Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations

BackgroundMutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans.
Methodology/Principal FindingsWe determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D β-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress.
Conclusions/SignificanceMutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.

**背景**：细丝蛋白A（filamin A, FLNa）是一类具有多种结合伴侣的关键细胞骨架蛋白，其发生突变可引发人类发育异常。
**方法/主要结果**：本研究解析了FLNa的第23个免疫球蛋白样重复结构域（IgFLNa23）的三维结构——该结构域可与FilGAP结合，而FilGAP是一种Rac特异性GTP酶激活蛋白，同时也是细胞极性与运动的调控因子；同时我们还探究了3种疾病相关突变对该蛋白互作的影响。通过核磁共振（NMR）结构分析与计算机模拟建模相结合的手段，我们明确了IgFLNa23的C、D β折叠链与FilGAP的C端32个氨基酸残基之间的互作界面细节。对预测得到的关键界面残基进行诱变实验，验证了两种蛋白之间的结合约束机制。我们制备了特异性的FLNa功能丧失型表达构建体，并利用其在体内分析FLNa与FilGAP互作的重要性。定点诱变实验结果显示，破坏FLNa与FilGAP的互作界面会扰乱细胞铺展过程。FilGAP无法与FLNa的同源蛋白FLNb或FLNc结合，这证实了该互作对于人类FLNa突变致病的关键意义。形成稳定的蛋白复合物需要两种互作蛋白均发生二聚化，且结合表面需正确对齐；而FLNa的第23与24个重复结构域之间的柔性铰链结构域可促进这一过程。与人类发育异常相关的FLNa突变会破坏其与FilGAP的互作，并削弱FLNa/丝状肌动蛋白（F-actin）网络在高机械应力下的弹性。
**结论与意义**：基于结构指导的诱变分析可制备用于探究FLNa特异性细胞互作的研究工具。疾病相关的FLNa突变对FLNa的功能具有明确的影响。