Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cat-ions other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion -pump-ing characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence proton pumping is observed. Our data reveal spe-cific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spec-troscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.

Automated summary

This study presents an extensive chemical shift resonance assignment of Krokinobacter eikastus rhodopsin 2 (KR2) within lipid bilayers using solid-state NMR spectroscopy. The H180A mutation was found to silence the response of KR2 to oxidized sodium, while proton pumping was observed in the absence of sodium. The study reveals specific long-range effects of the mutation along the sodium transfer pathway.