Tissue inhibitors of metalloproteinases (TIMPs) inhibit pathological hydrolysis of connective tissue by matrix metalloproteinases (MMPs) which accompanies arthritis, cancer, and degenerative eye diseases. Little is known of how TIMPs bind and inactivate MMPs with subnanomolar affinity. Availability of three-dimensional structures of a TIMP and of its complex with a representative metalloproteinase, in solution, would add understanding of the structural basis of high affinity. Structural NMR characterization will aid rational development of TIMP-derived inhibitors in long-range efforts to develop better, more selective inhibitors of MMPs.
The specific aims are: (1) Refine the NMR structure of the inhibitory domain of human TIMP-1 (N-TIMP-1) to high resolution. (2) Map the surface of N-TIMP-1 perturbed by human stromelysin-1 catalytic domain (MMP-3(DC)), using NMR to create a """"""""footprint"""""""". (3) Measure and interpret close contacts between N-TIMP-1 and MMP-3(DC) needed to orient them about their binding interface. (4) Identify and correct for conformational changes which occur in the solution structures of MMP-3(DC) and of N-TIMP-1 upon complexation. (5) Dock the revised N-TIMP-1 and MMP-3(DC) solution structures. (6) Compare DCp of the association of N-TIMP-1 with MMP-3(DC) with the buried interfacial surface area and polarity found in the solution structure. (7) Compare N-TIMP-1 backbone dynamics in the absence and presence of MMP-3(DC). (8) Probe the means by which the Thr2-to-Leu substitution of N-TIMP-1 introduces selectivity between MMP-3(DC) and MMP-1(DC) (collagenase catalytic domain), using titration calorimetry and NMR.
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