Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.

Tatyana Mamonova,Qiangmin Zhang,Peter A Friedman,Zimei Bu,Alessandro Bisello,Jahan Ali Khajeh,Peter Schuck

doi:10.1371/journal.pone.0129554

Tatyana Mamonova, Qiangmin Zhang + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0129554

Copy DOI

Journal: PloS one	Publication Date: Jun 12, 2015
Citations: 47	License type: CC BY 4.0

Affiliation: University of Pittsburgh, City College of New York

Abstract

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.

Highlights

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1), known as the 50-kDa ezrin-binding protein EBP50, is a multi-domain scaffolding protein that coordinates the assembly and trafficking of transmembrane receptors and ion channels [1,2,3]
NHERF1 binds an extensive set of proteins including the parathyroid hormone receptor (PTHR), the β2-adrenergic receptor (β2-AR), the cystic fibrosis transmembrane regulator (CFTR), the P2Y1 receptor, and the thromboxane A2 receptor, among others, that harbor a PDZ ligand
By performing extensive all-atom molecular dynamics (MD) simulations of the 22-residue carboxy-terminal tail of NPT2A and NHERF1 PDZ domains we identified the specific determinants of PDZ1-NPT2A interactions

Summary

Introduction

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1), known as the 50-kDa ezrin-binding protein EBP50, is a multi-domain scaffolding protein that coordinates the assembly and trafficking of transmembrane receptors and ion channels [1,2,3]. NHERF1 binds an extensive set of proteins including the parathyroid hormone receptor (PTHR), the β2-adrenergic receptor (β2-AR), the cystic fibrosis transmembrane regulator (CFTR), the P2Y1 receptor, and the thromboxane A2 receptor, among others, that harbor a PDZ ligand. Canonical, interactions occur through the GYGF core-binding motif of NHERF1 PDZ domains and the carboxy-terminal hydrophobic residue at ligand position 0. Another canonical interaction occurs between Ser/Thr at ligand position -2 and the structurally conserved His (PDZ1) or His212 (PDZ2) [4,10,13]. We applied a two-pronged approach of molecular dynamics simulation and experimental measurements

Objectives

Methods

Results

Conclusion