Allosteric Effects of the Proton Donor on the Microbial Proton Pump Proteorhodopsin

Abstract

Proteorhodopsin (PR) is a microbial proton pump that is ubiquitous in marine environments and may play an important role in the oceanic carbon cycle. Photoisomerization of the retinal chromophore in PR leads to a series of proton transfers between specific acidic amino acid residues and the Schiff base of retinal, culminating in a proton motive force to facilitate ATP synthesis. The proton donor in a similar retinal protein, bacteriorhodopsin, acts as a latch to allow the influx of bulk water. However, it is unclear if the proton donor in PR, E108, utilizes the same latch mechanism to become internally hydrated. Here, we used molecular dynamics simulations to model the changes in internal hydration of the blue variant of PR during photoactivation with the proton donor in protonated and deprotonated states. We find that there is a stark contrast in the levels of internal hydration of the cytoplasmic half of PR based on the protonation state of E108. Instead of a latch mechanism, deprotonation of E108 acts as a gate, taking advantage of a nearby polar residue (S61) to promote the formation of a stable water wire from bulk cytoplasm to the retinal-binding pocket over hundreds of nanoseconds. No large-scale conformational changes occur in PR over the microsecond timescale. This subtle yet clear difference in the effect of deprotonation of the proton donor in PR may help explain why the photointermediates that involve the proton donor (i.e., M and N states) have timescales that are orders of magnitude different from the archaeal proton pump, bacteriorhodopsin. In general, our study highlights the importance of understanding how structural fluctuations lead to differences in the way that retinal proteins accomplish the same task.

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

“…This specific position is documented to cause a ~20 nm bathochromic shift upon a glutamine (Q) to leucine (L) mutation. [13,15,[24][25][26][27][28][16][17][18][19][20][21][22][23] Experimental and computational studies have been performed to elucidate the exact nature of the Q105L color-tuning mechanism. [13,22,23,25,26,28,29] It was found that the color switching Q105L mutation affects the C 14 À C 15 bond length of the retinal polyene chain.…”

“…[13,15,[24][25][26][27][28][16][17][18][19][20][21][22][23] Experimental and computational studies have been performed to elucidate the exact nature of the Q105L color-tuning mechanism. [13,22,23,25,26,28,29] It was found that the color switching Q105L mutation affects the C 14 À C 15 bond length of the retinal polyene chain. [21,22] In general, the bond length alternation (BLA) or bond order alternation (BOA), correlate strongly with the absorption maxima of rhodopsins.…”

“…[10] The possibility to use experimentally resolved crystallographic structures, rather than homology models, as well as the availability of key mutants, serves as a great starting point for computational studies. [22,23] The aim of this study is to use hybrid QM/MM methods to investigate the origin of the spectral tuning in the BPR which cannot be explained by the external point charge model by Honig and coworkers. To address this question, we have used spectra constructed from 100 structures sampled from hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations.…”

“…The residue in position 105 has been experimentally identified to be a key residue responsible for the spectral shift. This specific position is documented to cause a ∼20 nm bathochromic shift upon a glutamine (Q) to leucine (L) mutation [13,15,24–28,16–23] . Experimental and computational studies have been performed to elucidate the exact nature of the Q105L color‐tuning mechanism [13,22,23,25,26,28,29] .…”

“…Two important mutated variants of HOT75BPR (or simply BPR) were crystallized: the counterion mutation D97N (PDB: 4KLY) and the double mutant D97N/Q105L (PDB: 4KNF) [10] . The possibility to use experimentally resolved crystallographic structures, rather than homology models, as well as the availability of key mutants, serves as a great starting point for computational studies [22,23] …”