Increased Backbone Mobility in β-Barrel Enhances Entropy Gain Driving Binding of N-TIMP-1 to MMP-3

The high-affinity inhibition of stromelysin 1 (MMP-3) by tissue inhibitor of metalloproteinases 1 (TIMP-1) helps control tissue remodeling and tumor development. The interaction of N-TIMP-1 with the catalytic domain of MMP-3 has been investigated by titration calorimetry and 15N NMR. Their unfavorable enthalpy of binding of +6.5 kcal mol−1 is unusual among protein–protein associations, deviates from structure-based prediction, and is compensated by a net entropy increase providing at least 18 kcal mol−1 of favorable free energy of binding at a 1 M reference state. The small heat capacity of binding agrees well with the heat capacity predicted from 65% of the surface buried on binding being polar, and suggests that the hydrophobic effect can account for only part of the entropy of binding. Using NMR, binding-induced changes in the backbone of N-TIMP-1 were checked as one possible source of conformational entropy changes. MMP binding slightly increases rigidity in some contact sites in TIMP-1 but increases mobility remotely in the otherwise rigid β-barrel core of N-TIMP-1, increasing 15N relaxation evidence of pico- to nanosecond and micro- to millisecond fluctuations of β-strands A–F. Residual dipolar couplings suggest dynamic deviations from X-ray coordinates of the complex. These suggest that the β-barrel has small backbone conformational fluctuations, while segments of strands βB, βE and βF might experience fluctuations only in their backbone environment. This is a distinctive example of affinity between two well-structured proteins being enhanced by increased conformational entropy in the reservoir of a folding core.