Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.钠氢交换调控因子1（Na+/H+ Exchanger Regulatory Factor-1, NHERF1）是一类含有2个PDZ结构域的支架蛋白，能够协同调控跨膜受体与离子通道的组装及转运过程。绝大多数携带C端识别基序的靶蛋白，可与两个PDZ结构域以近似等同的亲和力结合，因为二者拥有完全一致的核心结合基序。但部分底物——例如II型钠依赖性磷酸共转运蛋白（type II sodium-dependent phosphate co-transporter, NPT2A）——仅能特异性结合其中单一的PDZ结构域。本研究旨在明确介导NHERF1的PDZ结构域与NPT2A之间相互作用特异性的结构决定因素。通过联合开展全原子/显式溶剂分子动力学（Molecular Dynamics, MD）模拟、生物诱变实验、荧光偏振（Fluorescent Polarization, FP）结合测定以及等温滴定量热法（Isothermal Titration Calorimetry, ITC），我们发现：除0位与-2位残基的经典相互作用外，NPT2A的-1位精氨酸，可与PDZ1结构域的谷氨酸43（Glu43）和组氨酸27（His27）形成关键结合作用，而PDZ2结构域并不存在这两个残基。实验中对PDZ1引入的Glu43Asp与His27Asn突变，会降低其与NPT2A的结合能力。与之相反，在PDZ2结构域中引入Asp183Glu与Asn167His突变，则可促进形成有利的相互作用，使其解离常数达到微摩尔级别。本研究揭示了PDZ结构域内部以及经典PDZ识别基序之外的全新决定因素，这些因素可介导NPT2A对两个PDZ结构域的选择性识别。该研究结果挑战了现有PDZ识别的通用范式，同时为药物开发提供了全新的靶点。