Structural examination of serpin-protein interactions

丝氨酸蛋白酶抑制剂-蛋白质相互作用的结构检查

• 批准号: 6863041
• 负责人: PETER G.W. GETTINS
• 金额: $ 38.75万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-27 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6863041
• 关键词: X ray crystallography; conformation; covalent bond; cysteine endopeptidases; enzyme mechanism; fluorescence resonance energy transfer; low density lipoprotein receptor; nuclear magnetic resonance spectroscopy; protease inhibitor; protein binding; protein protein interaction; protein structure function; proteolysis; serine proteinases; structural biology; subtilisins; thermodynamics

DESCRIPTION (provided by applicant): Although protein serine proteinase inhibitors of the Kunitz, Kazal, and Bowman-Birk families make do with lock-and-key type interactions to inhibit target proteinases, serpins have now been shown by x-ray crystallography, NMR spectroscopy and FRET to employ a remarkable conformational change-based mechanism to inhibit some of the same serine proteinases. This mechanism involves changes to both the serpin and the proteinase, with insertion of the serpin reactive center loop (RCL) into the center of its own b-sheet A, and translocation of the proteinase over 70A to the distal end of the serpin from its initial docking position, with concomitant distortion of the proteinase active site and a "crushing" of a large portion of the proteinase upon it reaching its final location. Whereas these studies have provided a structural explanation of how serpins inhibit serine proteinases, they have not explained why such a complex, error-prone, mechanism is employed. An unproven assumption is that the conformational changes that occur in both serpin and proteinase are exploited subsequently for other downstream events, whether recognition and signaling via a receptor such as LRP, or else rendering the proteinase incapable of revival, through cleavage while in the serpin-proteinase complex. We propose three specific aims that together will enable evaluation of the importance of the full proteinase-translocating, proteinase-crushing sequence in expression of the functions that make serpins the preferred class of proteinase inhibitors under many circumstances. Specific Aim 1 will determine the structural requirements and functional consequences of serpin and proteinase binding to the receptor LRP. Specific Aim 2 will determine the structure of the mosquito serpin AFXa, its mechanism of inhibition of human factor Xa and the properties of the AFXa-Xa complex in relation to LRP binding and signaling. Specific Aim 3 will determine the conformational changes that occur in caspases, cathepsins and subtilisin-like proteinases upon formation of covalent complexes with serpins. We will use thermodynamic measurements of binding affinity and structural approaches of NMR spectroscopy, FRET and x-ray crystallography that we have successfully used in the previous grant period and, where appropriate, correlate our findings with functional consequences, through a collaboration with Dr. Dudley Strickland.

描述（由申请人提供）：尽管 Kunitz、Kazal 和 Bowman-Birk 家族的蛋白丝氨酸蛋白酶抑制剂通过锁钥匙型相互作用来抑制靶蛋白酶，但 X 射线晶体学、NMR 光谱和 FRET 现已表明丝氨酸蛋白酶抑制剂采用显着的基于构象变化的机制来抑制某些相同的丝氨酸蛋白酶。该机制涉及丝氨酸蛋白酶抑制蛋白和蛋白酶的变化，将丝氨酸蛋白酶抑制蛋白反应中心环（RCL）插入其自身的b片A的中心，并且蛋白酶从其初始对接位置易位超过70A至丝氨酸蛋白酶抑制蛋白的远端，同时蛋白酶活性位点发生变形，并且在到达其最终位置时大部分蛋白酶被“压碎”。尽管这些研究提供了丝氨酸蛋白酶抑制剂如何抑制丝氨酸蛋白酶的结构解释，但它们没有解释为什么采用如此复杂、容易出错的机制。一个未经证实的假设是，丝氨酸蛋白酶抑制剂和蛋白酶中发生的构象变化随后被用于其他下游事件，无论是通过 LRP 等受体进行识别和信号传导，还是通过丝氨酸蛋白酶抑制剂-蛋白酶复合物中的裂解使蛋白酶无法复活。我们提出了三个具体目标，这些目标共同将能够评估完整的蛋白酶转位、蛋白酶破碎序列在功能表达中的重要性，这些功能使丝氨酸蛋白酶抑制剂在许多情况下成为首选的蛋白酶抑制剂。具体目标 1 将确定丝氨酸蛋白酶抑制剂和蛋白酶与受体 LRP 结合的结构要求和功能后果。具体目标 2 将确定蚊子丝氨酸蛋白酶抑制剂 AFXa 的结构、其抑制人因子 Xa 的机制以及 AFXa-Xa 复合物与 LRP 结合和信号传导相关的特性。具体目标 3 将确定半胱天冬酶、组织蛋白酶和枯草杆菌蛋白酶样蛋白酶在与丝氨酸蛋白酶抑制剂形成共价复合物时发生的构象变化。我们将使用我们在上一个资助期间成功使用的结合亲和力的热力学测量和核磁共振波谱、FRET 和 X 射线晶体学的结构方法，并在适当的情况下，通过与 Dudley Strickland 博士的合作，将我们的发现与功能结果联系起来。