Structural basis of focal adhesion targeting domain-mediated signaling in cardiac hypertrophy

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) exists in monomeric closed (c) or arm exchanged (ae) dimeric state. FAT interaction with Grb2 necessitates an intermediate open (o) state that interacts with Grb2 and activates signaling pathways leading to pathological cardiac hypertrophy. Targeted molecular dynamics (TMD) simulation was carried out in order to capture the structure of the intermediate formed by opening of Helix1 (H1) from monomeric cFAT leading to the formation of monomeric aeFAT. During TMD, H1 separated from the four helices bundle of cFAT, completely unfolded and performed a full turn before folding back to a helix inclined at an acute angle to the helical bundle in aeFAT. The entire transition can be described in six distinct intermediate structural stages. The most significant correlation of H1 motion was observed with Loop3 (L3) and is the likely reason for the complete disruption of the FAT interaction with paxillin during the transition. High-affinity analogs of the paxillin LD4 region can be a promising strategy to drive the equilibrium towards cFAT, thus antagonizing FAT-Grb2 association. During transition, the overall shift in orientation of all the four helices rejects paxillin binding and approves Grb2 association. Exposure and β-turn conformation of the YENV motif (residues 925-928) in oFAT-facilitated phosphorylation and Grb2 binding. Docking, MD simulation and conservation analysis of oFAT-Grb2 complex provided insight into the structural determinants of binding and specificity. Our work provides a structural basis for pharmacological modulation of dynamic conformational changes and interactions of FAT.黏着斑激酶（focal adhesion kinase, FAK）的黏着斑靶向（focal adhesion targeting, FAT）结构域存在单体闭合（c）或臂交换（arm exchanged, ae）二聚体两种构象状态。FAT与生长因子受体结合蛋白2（Grb2）的相互作用依赖于中间开放（o）态：该构象可结合Grb2并激活下游信号通路，最终引发病理性心肌肥厚。为捕捉由单体闭合FAT（cFAT）的螺旋1（Helix1, H1）解折叠、进而形成单体臂交换FAT（aeFAT）过程中形成的中间态结构，本研究开展了靶向分子动力学（targeted molecular dynamics, TMD）模拟。在TMD模拟过程中，H1从cFAT的四螺旋束中脱离、完全解折叠并完成一整圈旋转，随后重新折叠为与aeFAT内螺旋束呈锐角倾斜的螺旋结构。整个构象转变可划分为六个独立的中间结构阶段。研究观察到H1的运动与Loop3（L3）存在最强相关性，这极有可能是转变过程中FAT与桩蛋白（paxillin）的相互作用被完全破坏的核心原因。桩蛋白LD4区域的高亲和力类似物，有望成为将构象平衡导向cFAT的潜在策略，从而拮抗FAT与Grb2的结合。在构象转变过程中，四个螺旋的整体取向偏移会排斥桩蛋白的结合，同时促进Grb2的缔合。开放态FAT（oFAT）中YENV基序（残基925-928）的暴露及其β转角构象，可促进该基序的磷酸化并增强与Grb2的结合能力。针对oFAT-Grb2复合物开展的分子对接、MD模拟及保守性分析，为该结合的结构决定因素与结合特异性提供了关键见解。本研究为动态构象变化及FAT相互作用的药理学调控提供了坚实的结构基础。