Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging.

Hirschi, Stephan; Lemmin, Thomas; Ayoub, Nooraldeen; Kalbermatter, David; Pellegata, Daniele; Ucurum, Zöhre; Gertsch, Jürg; Fotiadis, Dimitrios (2024). Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging. Nature Communications, 15(1), p. 6950. Springer Nature 10.1038/s41467-024-50960-3

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Microbial ion-pumping rhodopsins (MRs) are extensively studied retinal-binding membrane proteins. However, their biogenesis, including oligomerisation and retinal incorporation, remains poorly understood. The bacterial green-light absorbing proton pump proteorhodopsin (GPR) has emerged as a model protein for MRs and is used here to address these open questions using cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations. Specifically, conflicting studies regarding GPR stoichiometry reported pentamer and hexamer mixtures without providing possible assembly mechanisms. We report the pentameric and hexameric cryo-EM structures of a GPR mutant, uncovering the role of the unprocessed N-terminal signal peptide in the assembly of hexameric GPR. Furthermore, certain proteorhodopsin-expressing bacteria lack retinal biosynthesis pathways, suggesting that they scavenge the cofactor from their environment. We shed light on this hypothesis by solving the cryo-EM structure of retinal-free proteoopsin, which together with mass spectrometry and MD simulations suggests that decanoate serves as a temporary placeholder for retinal in the chromophore binding pocket. Further MD simulations elucidate possible pathways for the exchange of decanoate and retinal, offering a mechanism for retinal scavenging. Collectively, our findings provide insights into the biogenesis of MRs, including their oligomeric assembly, variations in protomer stoichiometry and retinal incorporation through a potential cofactor scavenging mechanism.

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