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Structural insights into light-driven anion pumping in cyanobacteria.

ABSTRACT: Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na⁺, K⁺, Cl^-, I^-, NO₃^-. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics.

SUBMITTER: Astashkin R

PROVIDER: S-EPMC9617919 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Structural insights into light-driven anion pumping in cyanobacteria.

Astashkin R R Kovalev K K Bukhdruker S S Vaganova S S Kuzmin A A Alekseev A A Balandin T T Zabelskii D D Gushchin I I Royant A A Volkov D D Bourenkov G G Koonin E E Engelhard M M Bamberg E E Gordeliy V V

Nature communications 20221029 1

Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl-, I-, NO3-. The only characterized MR proposed to pump sulfate in addition to halides belongs to t ...[more]

PMID: 36309497

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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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Structural insights into light-driven anion pumping in cyanobacteria.

Publications

Structural insights into light-driven anion pumping in cyanobacteria.

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