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Identification of intermediate conformations in the photocycle of the light-driven sodium-pumping rhodopsin KR2.

ABSTRACT: 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.

SUBMITTER: Tsujimura M

PROVIDER: S-EPMC8039564 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H(+)/Na(+) pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na(+) and H(+) transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na(+) pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na(+)-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na(+) transport and electrogenic H(+) transport, respectively, in the presence and absence of Na(+). The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na(+) transport by IAR requires Na(+) only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H(+)/Na(+) pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.

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Identification of intermediate conformations in the photocycle of the light-driven sodium-pumping rhodopsin KR2.

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