Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.
Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.
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DOI：
10.1371/journal.pone.0129554
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Friedman PA
中科院分区：
文献类型：
--
作者：
Mamonova T;Zhang Q;Khajeh JA;Bu Z;Bisello A;Friedman PA
Na+/H+交换调节因子-1（NHERF 1）是一种支架蛋白，含有2个PDZ结构域，可协调跨膜受体和离子通道的组装和运输。大多数含有C-末端识别基序的靶蛋白或多或少等同地结合到含有相同核心结合基序的PDZ结构域。然而，一些底物如II型钠依赖性磷酸盐共转运蛋白（NPT 2A）仅独特地结合一个PDZ结构域。我们试图确定负责NHERF 1 PDZ结构域和NPT 2A之间相互作用特异性的结构决定因素。通过进行全原子/显式溶剂分子动力学（MD）模拟，结合生物诱变，荧光偏振（FP）结合试验和等温滴定量热法（ITC），我们发现，除了0和-2位残基的典型相互作用外，在NPT 2A的-1位的Arg在与PDZ 1的Glu 43和His 27的结合中起关键作用，而这在PDZ 2中是不存在的。实验引入的PDZ 1突变（Glu 43 Asp和His 27 Asn）降低了与NPT 2A的结合。相反，在PDZ 2中引入Asp 183 Glu和Asn 167 His突变促进了有利的相互作用的形成，从而产生微摩尔KD。结果描述了新的决定因素内的PDZ域和外的典型的PDZ识别基序，负责两个PDZ域之间的NPT 2A的歧视。这些结果挑战了PDZ识别的一般范式，并为药物开发提供了新的靶点。
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 K Ds. 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.
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发表时间：
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