NMR of Transmembrane and Intracellular Domains of Human Nicotine Acetylcholine Receptor A7 Subunit

Abstract

Structure determination of mammalian membrane proteins is challenging due to low expression level, unpredictable folding, and poor protein stability. We addressed these critical issues using a novel approach to break hydrophobic patches by minimal mutagenesis in transmembrane (TM) domains. For a 300-residue sequence encompassing the entire TM and long intracellular (IC) loop domains of the human nicotinic acetylcholine receptor (nAChR) α7 subunit, 16 mutation sites were identified and experimentally implemented. The resultant mutant (nAChRα7TM-IC) was expressed with a typical yield of 5 mg/L in M9 medium for NMR investigation. Replacing IC loop with a 5-Gly linker (nAChRα7TM-5G) increased the yield to 10 mg/L in M9. Both nAChRα7TM-IC and nAChRα7TM-5G folded into stable structures in ∼1% empigen with helical content of 41% and 50%, respectively, determined by circular dichroism. Thus, the helical content of IC can be calculated to be 29% at least, in agreement with sequence-based predictions. Exceptionally high-resolution and well-dispersed NMR spectra, comparable to those of soluble proteins of similar size, were obtained for nAChRα7TM-5G, allowing for spectral assignment and structure calculation. Reasonably high-resolution NMR spectra of nAChRα7TM-IC were also acquired at 900 MHz, with majority of TM-domain peaks overlapping with those in nAChRα7TM-5G, suggesting that the presence of IC domain did not greatly change the structure of TM domains. Backbone dynamics analyses of the two proteins using our newly developed histogram method, which does not require full spectral assignment, showed that the presence of IC dramatically affected the intrinsic dynamics of TM domains. Details of NMR structure characterization of TM and IC domains of nAChR will be presented. The approach is readily applicable to other receptor proteins, opening a new avenue for structural investigation of membrane proteins. Supported by NIH (R37GM049202, R01GM056257, R01GM069766, and P01GM055876).