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

Area of Science:

• Membrane protein biogenesis
• Microbial rhodopsins
• Structural biology

Background:

• Microbial ion-pumping rhodopsins (MRs) are vital retinal-binding membrane proteins, but their assembly and retinal incorporation are poorly understood.
• Proteorhodopsin (PR), a green-light absorbing proton pump, serves as a model for studying MR biogenesis.
• Existing research presents conflicting data on PR stoichiometry (pentamer vs. hexamer) and lacks mechanistic explanations.

Purpose of the Study:

• To elucidate the biogenesis of microbial ion-pumping rhodopsins, focusing on oligomeric assembly and retinal incorporation.
• To resolve conflicting reports on proteorhodopsin stoichiometry and investigate the underlying assembly mechanisms.
• To explore the mechanism of retinal scavenging in bacteria lacking endogenous retinal biosynthesis.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine high-resolution structures of proteorhodopsin mutants.
• Molecular dynamics (MD) simulations to investigate protein assembly, dynamics, and cofactor binding.
• Mass spectrometry to analyze protein composition and interactions.

Main Results:

• Determined pentameric and hexameric cryo-EM structures of a GPR mutant, revealing the N-terminal signal peptide's role in hexamer formation.
• Identified decanoate as a potential retinal placeholder in the chromophore binding pocket of retinal-free proteoopsin.
• MD simulations provided insights into decanoate-retinal exchange, suggesting a cofactor scavenging mechanism.

Conclusions:

• The N-terminal signal peptide influences proteorhodopsin oligomeric assembly, explaining stoichiometric variations.
• Decanoate acts as a temporary placeholder, facilitating retinal scavenging by proteorhodopsin-expressing bacteria.
• This study provides a comprehensive understanding of microbial ion-pumping rhodopsin biogenesis, including assembly and cofactor acquisition.