Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin

Abstract

The green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

Molecular Origin of the Anomalous pH Effect in Blue Proteorhodopsin

“…22−24 In addition, the recent cryo-EM structure reported the pentamer structure for GPR. 25 Thus, a similar oligomeric structure is likely for VcBPR at pH 7.0. When the pH is lowered, the peak at the elution volume of 0.56 mL is reduced and shifting to higher elution volumes.…”

“…The SEC profile of VcBPR at pH 7.0 shows a single peak at the elution volume of 0.56 mL (Figure ). Atomic force microscopy (AFM) and X-ray crystallography of PRs reported pentamer or hexamer structure. − In addition, the recent cryo-EM structure reported the pentamer structure for GPR . Thus, a similar oligomeric structure is likely for VcBPR at pH 7.0.…”

Deciphering the Spectral Tuning Mechanism in Proteorhodopsin: The Dominant Role of Electrostatics Instead of Chromophore Geometry

“…[39] Earlier studies have relied on homology models based on the amino acid sequence of the Monterey Bay GPR (PDB code: 2L6X) using bacteriorhodopsin as a template. [40][41][42][43] However, homology models can often impede predictions due to the sensitivity of the chromophore to small displacements of the surrounding side chains. [44][45][46] Moreover, the uncertainty about the protonation state of the D97 counterion in GPR (pKa value of ~7) can alter the result of the simulation.…”

“…A prerequisite for a computational study of the color tuning mechanism is a high‐resolution protein structure [39] . Earlier studies have relied on homology models based on the amino acid sequence of the Monterey Bay GPR (PDB code: 2L6X) using bacteriorhodopsin as a template [40–43] .…”

“…A prerequisite for a computational study of the color tuning mechanism is a high‐resolution protein structure [39] . Earlier studies have relied on homology models based on the amino acid sequence of the Monterey Bay GPR (PDB code: 2L6X) using bacteriorhodopsin as a template [40–43] . However, homology models can often impede predictions due to the sensitivity of the chromophore to small displacements of the surrounding side chains [44–46] .…”