A Conformational Switch in the Scaffolding Protein NHERF1 Controls Autoinhibition and Complex Formation

Abstract

The mammalian Na؉ /H ؉ exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.

Extensions of PDZ domains as important structural and functional elements

“…3B), the second PDZ domain (PDZ2) is considerably more stable in denaturing conditions and also able to bind a CFTR peptide with increased (~10-fold higher) affinity. Similar to PSD-95 PDZ3, the extension does not seem to have a large impact on the fold of the canonical domain; superimposition of the structures of an extended and truncated form show that there is little deviation in the backbone conformation (Bhattacharya et al, 2010). It certainly seems that the NHERF1 PDZ2 extension and PSD-95 PDZ3 extension have many conserved structural and functional features.…”

Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding

“…15 Their results showed that residues 0 to -5 (See The additional C-terminal extension is not unique for PDZ3. 25,26 In MAGI PDZ1 for example, an extended C-terminal loop makes direct interactions with Arg -4 , Arg -5 and Thr -6 of the peptide ligand and mutation in this C-terminal loop decreases the binding affinity drastically. 27 Thus, from previous work 11,12,14,24,27 together with the present data we conclude that some PDZ domains contain certain structural elements at their C-terminus, which have a direct effect on binding affinity.…”

NMR reveals novel mechanisms of protein activity regulation

“…18,19 Autoinhibition is increasingly seen in proteins involved in a diverse set of biological phenomena. [15][16][17][20][21][22][23] Mechanisms that are known to relieve autoinhibition include posttranslational modification, proteolysis, and addition of proteins or small ligands. The mechanisms and especially the thermodynamics and kinetics of the activation process in autoregulated proteins are generally poorly understood.…”