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. Light-driven sodium-pumping rhodopsins are unique ion transporters. Here, authors present a characterization of such rhodopsins with a modified active center allowing for efficient sodium transport under various environmental conditions.

The primary goal of our work is to provide structural insights into the influence of the hydrophobic lipid environment on the membrane proteins (MPs) structure and function. Our work will not cover the well-studied hydrophobic mismatch between the lipid bilayer and MPs. Instead, we will focus on the less-studied direct molecular interactions of lipids with the hydrophobic surfaces of MPs. To visualize the first layer of amphiphiles surrounding MPs and analyze their interaction with the proteins, we use the available highest-quality crystallographic structures of microbial rhodopsins. The results of the structure-based analysis allowed us to formulate the hypothetical concept of the role of the nearest layer of the lipids as an integral part of the MPs that are important for their structure and function. We then discuss how the lipid-MPs interaction is influenced by exogenous hydrophobic molecules, noble gases, which can compete with lipids for the surface of MPs and can be used in the systematic approach to verify the proposed concept experimentally. Finally, we raise the problems of currently available structural data that should be overcome to obtain a more profound picture of the lipid-MP interactions.

Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.

Crystallization of bacteriorhodopsin (bR) in the lipidic cubic phase using n-octyl-β-D-glucoside (OG) and its more stable and inexpensive analogue n-octyl-β-D-thioglucoside (OTG) was comparatively analyzed [1]. It was shown that bacteriorhodopsin is efficiently crystallized in OTG in the same detergent concentration range as in OG. However, x-ray diffraction analysis shows that bR crystals in OG are characterized by a better resolution (1.35 Å) than bR crystals in OTG (1.45 Å).