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.

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.这种机制涉及对SERPIN和蛋白酶的变化，将Serpin反应性中心环（RCL）插入其自身B-表A的中心，以及将70a上方的蛋白酶迁移到Serpin的远端，从最初的docking位置与蛋白酶的最终造成蛋白酶的位置相同，并在蛋白酶的最终位置上脱离了蛋白酶，并将其最终的位置覆盖。尽管这些研究提供了关于SERPINS如何抑制丝氨酸蛋白酶的结构解释，但它们尚未解释为什么采用这种复杂，容易出错的机制。一个未经证实的假设是，丝丁酶和蛋白酶中发生的构象变化随后被剥削为其他下游事件，无论是通过LRP等受体识别和信号传导，还是在Serpin-蛋白酶复合物中通过断裂，使蛋白酶无法复兴的蛋白酶。我们提出了三个特定的目的，即将共同评估在许多情况下使SERPINS成为首选蛋白酶抑制剂类别的功能表达中的完整蛋白酶转移，蛋白酶灌溉序列的重要性。具体目标1将确定SERPIN和蛋白酶与受体LRP结合的结构要求和功能后果。具体的目标2将确定蚊子Serpin AFXA的结构，其抑制人体因子Xa的机制以及与LRP结合和信号传导相关的AFXA-XA复合物的特性。特定的目标3将确定胱天蛋白酶，组织蛋白酶和枯草蛋白酶样蛋白酶在与SERPINS共价复合物形成后发生的构象变化。我们将使用NMR光谱，FRET和X射线晶体学的结合亲和力和结构方法的热力学测量结果，我们在上一个赠款期间成功使用了，并且在适当的情况下，通过与Dudley Strickland博士的协作将我们的发现与功能后果相关联。