Molecular Basis of Voltage-Gated Proton Channel's Intracellular Trafficking

Abstract

determined the cryo-EM structures of zebrafish Otop1 (3.0 A ˚) and chicken OTOP3 (3.3 A ˚), both embedded in lipid nanodiscs. The structures reveal that the Otopetrin family adopt a unique dimeric fold, with each subunit divided into two structurally related halves. Notably we observe cholesterol(-like) molecules occluding the wide central tunnel formed between the two subunits. Using all-atom molecular dynamics simulations, we show that cholesterol molecules at the resolved positions within the central tunnel represent stable binding poses, thereby excluding water and other solutes. We then examined the putative proton conducting pathways in our MD simulations, using areas susceptible to water penetration as a first approximation. The two halves of both Otop1 and OTOP3 are partially hydrated as expected, due to their vestibular-shaped openings to the cytoplasmic side, and the presence of polar and charged residues. Surprisingly, a third region of water penetration is found along the intrasubunit interface between the two halves in a subunit. In functional studies, a key glutamate found in this region is intolerant to charge-neutralisation mutations. These results also correlate to the extent of hydration in this region in the simulations. Altogether, we show that there might be three potential pathways (or their combinations) contributing to proton currents in Otopetrins. We also study novel mechanically-activated (MA) ion channels found in mammals and plants, via a combination of structural and computational studies. Recently we solved the structures of OSCA channels in plants and work is underway for other MA channels.