Structure of the Na,K-ATPase regulatory protein FXYD2b in micelles: Implications for membrane–water interfacial arginines

Abstract

FXYD2 is a membrane protein responsible for regulating the function of the Na,K-ATPase in mammalian kidney epithelial cells. Here we report the structure of FXYD2b, one of two splice variants of the protein, determined by NMR spectroscopy in detergent micelles. Solid-state NMR characterization of the protein embedded in phospholipid bilayers indicates that several arginine side chains may be involved in hydrogen bond interactions with the phospholipid polar head groups. The structure and the NMR data suggest that FXYD2b could regulate the Na,K-ATPase by modulating the effective membrane surface electrostatics near the ion binding sites of the pump.

Structural determination of virus protein U from HIV-1 by NMR in membrane environments

“…Dipolar couplings and torsion angle were used in the initial structure determination. In the second and third steps of the calculations distance restraints between the residues and the plane distance restraints (53) were added with kdih = 400 kcal mol −1 rad −2 . 1 H- 15 N krdc was ramped from 0.01 to 2 kcal mol −1 rad −2 , 1 H- 13 C krdc was ramped from 0.01 to 1 kcal mol −1 rad −2 , know was ramped from 2 to 40 kcal mol −1 Å −2 , and kplane was ramped from 0.01 to 5 kcal mol −1 Å −2 .…”

Applications of NMR to membrane proteins

“…For example, the 1 H/ 15 N PISEMA (polarization inversion with spin exchange and the magic angle) spectra of the human Na,K-ATPase regulatory protein FXYD2 in oriented bilayers [132] show signals from amide sites in a wheel-like pattern that reflects the ∼20° orientation of the transmembrane helix axis relative to the membrane normal (Fig. 4A, C).…”

Backbone structure of Yersinia pestis Ail determined in micelles by NMR-restrained simulated annealing with implicit membrane solvation

“…Such conformations are not expected to exist in the native lipid bilayer membrane environment where the loops are known to engage ligands in the water phase. Previous NMR structure calculations of membrane proteins have sought to eliminate such anomalous conformations by establishing an artificial water-membrane boundary through the use of harmonic coordinate restraints (Teriete et al 2007) or plane distance restraints (Xu et al 2008; Fox et al 2014; Gong et al 2015), or with an empirical potential based on the preferred membrane insertion depth of each amino acid (Shi et al 2009). However, these approaches do not provide a physical representation of the water-membrane environment.…”