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Mechanism by which water and protein electrostatic interactions control proton transfer at the active site of channelrhodopsin.

ABSTRACT: Channelrhodopsins are light-sensitive ion channels whose reaction cycles involve conformation-coupled transfer of protons. Understanding how channelrhodopsins work is important for applications in optogenetics, where light activation of these proteins triggers changes in the transmembrane potential across excitable membranes. A fundamental open question is how the protein environment ensures that unproductive proton transfer from the retinal Schiff base to the nearby carboxylate counterion is avoided in the resting state of the channel. To address this question, we performed combined quantum mechanical/molecular mechanical proton transfer calculations with explicit treatment of the surrounding lipid membrane. The free energy profiles computed for proton transfer to the counterion, either via a direct jump or mediated by a water molecule, demonstrate that, when retinal is all-trans, water and protein electrostatic interactions largely favour the protonated retinal Schiff base state. We identified a conserved lysine group as an essential structural element for the proton transfer energetics in channelrhodopsins.

SUBMITTER: Adam S

PROVIDER: S-EPMC6080761 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Mechanism by which water and protein electrostatic interactions control proton transfer at the active site of channelrhodopsin.

Adam Suliman S Bondar Ana-Nicoleta AN

PloS one 20180807 8

Channelrhodopsins are light-sensitive ion channels whose reaction cycles involve conformation-coupled transfer of protons. Understanding how channelrhodopsins work is important for applications in optogenetics, where light activation of these proteins triggers changes in the transmembrane potential across excitable membranes. A fundamental open question is how the protein environment ensures that unproductive proton transfer from the retinal Schiff base to the nearby carboxylate counterion is av ...[more]

PMID: 30086158

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Project description:The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamide ribonucleotide (GAR), a process that is pH-dependent with pK(a) of approximately 8. Experimental studies of pH-rate profiles of wild-type and site-directed mutants of GART have led to the proposal that His108, Asp144, and GAR are involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formyl group of fTHF. This model implied a protonated histidine with pK(a) of 9.7 and a neutral GAR with pK(a) of 6.8. These proposed unusual pK(a)s have led us to investigate the electrostatic environment of the active site of GART. We have used Poisson-Boltzmann-based electrostatic methods to calculate the pK(a)s of all ionizable groups, using the crystallographic structure of a ternary complex of GART involving the pseudosubstrate 5-deaza-5,6,7,8-THF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs have been constructed to elucidate pairwise electrostatic interactions between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a reconstructed pseudocofactor with an attached formyl group, in an environment with optimal local van der Waals interactions (locally minimized) that imitates closely the catalytic reactants, has been used for pK(a) calculations. Strong electrostatic coupling among catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We show that a proton can be exchanged between GAR and His108, depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108, catalysis could be possible. Using the formylated locally minimized construct of GART, a high pK(a) for His108 was calculated, indicating a protonated histidine, and a low pK(a) for GAR(NH(2)) was calculated, indicating that GAR is in neutral form. Our results are in qualitative agreement with the current mechanistic picture of the catalytic process of GART deduced from the experimental data, but they do not reproduce the absolute magnitude of the pK(a)s extracted from fits of k(cat)-pH profiles, possibly because the static time-averaged crystallographic structure does not describe adequately the dynamic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pK(a) of GAR(NH(2)) is produced by the theoretical mutations of His108Ala and Asp144Ala, which is not in agreement with the observed insensitivity of the pK(a) of GAR(NH(2)) modeled from the experimental data using similar mutations. Finally, we show that important three-way electrostatic interactions between highly conserved His137, with His108 and Asp144, are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed computational and experimental work is necessary.

Dataset Information

Mechanism by which water and protein electrostatic interactions control proton transfer at the active site of channelrhodopsin.

Publications

Mechanism by which water and protein electrostatic interactions control proton transfer at the active site of channelrhodopsin.

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