Solution Structure of the Cytoplasmic Region of Na+/H+ Exchanger 1 Complexed with Essential Cofactor Calcineurin B Homologous Protein 1

Masaki Mishima,Shigeo Wakabayashi,Chojiro Kojima

doi:10.1074/jbc.m604092200

Abstract

Na+/H+ exchanger 1 (NHE1) regulates intracellular pH, Na+ content, and cell volume. Calcineurin B homologous protein 1 (CHP1) serves as an essential cofactor that facilitates NHE1 exchange activity under physiological conditions by direct binding to the cytoplasmic juxtamembrane region of NHE1. Here we describe the solution structure of the cytoplasmic juxtamembrane region of NHE1 complexed with CHP1. The region of NHE1 forms an amphipathic helix, which is induced by CHP1 binding, and CHP1 possesses a large hydrophobic cleft formed by EF-hand helices. The apolar side of the NHE1 helix participates in extensive hydrophobic interactions with the cleft of CHP1. We suggest that helix formation of the cytoplasmic region of NHE1 by CHP1 is a prerequisite for generating the active form of NHE1. The molecular recognition detailed in this study also provides novel insight into the target binding mechanism of EF-hand proteins.

Highlights

Naϩ/Hϩ exchangers comprise a family of countertransport proteins that catalyze the electroneutral exchange of Naϩ and Hϩ
We suggest that helix formation of the cytoplasmic region of Na؉/H؉ exchanger 1 (NHE1) by Calcineurin B homologous protein 1 (CHP1) is a prerequisite for generating the active form of NHE1
Structure Determination—To better understand the mechanism pertaining to CHP1-regulated NHE1 activity, the solution structure of unmyristoylated CHP1 complexed with the cytoplasmic region [503–545] of NHE1 was determined by NMR spectroscopy

Summary

Further purification was performed by gel filtration using

0.9 mM protein in 50 mM Tris-d7 buffer (pH 6.9), 1 mM dithiothreitol-d10, 30 mM KCl in H2O/2H2O (9:1) or 2H2O. Resonance assignments for 1HN, 15N, 13C␣, 13C␤, and 13CЈ nuclei for the CHP1-NHE1 complex were obtained through the following 2H-decoupled, triple resonance spectra applied to a fractionally deuterated 15N/13C-labeled sample: three-dimensional HNCACB/HN(CO)CACB [24] and three-dimensional HN(CA)CO/HNCO experiments [25, 26]. The initial structure calculations were performed by iterative automated assignment of the NOE spectra using. Derived distance restraints obtained from the manual and the CANDID-assisted assignments from the 15N- or 13C-edited NOE data. Wildtype and CHP1 mutant GST fusion proteins, in addition to GST alone, were incubated for 30 min at 20 °C with NHE1 peptide in binding buffer containing 50 mM HEPES (pH 7.5), 20 mM CHAPS, 10% glycerol, 1 mM Pefabloc, and 1 mM dithiothreitol. Quantification was represented as the average value of experiments performed in triplicate

RESULTS AND DISCUSSION

HSQC spectra of

It is interesting to note that both

The interface formed between