intrinsic enthalpy change (⌬H int ), and ionization change (N Hϩ ) for each interaction were calculated as described below.
Correlation of Thermodynamics with Structure-As discussed previously (5, 6) the ⌬C p o value for a protein-protein interaction is generally considered to be related to changes in nonpolar and polar-accessible surface area, ⌬ASA np and ⌬ASA pol (Equations 1 and 2) on complex formation (where surface burial has a negative sign), The parameterizations of the coefficients for changes in nonpolar and polar surface used here were a ϭ 0.28 Ϯ 0.12 cal mol Ϫ1 K Ϫ1 Å Ϫ2 and b ϭ Ϫ0.09 Ϯ 0.30 cal mol Ϫ1 K Ϫ1 Å Ϫ2 (36). The enthalpy of binding (⌬H o ) at 60°(35) was calculated using the relationship, where c is Ϫ7.27cal mol Ϫ1 Å Ϫ2 , and -is 29.16 cal mol Ϫ1 Å Ϫ2 (4). ⌬H o at 25°C is then calculated using the calculated value for ⌬C p. Polar and apolar surface areas in the interfaces of N-TIMP⅐MMP complexes were measured from atomic coordinates using InterProSurf (37).

Results and Discussion
Effect of the T98L Mutation in NT1 on Its Interactions with MMP3 and MMP14 -For the interactions of NT1, NT2, and the T98L mutant of NT1 with MMP14, enthalpies of binding (⌬H obs ) were determined by isothermal titrations at 291 K ( Fig.  2) in buffers with different enthalpies of ionization (Pipes, Hepes, Mops, Bes, and Aces). These values were analyzed by linear regression analysis of the plot of ⌬H obs against ⌬H ion using the relationship where ⌬H ion is the enthalpy of ionization of the buffer, N Hϩ is the number of protons taken up (positive values) or released to the buffer during the protein-protein interaction, and ⌬H int o is the enthalpy change independent of buffer (5, 6). These analyses ( Fig. 3A) indicate that there is negligible release of protons for the interactions of WT NT1 and NT2 with MMP14 (N Hϩ of ϩ0.06 Ϯ 0.00 and ϩ0.08 Ϯ 0.01, respectively), whereas the interaction with the NT1 T98L mutant releases one proton (N Hϩ of 1.02 Ϯ 0.10). The interactions all have highly unfavorable ionization-independent enthalpy changes, ranging from 9 kcal/mol for NT1 to 14.7 kcal/mol for the NT1 T98L mutant (Table 1). Their free energies of binding (⌬G) were calculated from the K i values determined by inhibition kinetics at 298 using ⌬G o ϭ RTln(1/K i ) (see Table 3). ⌬H obs values from titrations in HEPES buffer at 291, 298, 303, and 310 K ( The value of ⌬C p o is provided by the slope of a plot of ⌬H obs against temperature (Fig. 4A). The ⌬G o values were determined at 298 K, but the ⌬H int values were measured at 291 K. Therefore, to obtain a set of parameters at the same temperature, ⌬C p for the interaction was used to calculate the ⌬H int at 298 K from the value at 291 K (Table 3). T⌬S int values for the mutants were calculated from ⌬G and ⌬H int at 298 K (Table 3). These results indicate that the weaker binding of NT1 to MMP14 relative to NT2 reflects a 7 kcal/mol lower T⌬S int of binding. The increased affinity arising from the T98L mutation results from an increase in T⌬S int that is partly offset by an (unfavorable) increase in the ⌬H int of binding (Table 3).
Further analysis clarified the source of the entropy changes that affect the binding of these N-TIMP variants with MMP14. The overall entropy change, ⌬S int , includes contributions from the interacting proteins (⌬S protein ) and solvent (⌬S solv ), of which the latter can be estimated from the ⌬C p o for the interaction using the relationship where Ts* is the reference temperature (385 K) at which the hydrophobic contribution to ⌬S is zero (4,35). ⌬S protein includes negative, unfavorable effects arising from the loss of translational and rotational freedom T(⌬S trans ϩ ⌬S rot ) on complex formation, which has been estimated to be ϳ3 (Ϯ2.4) kcal/mol for the NT1/MMP3cd interaction (5) and is expected to be similar for other N-TIMP⅐MMP interactions (6). It also includes the change in conformational entropy, ⌬S conf , that encompasses an unfavorable loss of entropy resulting from   MAY 20, 2016 • VOLUME 291 • NUMBER 21

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increased rigidity of the interaction sites of the two proteins in their complex. However, it may also contain favorable entropy increases resulting from increased dynamics in regions distant from the interaction sites in the bound protein populations (5, 6). ⌬C p o values for the interactions with MMP-14 are large and negative and lead to estimates of 32-47 kcal/mol for increases in T⌬S solv for the interactions with NT1, NT2, and NT1 T98L (Table  3). Using these values we estimate that the binding of either NT1 or NT1 T98L to MMP-14c results in energetic costs reflecting conformational entropy changes (T⌬S conf ) of Ϫ14.7 and Ϫ24.0 kcal/ mol for NT1 and NT1 T98L, respectively ( Table 3). The interaction with NT2 is associated with an insignificant change in T⌬S conf (1.2 Ϯ 1.4 kcal/mol). These results show that the weak binding of NT1 to MMP-14c arises from a large conformational entropy penalty. Surprisingly, the T98L mutation increases this penalty, but this increase is exceeded by the enhanced hydrophobic effect (T⌬S solv ), consistent with the hydrophilic to hydrophobic T98L substitution in the NT1 reactive site.
The T98L mutation in NT1 also increases the affinity for MMP-3 by a factor of 4 (15), but the thermodynamic origins of this are different. A more unfavorable ⌬H int (8.2 versus 6.0 kcal/ mol) is exceeded by an increase in T⌬S int (Table 3). Unexpectedly, ⌬H obs for binding to MMP3c increases with temperature (Fig. 4B), indicating a positive ⌬C p value of 124 cal/mol/K. From this we estimate a value of Ϫ10.6 kcal/mol for T⌬S solv , an unfavorable contribution to ⌬G that is compensated by an estimated increase in T⌬S conf of 33 kcal/mol. In contrast, the interaction of MMP3c with WT NT1, characterized previously, has a ⌬C p of Ϫ50, a favorable T⌬S solv of 3.8 kcal/mol, and a smaller T⌬S conf of 17.4 kcal/mol (5).   Interaction of the NT2 S2D and S2DS4A Mutants with MMP-1c-The interactions of these two mutants were characterized with only MMP1c because they do not inhibit MMP3c or MMP14c (18). Linear regression analysis of ⌬H obs values for isothermal titrations at 291 K in buffers with different enthalpies of ionization (Table 4; Figs. 3 and 5) indicates fractional release and uptake of protons, (N Hϩ ) of Ϫ0.47 and ϩ0.46, respectively, for the S2D and S2D/S4A mutants compared with a negligible uptake of ϩ0.14 previously determined for WT NT2. This analysis also shows that the ionization-independent ⌬H int for the single site mutant, S2D, is 9.5 kcal/mol, much more unfavorable than that for the double mutant, S2D/S4A (1.5 kcal/mol; Table 4), contributing to the weaker binding of S2D shown by ⌬G values (calculated from the K i values) of Ϫ10.2 kcal/mol for S2D and Ϫ14.5 kcal/mol for S2D/S4A (see Table 6). As previously discussed, ⌬H obs values from titrations at different temperatures ( Table 5, Fig. 4B) were used to determine ⌬C p o . This in turn was used to calculate the ⌬H int at 298 K and to estimate ⌬S solv . Values of T⌬S int for both mutants, calculated from ⌬G and ⌬H int values at 298 K, show that the entropy contribution for S2D is ϳ2 kcal/mol greater than for the S2D/S4A mutant. The thermodynamic parameters of S2D/ S4A are similar to those for WT NT2, but the weaker binding of the S2D mutant reflects a larger enthalpy penalty that is only partly compensated by the increase in T⌬S int ( Table 6). The source of the entropy driving the interactions WT NT2 and the mutants with MMP1c was determined as described in the previous section using solvent entropy changes (⌬S solv ) estimated from the heat capacity changes for the interactions. The values of T⌬S conf at 298 K indicate that conformational entropy increases contribute Ϫ9.3 and Ϫ11.0 kcal/mol to ⌬G for interactions of S2D and S2DS4A with MMP1c, contrasting with WT NT2, which has an unfavorable T⌬S conf of Ϫ3.4 kcal/mol, presumably derived from increased rigidity in the interaction interface. The thermodynamic parameters previously determined for the interaction of NT1 with MMP1c are also given in Table 6.    MAY 20, 2016 • VOLUME 291 • NUMBER 21

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Relationship of Thermodynamic Profiles to Structures-Like previously studied N-TIMP⅐MMP interactions (5, 6) the present interactions have minimal to strongly positive ⌬H int values and are driven by large entropy increases. This helps to explain how TIMPs inhibit numerous matrixins whose active sites are adapted to binding diverse biological targets.
Crystallographic structures relevant to the interactions with MMP14 are the TIMP2⅐MMP14c complex, pdb 1BUV, and the complex of the V4A/P6V/T98L of NT1 with MMP14c, pdb 3MA2 (24,26). A model of the T98L NT1 mutant with MMP3c was generated from pdb 1UEA (23) by truncating residues 126 -180, changing Thr-98 to Leu followed by energy minimization. The V4A, P6V, and T98L mutations in NT1 individually lower the K i for MMP14c 2-3 fold, suggesting that they contribute additively to the ϳ10-fold increase in affinity of the triple mutant (14) without a major conformational change, supporting the use of the 3MA2 structure (26) as a template. The areas of apolar and polar surface in the interfaces of the models were analyzed to predict the values of ⌬C p and ⌬H (at 25°C) for the interaction (27,28) for comparison with those determined experimentally (Table 7). These values show reasonable agreement for the MMP14c complexes, but the experimentally determined ⌬C p values for the MMP3c complexes stand out as being far less negative than those calculated from the structures.
We previously developed models of complexes of NT2 and its S2DS4A mutant with MMP1c (18). These suggested that the double mutation results in more extensive contacts of NT2 Asp-2 with the S1Ј pocket of MMP1c relative to Ser2 in WT NT2 (18). The selective inhibition of MMP1c by the S2D/S4A and S2D mutants appears to result from the unique presence of R114 in MMP1c (see Fig. 1), which is replaced by uncharged residues in other human MMPs except for MMP21 (Lys) and MMP23 (His). In contrast, the model of the complex of the NT2 S2D/S4A mutant with MMP-3c shows increased separation in the S1Ј pocket, suggesting that charge repulsion between Asp-2 of the inhibitor and the catalytic site Glu-202 of MMP-3c is responsible for the extremely weak binding (18). In the complex of the S2D/S4A mutant with MMP1c, favorable electrostatic interactions between Asp-2 of the NT2 mutant and Arg214 in MMP-1 allows high affinity binding. The greater polarity of Asp as compared with S2 of WT NT2 also results in less negative ⌬C p values for the interactions of this mutant with MMP1. This leads to lower (estimated) T⌬S solv values and increased values for T⌬S conf implying that the reduced hydrophobic effect is   compensated by enhanced conformational dynamics. We propose that structural adjustments in the interaction interface resulting from the insertion of the negatively charged Asp-2 are transmitted through the protein structures to more distant sites, reducing stability and increasing conformational dynamics (5).
Conformational Dynamics and Selectivity in MMP Inhibition-The disagreement between the experimental and calculated values for ⌬C p for the NT1/MMP3c interaction was pre-viously attributed to large conformational differences the structures of the both proteins in their free and bound states (19,22,23). As a result, the areas of polar and apolar surface quantified from the interaction interface in the complex differ from those buried during the interaction process (5,6). In contrast, in the NT1⅐MMP1c complex, the structure of MMP1c shows only minor differences when compared with the structure of the MMP1c (11). These analyses are misleading because they treat the crystallographic structures as static models, whereas they are the average structures of assemblies of conformers (38,39). The process of complex formation results in the selection of populations of conformers that differ from those in the free state; the selected populations differ in conformational dynamics, solvent interactions, and structure. Fig. 6 compares the structures of the NT1 components from the crystallographic structures of various MMP complexes and with two chains from the solution NMR structure of free NT1 (11,22,23,26,27) showing large differences, particularly in the loops between ␤-strands A and B and B and C where missing electron density indicates local unfolding. This may expose apolar groups, making a positive contribution to ⌬C p . The core of the reactive site, including the N-terminal five residues is similar in the different complexes but differs from the NMR structure of the free protein (Fig. 6).
Eftink et al. (40) have shown that if an interacting protein has two conformational states that both interact with a binding partner, indicating non-mandatory coupling between binding and conformational change, ⌬C p can have positive or negative values depending on parameters relating to the conformational transition (40). This may explain the "anomalous" ⌬C p values for N-TIMP interactions with MMP3c, including the positive ⌬C p for the interaction of MMP3c with the NT1 T98L mutant.
Previously reported dynamics simulations with NT1 and its T98L mutant suggested that the mutation reduced the flexibility of its interface for MMP-14, including the highly flexible N-terminal region, leading to a reduced ⌬S conf penalty for binding (26). Our experimental data indicate that WT NT1 binding carries a large entropic penalty that reduces T⌬S conf by 12 kcal/ mol. The NT1 T98L mutation increases this penalty by 8 kcal/ mol, and the increased K a comes from an increase in solvent entropy (T⌬S solv ), estimated from the more negative ⌬C p . The reduced flexibility in free NT1 indicated by dynamics simulations appears to be negated by a greater loss of conformational entropy in the NT1⅐MMP14 complex that may arise from additional interactions between Leu-98 of the NTIMP and the MMPs that are not present in the complex with WT NT1. Such interactions lead to a more negative ⌬C p , reflecting an increase in the hydrophobic effect. Fig. 7A shows the strong negative correlation between ⌬S solv (derived from ⌬C p ) and ⌬S conf for all currently characterized N-TIMP⅐MMP interactions (adjusted R 2 ϭ 0.975). The combined data for the interactions of three MMPs with WT and mutant forms of NT1 and NT2 do not include all possible mutant N-TIMP⅐MMP combinations. However, the data for the interactions of the WT N-TIMPs with the three MMPs show a similar correlation between ⌬S conf and⌬S solv . One explanation of ⌬S solv /⌬S conf compensation is that ⌬S conf is calculated from ⌬S solv and ⌬S int ; T⌬S int for the 10 currently characterized interactions has a low variance (57.7 Ϯ 9.4 cal/mol) so that the apparent compensation between ⌬S solv and ⌬S conf is not unexpected. In Fig. 7A, the data points for interactions with each MMP are grouped together, and the graphical summary of thermodynamic parameters in Fig. 7B shows that interactions involving MMP3 have the highest ⌬S conf and lowest ⌬S solv values, whereas those involving MMP14 have the lowest (mostly negative) ⌬S conf and highest ⌬S solv . Interactions between NTIMPs and MMP1 have intermediate values of ⌬S conf and ⌬S solv and the least unfavorable ⌬H int . This is consistent with our previous observation, based on less data, that the character of the MMP has a major influence on the proportions of ⌬S conf and ⌬S solv (6), apparently reflecting intrinsic differences in structure and flexibility between the MMPs. Interactions with MMP14 bury more nonpolar surface than those with MMP1, resulting in a more positive ⌬S solv . MMP3c has a higher conformational flexibility than MMP1c and MMP14c (41), reflected in conformational changes in complexes with inhibitors and TIMP-1 (23,42). Possibly, these differences between MMPs might be exploited in designing more selective TIMP mutants or other inhibitors, but this could be limited by the observed compensation between ⌬S conf and ⌬S solv .
Conclusions-Specific protein-protein interactions are pivotal for the control of numerous biological processes including those mediated by regulated proteolysis. Although the four human TIMPs are high affinity metalloproteinase inhibitors, they show little selectivity in their interactions with the 23 human MMPs. This is consistent with entropy-driven binding, which encompasses free energy contributions from the relatively nonspecific hydrophobic effect (solvent entropy changes) and/or changes in conformational entropy. The mutually compensating ⌬S solv and ⌬S conf , estimated using the key thermodynamic parameter, ⌬C p (44), are important in modulating NTIMP⅐MMP interactions. The character of the MMP strongly affects their proportions, and the magnitude of ⌬H int (Fig. 7B). This is illustrated by the T98L mutation in NT1, which enhances binding to MMP-3 by increasing ⌬S conf and to MMP-14 by enhancing ⌬S solv . The highly conserved N-terminal regions of TIMPs have a high level of flexibility in the free protein (22), a common feature of multi-specific protein interaction sites (45). Mutations in the N terminus have major effects on avidity and selectivity for MMPs. In previous studies, NTIMP mutants identified as having the greatest selectivity against MMP-1 are the NT1 T2R (14) and T2R/V4I mutants (16), whereas that with the greatest selectivity for MMP-1 is the NT2 S2D/S4A mutant (18). These mutations alter electrostatic interactions between the NTIMP and the unique Arg-214 in the MMP S1Ј site, suggesting that the N-terminal region of TIMPs and unique active site features of MMPs are both keys to developing selective inhibitors.