Hirschi, StephanStephanHirschiLemmin, Thomas MaxThomas MaxLemminAyoub, Nooraldeen Fathi NooraldeenNooraldeen Fathi NooraldeenAyoub0000-0003-3146-242XKalbermatter, David AndreasDavid AndreasKalbermatter0000-0002-8842-5454Pellegata, DanieleDanielePellegataUcurum Fotiadis, ZöhreZöhreUcurum FotiadisGertsch, JürgJürgGertschFotiadis, Dimitrios JoséDimitrios JoséFotiadis2024-10-262024-10-262024-08-13https://boris-portal.unibe.ch/handle/20.500.12422/179736Microbial 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.en500 - Science::570 - Life sciences; biology600 - Technology::610 - Medicine & healtharticle10.48350/1996853913815910.1038/s41467-024-50960-3