Modeling and Experimental Study of NHERF1 PDZ Domain Specificity

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. PDZ domains bind a short sequence of amino acids at the C-terminus of their targets. Molecular dynamics (MD) simulations characterized interactions between NHERF1 PDZ1 or PDZ2 and the C-terminus of the type II sodium-dependent phosphate cotransporter (NPT2a), the parathyroid hormone receptor (PTHR), and NHERF1 ezrin-binding domain (EBD) (self-associated conformation). Modeling and earlier results determined optimal peptide length for experimental binding. NPT2a and PTHR (22 and 9 residues, respectively) peptides were used for fluorescent polarization measurements of solution-state affinities with isolated PDZ domains or intact protein. Mutations in the core-binding sequence permitted analysis of binding to a single PDZ domain in full-length NHERF1. The Kd for interaction of NPT2a with PDZ1 or PDZ2 in full-length NHERF1 was 1.6μM and 33μM, respectively. Only isolated PDZ1 interacted with NPT2a (Kd=11μM). Isolated PDZ1 and PDZ2 interact with PTHR with Kd's of 2.9μM and 1.3μM, respectively. In the context of full-length NHERF1 PTHR binds PDZ1 and PDZ2 with a Kd of 1.7μM and 2.2μM, respectively. We conclude that variances are evident between measurements of isolated PDZ domains and full-length NHERF1. The lower affinity of the PDZ2 domain in full-length NHERF1 compared to isolated PDZ2 is due to adoption of the self-associated conformation. Graded occupancy of PDZ domains may impart differences in NHERF1 signaling and trafficking. We suggest that energetic barriers separating NHERF1 self-associated and open states in the cellular environment may be reduced by phosphorylation. MD simulations predict that upstream residues in the C-terminus contribute to the binding and specificity of interaction with PDZ domains. The results provide novel molecular insights into the recognition of major PDZ binding partners.