A subgroup of light-driven sodium pumps with an additional Schiff base counterion.

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

Novel pH-Sensitive Microbial Rhodopsin from Sphingomonas paucimobilis.

This work provides the first characteristics of the rhodopsin SpaR from Sphingomonas paucimobilis, aerobic bacteria associated with opportunistic infections. The sequence analysis of SpaR has shown that this protein has unusual DTS motif which has never reported in rhodopsins from Proteobacteria. We report that SpaR operates as an outward proton pump at low pH; however, proton pumping is almost absent at neutral and alkaline pH. The photocycle of this rhodopsin in detergent micelles slows down with an increase in pH because of longer Schiff base reprotonation. Our results show that the novel microbial ion transporter SpaR of interest both as an object for basic research of membrane proteins and as a promising optogenetic tool.