Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly
Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly
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DOI：
10.1074/jbc.ra119.008218
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发表时间：
2019-07-19
影响因子：
4.8
通讯作者：
Bu, Zimei
中科院分区：
文献类型：
--
作者：
Bhattacharya, Shibani;Stanley, Christopher B.;Bu, Zimei
Na+/H+交换调节辅因子1（NHERF 1）蛋白调节几种跨膜G蛋白偶联受体（GPCR）和离子转运蛋白与膜-细胞骨架衔接蛋白ezrin的组装和细胞内运输。在这里，我们应用溶液NMR和小角中子散射（SANS）来表征全长NHERF 1和与磷酸盐稳态受损有关的疾病相关变体的结构。使用NMR，我们绘制了NHERF 1的模块化结构，该结构由两个结构独立的PDZ结构域组成，它们通过一个灵活的无序连接体连接。我们观察到NHERF 1的超长和无序的C-末端尾具有1型PDZ结合基序，其与近端的第二PDZ结构域弱相互作用以形成动态自抑制结构。使用集成优化的SANS数据分析，我们提取的结构从广泛的构象空间采样的柔性链的分子大小分布。我们的研究结果表明，NHERF 1是一个分散的集合可变PDZ结构域的配置和无序的C-末端尾巴。三种疾病变体（L110 V，R153 Q和E225 K）的联合NMR/SANS数据分析显示，与WT蛋白相比，局部PDZ结构域结构和全局构象存在显着差异。此外，我们表明，取代影响NHERF 1结合ezrin和G蛋白偶联受体激酶6A（GRK 6A）的C-末端肽的亲和力和动力学。这些发现提供了重要的洞察NHERF 1的内在灵活性的疾病相关的点突变，改变信号复合物的动态组装的调制。
The Na+/H+ exchange regulatory cofactor 1 (NHERF1) protein modulates the assembly and intracellular trafficking of several transmembrane G protein-coupled receptors (GPCRs) and ion transport proteins with the membrane-cytoskeleton adapter protein ezrin. Here, we applied solution NMR and small-angle neutron scattering (SANS) to structurally characterize full-length NHERF1 and disease-associated variants that are implicated in impaired phosphate homeostasis. Using NMR, we mapped the modular architecture of NHERF1, which is composed of two structurally-independent PDZ domains that are connected by a flexible, disordered linker. We observed that the ultra-long and disordered C-terminal tail of NHERF1 has a type 1 PDZ-binding motif that interacts weakly with the proximal, second PDZ domain to form a dynamically autoinhibited structure. Using ensemble-optimized analysis of SANS data, we extracted the molecular size distribution of structures from the extensive conformational space sampled by the flexible chain. Our results revealed that NHERF1 is a diffuse ensemble of variable PDZ domain configurations and a disordered C-terminal tail. The joint NMR/SANS data analyses of three disease variants (L110V, R153Q, and E225K) revealed significant differences in the local PDZ domain structures and in the global conformations compared with the WT protein. Furthermore, we show that the substitutions affect the affinity and kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein-coupled receptor kinase 6A (GRK6A). These findings provide important insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes.