Structure and mechanism of the human NHE1-CHP1 complex

Yanli Dong,Xuejun C Zhang,Yan Zhao,Annie Boucher,Bin Li,Dusik Kim,Alina Ilie,Yiwei Gao,John Orlowski

doi:10.1038/s41467-021-23496-z

Abstract

Sodium/proton exchanger 1 (NHE1) is an electroneutral secondary active transporter present on the plasma membrane of most mammalian cells and plays critical roles in regulating intracellular pH and volume homeostasis. Calcineurin B-homologous protein 1 (CHP1) is an obligate binding partner that promotes NHE1 biosynthetic maturation, cell surface expression and pH-sensitivity. Dysfunctions of either protein are associated with neurological disorders. Here, we elucidate structures of the human NHE1-CHP1 complex in both inward- and inhibitor (cariporide)-bound outward-facing conformations. We find that NHE1 assembles as a symmetrical homodimer, with each subunit undergoing an elevator-like conformational change during cation exchange. The cryo-EM map reveals the binding site for the NHE1 inhibitor cariporide, illustrating how inhibitors block transport activity. The CHP1 molecule differentially associates with these two conformational states of each NHE1 monomer, and this association difference probably underlies the regulation of NHE1 pH-sensitivity by CHP1.

Highlights

Sodium/proton exchanger 1 (NHE1) is an electroneutral secondary active transporter present on the plasma membrane of most mammalian cells and plays critical roles in regulating intracellular pH and volume homeostasis
To identify the Na+ binding site, we first prepared the sample in the presence of 150 mM NaCl at pH 7.5, and the corresponding complex structure is termed as NHE1-CHP1Na/7.5
In addition to discernable features of transmembrane helix density, two-dimensional (2D) classification revealed some “crab”-shaped classes featuring splayed and blurred densities protruding from one side of the nanodisc which are presumably due to Calcineurin B-homologous protein 1 (CHP1) association from the cytoplasm

Summary

Introduction

Sodium/proton exchanger 1 (NHE1) is an electroneutral secondary active transporter present on the plasma membrane of most mammalian cells and plays critical roles in regulating intracellular pH and volume homeostasis. One of the four classes featuring clearly resolved transmembrane helices was selected for focused 3D reconstructions of the transmembrane domain (TMD) of NHE1 and yielded an EM map of 3.3-Å resolution, which is rich in features, including densities for sidechains and associated lipid molecules, allowing us to build a de novo model of the NHE1 dimer (Supplementary Fig. 2 and Table 1).

Results

Conclusion