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Title: 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. We applied in this report 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 andmore » kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein–coupled receptor kinase 6A (GRK6A). Significant insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes are provided in this work.« less

Authors:
 [1]; ORCiD logo [2]; ORCiD logo [2]; ORCiD logo [3];  [4]
  1. New York Structural Biology Center, NY (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  3. Univ. of Pittsburgh, PA (United States)
  4. City Univ. (CUNY), NY (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22), National Institutes of Health (NIH); National Science Foundation (NSF); U.S. Dept. of Defense
OSTI Identifier:
1545195
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 294; Journal Issue: 29; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; scaffolding protein; PDZ domain; G protein-coupled receptor kinase 6A (GRK6A); intrinsically disordered protein; NHERF1; NMR; small-angle neutron scattering (SANS); SLC9A3 regulator 1 (SLC9A3R1); intracellular transport

Citation Formats

Bhattacharya, Shibani, Stanley, Christopher B., Heller, William T., Friedman, Peter A., and Bu, Zimei. Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly. United States: N. p., 2019. Web. doi:10.1074/jbc.ra119.008218.
Bhattacharya, Shibani, Stanley, Christopher B., Heller, William T., Friedman, Peter A., & Bu, Zimei. Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly. United States. doi:10.1074/jbc.ra119.008218.
Bhattacharya, Shibani, Stanley, Christopher B., Heller, William T., Friedman, Peter A., and Bu, Zimei. Thu . "Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly". United States. doi:10.1074/jbc.ra119.008218. https://www.osti.gov/servlets/purl/1545195.
@article{osti_1545195,
title = {Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly},
author = {Bhattacharya, Shibani and Stanley, Christopher B. and Heller, William T. and Friedman, Peter A. and Bu, Zimei},
abstractNote = {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. We applied in this report 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). Significant insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes are provided in this work.},
doi = {10.1074/jbc.ra119.008218},
journal = {Journal of Biological Chemistry},
number = 29,
volume = 294,
place = {United States},
year = {2019},
month = {6}
}

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