Oral Presentation 18th International Congress on Photobiology 2024

Where do microbial rhodopsins come from? What are they? Where are they going? (#56)

Keiichi Inoue 1
  1. The Institute for Solid State Physics, The University of Tokyo, Kashiwa, CHIBA, Japan

Microbial rhodopsins are photoreceptive heptahelical membrane proteins that use an all-trans-retinal chromophore covalently bound to a conserved lysine residue in the seventh transmembrane helix through a protonated Schiff-base linkage. Upon Light absorption, the retinal undergoes all-trans-to-13-cis photoisomerization, initiating a photocyclic reaction accompanying a series of conformational change of the protein, thereby eliciting the biological function. While most microbial rhodopsins act as outward light-driven H+ pump, we discovered a new outward Na+-pumping rhodopsin, KR2, in the genome of the marine flavobacterium, Krokinobacter eikastus [1]. The structural and spectroscopic analyses revealed that intramolecular proton transfers are coupled with the Na+ transport through transiently neutralizing the positive charge of the protonated retinal Schiff base [2-3]. Through a comparison of amino acid sequences between KR2 and outward H+ pumps, we found that three residues in the third transmembrane helix play an crucial role in determining the substrate ion species and transport direction; these residues are referred to as motif residues. By focusing microbial rhodopsin genes with motif residues different from known groups, we identified two types of inward H+-pumping microbial rhodopsins [4-6]. X-ray structural analysis of these inward H+ pumps indicated that a single, and fewer in number compared to outward H+ pumps, counterion and a unique acidic residue on the cytoplasmic side facilitate the transport of H+ in the opposite direction to that of outward H+ pumps [7]. In 2018, heliorhodopsin, which forms a new family distinct from canonical microbial rhodopsins and has an inverted protein orientation with N- and C-termini facing the cytoplasmic and extracellular sides, respectively, was discovered through functional metagenomics [8]. Structural analysis showed a large fenestration between the fourth and fifth helices in heliorhodopsin, and biochemical assay suggested exogenous all-trans-retinal binds to the binding pocket in the protein through this fenestration [9]. Further genomic, metagenomic, and functional metagenomic surveys identified many new functional microbial rhodopsin groups [10,11]. These findings of new types of microbial rhodopsins are changing our traditional view of this protein family, which has been established over the past century. I will present our studies on these new microbial rhodopsins and offer perspectives on future research.

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  1. [1] Inoue et al., Nat. Commun. (2013); [2] Kato, Inoue et al., Nature (2015); [3] Inoue et al., Nat. Commun. (2019); [4] Inoue et al., Nat. Commun. (2016); [5] Inoue et al., Sci. Adv. (2020); [6] Marín et al., JACS (2023); [7] Higuchi et al., PNAS (2021); [8] Pushkarev, Inoue et al., Nature (2018); [9] Shihoya, Inoue et al., Nature (2019); [10] Rozenberg et al., PNAS (2022); [11] Chazan et al., Nature (2023);