Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping

Abstract

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs reportedly work as light-driven inward H+ pumps, as xenorhodopsin. Here we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 in SzR are shorter than those in the other microbial rhodopsins. Thus, E81 is located near the cytosol, playing a critical role in the inward H+ release. We suggested the H+ is not metastably trapped in E81 and released through the water-mediated transport network from the retinal Schiff base to the cytosol. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.

Structural characterization of proton-pumping rhodopsin lacking a cytoplasmic proton donor residue by X-ray crystallography

“…In addition, cryo-trapping and femtosecond time-resolved X-ray crystallographic structural analyses have observed the rotation of the side chain of Leu93 during the photocycle (37,38). The mutation of homologous leucine residue in light-driven inward H + pumping rhodopsin, schizorhodopsin, results in the loss of function and direct connection between RSB and cytoplasmic solvent, even in the dark (39). These results indicate the importance of this leucine residue in H + transport.…”

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

“…The Arg conformations have been analyzed for C1C2 (PDB: 3UG9) (Kato et al, 2012), CrChR2 (PDB: 6EID) (Volkov et al, 2017), C1Chrimson (PDB: 5ZIH) (Oda et al, 2018), GtACR1 (PDB: 6CSM) (Kim et al, 2018), ChRmine (this study), HsBR (PDB: 5ZIM) (Hasegawa et al, 2018), HwBR (PDB: 4QID), cruxrhodopsin-3 (PDB: 4JR8) (Chan et al, 2014), deltarhodopsin (PDB: 4FBZ) (Zhang et al, 2013), GR (PDB: 6NWD) (Morizumi et al, 2019), Archaerhodopsin-1 (PDB: 1UAZ) (Enami et al, 2006), Archaerhodopsin-2 (PDB: 2EI4) (Yoshimura and Kouyama, 2008), PR from the Mediterranean Sea at a depth of 12 m (Med12BPR, PDB: 4JQ6) (Ran et al, 2013), PR from the Pacific Ocean near Hawaii at a depth of 75 m (HOT75BPR, PDB: 4KLY) (Ran et al, 2013), CsR (PDB: 6GYH) (Fudim et al, 2019), HsHR (PDB: 1E12) (Kolbe et al, 2000), NpHR (PDB: 3A7K) (Kouyama et al, 2010), ClR (PDB: 5ZTK) (Yun et al, 2021), KR2 (PDB: 3X3C) (Kato et al, 2015a), and schizorhodopsin 4 (PDB: 7E4G) (Higuchi et al, 2021).…”

“…(B) An unrooted phylogenetic tree was drawn for representative microbial rhodopsins using the neighbor-joining method (Saitou and Nei, 1987), and 1,000 bootstrap replicates. Evolutionary analyses were conducted in MEGA7 (Kumar et al, 2016) (C) Superposed Schiff base region of ChRmine (red) and representative microbial rhodopsins (CrChR2 [Volkov et al, 2017], C1Chrimson [Oda et al, 2018], GtACR1 [Kim et al, 2018], schizorhodopsin 4 [SzR4] [Higuchi et al, 2021], KR2 [Kato et al, 2015b], HsHR [Kolbe et al, 2000], NpSRII [Gordeliy et al, 2002], heliorhodopsin [Shihoya et al, 2019]), displayed with high transparency except for ChRmine. ).…”

The expanding Asgard archaea invoke novel insights into Tree of Life and eukaryogenesis

“…The ESCRT machinery as components of the ubiquitin system has one of the most conserved genomic neighborhoods in the genomes of Asgard archaea, implying signatures of vertical inheritance of ESCRT machinery from the ancestor 45 . The physiological functions of the ESCRT genes in Asgard archaea have been studied [58][59][60][61] . Collectively, this posits that these cell structure and trafficking machinery involving ESPs are functionally active in Asgard archaea, resembling activities in eukaryotic cells.…”