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

Abstract

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.

Keywords: calorimetry; conformational change; enzyme inhibitor; matrix metalloproteinase (MMP); protein dynamic; protein engineering; protein-protein interaction; thermodynamics; tissue inhibitor of metalloproteinase (TIMP); zinc.

FIGURE 1.

A schematic view of the interactions between the core of the NT1 interaction ridge and the active site and substrate binding subsites of MMP1. The N-terminal five residues of TIMP-1 are colored cyan, residues 67–70 and 98–99 are gray, the Cys-1–Cys-70 and Cys-3–Cys-99 disulfide bonds are yellow, oxygen atoms are red, and nitrogen atoms are blue. Residues 214–221 of MMP1 are represented by the blue ribbon, and the catalytic Zn²⁺ is represented by a purple sphere.

FIGURE 2.

Titration of different N-TIMPs by MMP14c in HEPES buffer, pH 7.4, at 310 K. A, aliquots (20 μl) of MMP14c (120 μm) were injected into NT1 (23 μm). B, aliquots (20 μl) of MMP14c (120 μm) were injected into NT1 T98L mutant (22 μm). C, aliquots (20 μl) of MMP14c (148 μm) were injected into NT2 (16 μm). The heats of binding were measured as described under “Experimental Procedures.”

FIGURE 3.

Plot of observed enthalpies of binding against buffer ionization enthalpies for the interactions of MMP1c and MMP14c with different N-TIMPs. A, titrations of NT2 (circles), NT1 (squares), and NT1 T98L mutant (triangles) with MMP-4c. B, titrations of NT2 S2D/S4A mutant (circles) and S2D mutant (squares) with MMP1c. Data for the titration of WT NT2 with MMP1c, taken from Wu et al. (6), are represented by the line with no data points. Titrations were generally not repeated because of the large amounts of purified proteins required for each experiment (∼1500 μg of MMP and 700 μg of N-TIMP). However, data from four titrations, conducted in different buffers, were analyzed by linear regression analysis to determine ΔH_int^o and N_H+. Also data from four titrations at different temperatures were similarly analyzed to determine ΔC_p for each MMP/inhibitor pair so that the two key parameters, ΔH_int^o and ΔC_p, for each NTIMP·MMP interaction were derived from four titrations.

FIGURE 4.

Plot of observed interaction enthalpies of binding against temperature (K) for different N-TIMP interactions with MMPs. A, data for the titration of NT2, NT1, and the T98L mutant of NT1 with MMP14c are displayed as circles, squares, and triangles, respectively. B, results for the titrations the NT2 S2D/S4A mutant (circles), NT2 S2D mutant (squares), and NT2 (no symbols and taken from Wu et al. (6)) with MMP1c. Results for the titration of MMP3c by the NT1 T98L are represented by filled circles. The lines were generated by linear regression analysis.

FIGURE 5.

Isothermal calorimetric titration of NT2 S2D/S4A mutant with MMP1c in different buffers at 291K. Left, aliquots (20 μl) of MMP1c (150 μm) were injected into NT2 (30 μm) in ACES buffer, pH 7.4. Right, aliquots (20 μl) of MMP1c (150 μm) were injected into NT2 (30 μm) in HEPES buffer, pH 7.4. The heats of binding were measured as described under “Experimental Procedures.”

FIGURE 6.

Superimposed ribbon structures of NT1 extracted from crystallographic structures of different complexes and solution NMR structures of the free protein. The structures from the complexes are colored as follows: pink, MMP1c (PDB code 2jot); cyan, MMP3c (PDB code 1uea); blue, MMP14c (PDB code 3ma2); purple, MMP10c (PDB code 3v96). The free NT1 structures were chains 1, 20 and 29 from PDB code 1d2b and are colored light gray. The structures were superimposed and displayed using CHIMERA (43).

FIGURE 7.

Contributions of ΔH_int, ΔS_solv, and ΔS_conf to N-TIMP·MMP interactions. A, bar chart of values for different interactions. Black, ΔH_int; white, ΔS_solv; gray, ΔS_conf. B, linear inverse relationship between ΔS_solv and ΔS_conf for all characterized interactions. Data for interactions with MMP1c are denoted by circles, MMP3c by squares, and MMP14c by triangles; WT N-TIMPs are represented by open symbols and engineered N-TIMPs by filled symbols. The units of ΔS are cal/mol. The line, generated by regression analysis, represents the relationship: ΔS_conf = 67.6 − ΔS_solv.