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: 6999373
• 负责人: PETER G.W. GETTINS
• 金额: $ 37.84万
• 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/6999373
• 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利用锁和钥匙型相互作用来抑制靶蛋白酶，但现在已经通过X射线晶体学、NMR光谱学和FRET显示丝氨酸蛋白酶抑制剂采用显著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剂和蛋白酶的变化，丝氨酸蛋白酶抑制剂反应中心环（RCL）插入其自身b-片层A的中心，并且蛋白酶从其初始对接位置移位超过70 A至丝氨酸蛋白酶抑制剂的远端，伴随着蛋白酶活性位点的扭曲和大部分蛋白酶在其到达其最终位置时的“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这样一种复杂的、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，无论是通过受体如LRP的识别和信号传导，还是在丝氨酸蛋白酶抑制剂-蛋白酶复合物中通过切割使蛋白酶不能复活。我们提出了三个具体的目标，一起将使完整的蛋白酶易位，蛋白酶破碎序列在表达的功能，使丝氨酸蛋白酶抑制剂的首选类蛋白酶抑制剂在许多情况下的重要性进行评估。具体目标1将确定丝氨酸蛋白酶抑制剂和蛋白酶结合受体LRP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性质。特异性目的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。我们将使用热力学测量的结合亲和力和NMR光谱，FRET和X射线晶体学，我们已经成功地使用在以前的授权期间，并在适当的情况下，我们的研究结果与功能的后果，通过与达德利斯特里克兰博士合作的结构方法。