Red-shifting mutation of light-driven sodium-pump rhodopsin.
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ABSTRACT:
SUBMITTER: Inoue K
PROVIDER: S-EPMC6491443 | biostudies-literature | 2019 Apr
REPOSITORIES: biostudies-literature
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Project description:The conversion of light energy into ion gradients across biological membranes is one of the most fundamental reactions in primary biological energy transduction. Recently, the structure of the first light-activated Na+ pump, Krokinobacter eikastus rhodopsin 2 (KR2), was resolved at atomic resolution [Kato HE, et al. (2015) Nature 521:48-53]. To elucidate its molecular mechanism for Na+ pumping, we perform here extensive classical and quantum molecular dynamics (MD) simulations of transient photocycle states. Our simulations show how the dynamics of key residues regulate water and ion access between the bulk and the buried light-triggered retinal site. We identify putative Na+ binding sites and show how protonation and conformational changes gate the ion through these sites toward the extracellular side. We further show by correlated ab initio quantum chemical calculations that the obtained putative photocycle intermediates are in close agreement with experimental transient optical spectroscopic data. The combined results of the ion translocation and gating mechanisms in KR2 may provide a basis for the rational design of novel light-driven ion pumps with optogenetic applications.
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Project description:Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion-pumping characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.
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Project description:The light-driven rhodopsin KR2 transports Na+via the M- and O-states. However, the mechanisms by which the retinal regulates Na+ pumping is unknown, in part because KR2 adopts both pentamer and monomer forms in crystal structures and in part because these structures show differences in the protein conformation near the Schiff base, even when they are of the same intermediate state within the photocycle. A particular open question is the nature of the H-bond networks and protonation state in the active site, including Asp116. Here, we analyze the protonation state and the absorption wavelength for each crystal structure, using a quantum mechanical/molecular mechanical approach. In the pentamer ground state, the calculated absorption wavelength reproduces the experimentally measured absorption wavelength (530 nm). The analysis also shows that ionized Asp116 is stabilized by the H-bond donations of both Ser70 and a cluster of water molecules. The absorption wavelength of 400 nm in the M-state can be best reproduced when the two O atoms of Asp116 interact strongly with the Schiff base, as reported in one of the previous monomer ground state structures. The absorption wavelengths calculated for the two Na+-incorporated O-state structures are consistent with the measured absorption wavelength (∼600 nm), which suggests that two conformations represent the O-state. These results may provide a key to designing enhanced tools in optogenetics.
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Project description:The light-driven inward chloride ion-pumping rhodopsin Nonlabens marinus rhodopsin-3 (NM-R3), from a marine flavobacterium, belongs to a phylogenetic lineage distinct from the halorhodopsins known as archaeal inward chloride ion-pumping rhodopsins. NM-R3 and halorhodopsin have distinct motif sequences that are important for chloride ion binding and transport. In this study, we present the crystal structure of a new type of light-driven chloride ion pump, NM-R3, at 1.58 Å resolution. The structure revealed the chloride ion translocation pathway and showed that a single chloride ion resides near the Schiff base. The overall structure, chloride ion-binding site, and translocation pathway of NM-R3 are different from those of halorhodopsin. Unexpectedly, this NM-R3 structure is similar to the crystal structure of the light-driven outward sodium ion pump, Krokinobacter eikastus rhodopsin 2. Structural and mutational analyses of NM-R3 revealed that most of the important amino acid residues for chloride ion pumping exist in the ion influx region, located on the extracellular side of NM-R3. In contrast, on the opposite side, the cytoplasmic regions of K. eikastus rhodopsin 2 were reportedly important for sodium ion pumping. These results provide new insight into ion selection mechanisms in ion pumping rhodopsins, in which the ion influx regions of both the inward and outward pumps are important for their ion selectivities.
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