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.
| Original language | English |
|---|---|
| Pages (from-to) | 11297-11310 |
| Number of pages | 14 |
| Journal | Journal of Biological Chemistry |
| Volume | 294 |
| Issue number | 29 |
| DOIs | |
| State | Published - Jul 19 2019 |
Funding
Acknowledgments— The data collected at New York Structural Biology Center was made possible by ORIP/National Institutes of Health Facility Improvement Grants CO6RR015495 and S10OD016432 (800 MHz). The 900 MHz NMR spectrometers were purchased with funds from National Institutes of Health Grant P41GM066354, the Keck Foundation, New York State Assembly, the United States Department of Defense, and National Institutes of Health Grant 2G12 RR003060 from the NCRR to City College of New York. This study made use of NMRbox: National Center for Biomolecular NMR Data Processing and Analysis, a Biomedical Technology Research Resource (BTRR), supported by National Institutes of Health Grant P41GM111135 from NIGMS. A portion of the research conducted at ORNL’s Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, United States Department of Energy. We thank Carrie Gao for technical support during the SANS experiments. This work was supported by National Science Foundation Grant MCB-1817684 (to Z. B.) and National Institutes of Health Grant 1R01DK105811 (to P. A. F.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1–S7 and Tables S1 and S2. 1To whom correspondence may be addressed. E-mail: sbhattacharya@ nysbc.org. 2 To whom correspondence may be addressed. E-mail: [email protected]. 3 To whom correspondence may be addressed. E-mail: [email protected].