Tryptophan scanning mutagenesis reveals distortions in the helical structure of the δM4 transmembrane domain of theTorpedo californicanicotinic acetylcholine receptor

Abstract

The lipid-protein interface is an important domain of the nicotinic acetylcholine receptor (nAChR) that has recently garnered increased relevance. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, there is still a need to gain insight into the mechanism by which lipid-protein interactions regulate the function and conformational transitions of the nAChR. In this study, we extended the tryptophan scanning mutagenesis (TrpScanM) approach to dissect secondary structure and monitor the conformational changes experienced by the δM4 transmembrane domain (TMD) of the Torpedo californica nAChR, and to identify which positions on this domain are potentially linked to the regulation of ion channel kinetics. The difference in oscillation patterns between the closed- and open-channel states suggests a substantial conformational change along this domain as a consequence of channel activation. Furthermore, TrpScanM revealed distortions along the helical structure of this TMD that are not present on current models of the nAChR. Our results show that a Thr-Pro motif at positions 462-463 markedly bends the helical structure of the TMD, consistent with the recent crystallographic structure of the GluCl Cys-loop receptor which reveals a highly bent TMD4 in each subunit. This Thr-Pro motif acts as a molecular hinge that delineates two gating blocks in the δM4 TMD. These results suggest a model in which a hinge-bending motion that tilts the helical structure is combined with a spring-like motion during transition between the closed- and open-channel states of the δM4 TMD.

Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR

“…Given that β, δ, and ε each contains a homologous residue to αSer226 (βThr237, δSer240, and εSer235) on M1, we expected a similar degree of potentiation upon mutation of the αCys418 equivalent residue in each subunit (βThr456, δMet460, and εPhe446) to tryptophan. In contrast, tryptophan substitutions in β, δ, and ε, (βT456W, δM460W, and εF446W) led to only a 1.7-fold gain, a 1.6-fold loss, and a 1.8-fold gain of function, respectively, consistent with what is observed in the Torpedo nAChR ( 36 , 37 , 38 ). Furthermore, the βT237A mutation on M1 had no effect on the magnitude of the βT456W-induced response implying that the introduced tryptophan, βT456W, does not interact with βT237 to potentiate channel function.…”

Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis

“…We also substituted a few residues with the bulky hydrophobic residue tryptophan since this should also perturb the structure when introduced at buried locations (42). In earlier studies, tryptophan scanning mutagenesis was used to identify membrane-facing regions in helical segments of transmembrane proteins (43,44). We expressed wildtype (WT) and mutated derivatives on the surface of HEK293T cells and monitored their expression and binding to various antibodies by flow cytometry (33,45).…”