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Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor.

Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na + pumping is not yet understood. Here, we present a structure of the cationic (non H + ) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.

Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina ( Ns XeR) and suggest a mechanism of inward proton pumping. We demonstrate that the Ns XeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.

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.

A key and common process present in organisms from all domains of life is the maintenance of the ion gradient between the inside and the outside of the cell. The gradient is generated by various active transporters, among which are the light-driven ion pumps of the microbial rhodopsin family. Whereas the proton-pumping and anion-pumping rhodopsins have been known for a long time, the cation (sodium) pumps were described only recently. Following the discovery, high-resolution atomic structures of the pump KR2 were determined that revealed the complete ion translocation pathway, including the positions of the characteristic Asn-Asp-Gln (NDQ) triad, the unusual ion uptake cavity acting as a selectivity filter, the unique N-terminal α-helix, capping the ion release cavity, and unexpected flexibility of the retinal-binding pocket. The structures also revealed pentamerization of KR2 and binding of sodium ions at the interface. Finally, on the basis of the structures, potassium-pumping KR2 variants have been designed, making the findings even more important for optogenetic applications. In this Structural Snapshot, we analyse the implications of the structural findings for understanding the sodium translocation mechanism and application of the pump and its mutants in optogenetics. [ABSTRACT FROM AUTHOR]

Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists. Rhodopsin genes have been identified in some large double-stranded DNA viruses, but the structure and functions of viral rhodopsins remain unknown. Here authors present crystal structure and characterization of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) which forms a pentamer and is a weak proton pump. [ABSTRACT FROM AUTHOR]

The article presents comprehensive functional studies of the inward proton pumps of the noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. Topics discussed include the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and a mechanism of inward proton pumping and the suitability of inwardly directed proton pumps for light-induced remote control of neurons and as an alternative to the well-known cation-selective channel rhodopsins.