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Molecular mechanism of light-driven sodium pumping.

ABSTRACT: The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton 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.

SUBMITTER: Kovalev K

PROVIDER: S-EPMC7195465 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Molecular mechanism of light-driven sodium pumping.

Kovalev Kirill K Astashkin Roman R Gushchin Ivan I Orekhov Philipp P Volkov Dmytro D Zinovev Egor E Marin Egor E Rulev Maksim M Alekseev Alexey A Royant Antoine A Carpentier Philippe P Vaganova Svetlana S Zabelskii Dmitrii D Baeken Christian C Sergeev Ilya I Balandin Taras T Bourenkov Gleb G Carpena Xavier X Boer Roeland R Maliar Nina N Borshchevskiy Valentin V Büldt Georg G Bamberg Ernst E Gordeliy Valentin V

Nature communications 20200501 1

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton 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, rev ...[more]

PMID: 32358514

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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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Molecular mechanism of light-driven sodium pumping.

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Molecular mechanism of light-driven sodium pumping.

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