NMR Structure of Water-Solubilized Transmembrane Domain of Nicotinic Acetylcholine Receptor

Abstract

No high-resolution experimental structure of the transmembrane (TM) domains of the nicotinic acetylcholine receptor (nAChR) is currently available. We have addressed some critical issues related to structure determination by strategically mutating 22 hydrophobic, putatively lipid-facing residues to hydrophilic ones, so that the resulting TM domains have improved water solubility while maintaining the native fold. Two different designs based on the nAChR α1 subunit were attempted. The first (TM123-4) consisted of the four TM domains in their natural sequential arrangement, with an artificial linker between TM3 and TM4. The second (TM4-123) has TM4 moved before TM1. TM123-4 failed to be expressed by E. coli, suggesting possible cell toxicity. In contrast, TM4-123 was expressed with high yield and produced stable samples for structure characterization by high-resolution NMR. Monomeric TM4-123 structure was resolved to a backbone RMSD of 1.2 A. The overall structure of TM4-123 is similar to that of the cryo-EM model, except that the very short TM2-TM3 loop (VPLI) found in the cryo-EM structure was replaced by a significantly longer loop (LIPSTSSA) and shifted by 5 residues towards the TM2 domain. More interestingly, ‘TSSA’ in this loop exists in two conformations: a rigid helix and a flexible coil, and these two conformations are in slow exchange on the NMR timescale. Given that the interaction between the TM2-TM3 loop and the extracellular domain likely mediates channel gating, the two conformations in this important loop might represent the critical transitions required for the channel to open. The high-resolution structure of water-solubilized nAChR TM domains allows us to better understand the structural basis of channel function (Funded by NIH R37GM049202, R01GM056257, and P01GM055876).