Podoliak, E. | Search bioRxiv

bioRxiv2023

A novel 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 identified a new subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterized a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and showed 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 new subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

biophysics↗

bioRxiv2023

Flotillin-Associated rhodopsin (FArhodopsin), a widespread paralog of proteorhodopsin in aquatic bacteria with streamlined genomes

Microbial rhodopsins are often found more than once in a single genome (paralogs) that often have different functions. We screened a large dataset of open ocean single-amplified genomes (SAGs) for co-occurrences of multiple rhodopsin genes. Many such cases were found among Pelagibacterales (SAR11), HIMB59 and the Gammaproteobacteria Pseudothioglobus SAGs. These genomes always had a bona fide proteorhodopsin and a separate cluster of genes containing a second rhodopsin associated with a predicted flotillin coding gene and have thus been named flotillin-associated rhodopsins (FArhodopsins). They are quite divergent from the other proteorhodopsin paralog and contain either DTT, DTL or DNI motives in their key functional amino acids. FArhodopsins are mainly associated with the lower layers of the epipelagic zone. All marine FArhodopsins had the retinal binding lysine, but we found their relatives in freshwater metagenomes that lack this key amino acid. Alfa-fold predictions of marine FArhodopsins indicate that their retinal pocket might be very reduced or absent, hinting that they are retinal-less (blind). Freshwater FArhodopsins were more diverse than marine FArhodopsins, but we could not determine if they are present as paralogs of other rhodopsins, due to the lack of SAGs or isolates. Although the function of FArhodopsins could not be established, their conserved genomic context indicated involvement in the formation of membrane microdomains. The conservation of FArhodopsins in diverse and globally abundant microorganisms suggests that they may be important in the adaptation to the twilight zone of aquatic environments. IMPORTANCERhodopsins have been shown to play a key role in the ecology of aquatic microbes. Here we describe a group of widespread rhodopsins in aquatic microbes associated with dim light conditions. Their characteristic genomic context found in both marine and freshwater environments indicates a novel potential involvement in membrane microstructure that could be important for the function of the co-existing proteorhodopsin proton pumps. The absence or reduction of the retinal binding pocket points to drastically different physiology. In addition to their ecological importance, novel rhodopsins have biotechnological potential in the nascent field of optogenetics.

microbiology↗