TIMP/METALLOPROTEINASE STRUCTURE AND INTERACTIONS

描述：金属蛋白酶组织抑制剂（TIMPs）抑制 基质金属蛋白酶引起的结缔组织病理性水解 （MMPs），其伴随关节炎、癌症和退行性眼病。 关于TIMP如何结合并以亚纳摩尔浓度抑制MMP的研究知之甚少。 亲和力 TIMP的三维结构的可用性及其在临床上的应用 与代表性金属蛋白酶的复合物，在溶液中，将添加 了解高亲和力的结构基础。 结构核磁共振 表征将有助于合理开发TIMP衍生的抑制剂， 长期努力开发更好的，更具选择性的MMPs抑制剂。 具体目标是：（1）优化抑制剂的NMR结构 人TIMP-1（N-TIMP-1）结构域的高分辨率。 (2)映射表面 受人基质分解素-1催化结构域（MMP-3（DC））干扰的N-TIMP-1， 利用核磁共振来创建“足迹”。 (3)测量和解读密切接触者 在N-TIMP-1和MMP-3（DC）之间需要定位它们的结合 接口. (4)识别并纠正发生的构象变化 MMP-3（DC）和N-TIMP-1在络合后的溶液结构中。 (5)对接修改后的N-TIMP-1和MMP-3（DC）溶液结构。 （六） 比较N-TIMP-1与MMP-3（DC）结合的DCp与埋藏的 在溶液结构中发现的界面表面积和极性。 （七） 比较在MMP-3（DC）不存在和存在下N-TIMP-1骨架动力学。 (8)探讨N-TIMP-1的Thr 2-to-Leu取代的方法 引入MMP-3（DC）和MMP-1（DC）（胶原酶）之间的选择性 催化域），使用滴定量热法和NMR。

DESCRIPTION: 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.