Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

The light-gated ion channel channelrhodopsin 2 (ChR2) from is a major optogenetic tool. Photon absorption starts a well-characterized photocycle, but the structural basis for the regulation of channel opening remains unclear. We present high-resolution structures of ChR2 and the C128T mutant, which has a markedly increased open-state lifetime. The structure reveals two cavities on the intracellular side and two cavities on the extracellular side. They are connected by extended hydrogen-bonding networks involving water molecules and side-chain residues. Central is the retinal Schiff base that controls and synchronizes three gates that separate the cavities. Separate from this network is the DC gate that comprises a water-mediated bond between C128 and D156 and interacts directly with the retinal Schiff base. Comparison with the C128T structure reveals a direct connection of the DC gate to the central gate and suggests how the gating mechanism is affected by subtle tuning of the Schiff base's interactions.

One of the major and essential classes of transmembrane (TM) receptors, present in all domains of life, is sensor histidine kinases, parts of two-component signaling systems (TCSs). The structural mechanisms of TM signaling by these sensors are poorly understood. We present crystal structures of the periplasmic sensor domain, the TM domain, and the cytoplasmic HAMP domain of the nitrate/nitrite sensor histidine kinase NarQ in the ligand-bound and mutated ligand-free states. The structures reveal that the ligand binding induces rearrangements and pistonlike shifts of TM helices. The HAMP domain protomers undergo leverlike motions and convert these pistonlike motions into helical rotations. Our findings provide the structural framework for complete understanding of TM TCS signaling and for development of antimicrobial treatments targeting TCSs.

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only nonproton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR’s extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.