Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly

Abstract

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. Signal transduction in the biological milieu is a dynamic exchange of protein-protein interactions (1, 2), coordinated by a diverse family of scaffolding proteins (2-4). Structural mod-ularity of the scaffolds is fine-tuned for linking and transporting the different binding partners to the supramolecular signaling complexes. Well-known scaffold modules include Src homology 3, Src homology 2, PTB, WW, and the more abundant PDZ domains, which are ubiquitous for recruiting diverse protein targets (3, 4). As mediators of protein-protein interactions, PDZ domains have been shown to possess remarkable specificity for C-terminal binding motifs as well as internal peptide sequences of proteins (3). PDZ domains have emerged as key organizers of protein complexes at the plasma membrane, cytoplasmic tails of membrane proteins, and ion channels to promote their transport and localization to the cell surface for signaling (5). Full-length NHERF1 includes tandem PDZ domains, PDZ1 and PDZ2, and a C-terminal ezrin-binding domain (EBD) 4 juxtaposed with a PDZ-binding type 1 motif (Fig. 1). The EBD is a specific target of the FERM domain from ezrin (ezFERM), which disrupts the autoinhibition between the PDZ2 domain and the C-terminal tail in NHERF1 (6). Binding of ezrin with NHERF1 allosterically increases the PDZ domain ...

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