Atomistic Study of Intramolecular Interactions in the Closed-State Channelrhodopsin Chimera, C1C2

Abstract

Channelrhodopsins (ChR1 and ChR2) are light-activated ion channels that enable photomobility of microalgae from the genus Chlamydomonas. Despite common use of ChR2 in optogenetics for selective control and monitoring of individual neurons in living tissue, the protein structures remain unresolved. Instead, a crystal structure of the ChR chimera (C1C2), an engineered combination of helices I-V from ChR1, without its C-terminus, and helices VI-VII from ChR2, is used as a template for ChR2 structure prediction. Surprisingly few studies have focused in detail on the chimera. Here, we present atomistic molecular dynamics studies of the closed-state, non-conducting C1C2 structure and protonation states. A new and comprehensive characterization of interactions in the vicinity of the gating region of the pore, namely between residues E90, E123, D253, N258, and the protonated Schiff base (SBH), as well as nearby residues K93, T127, and C128, indicates that the equilibrated C1C2 structure with both E123 and D253 deprotonated closely resembles the available crystal structure. In agreement with experimental studies on C1C2, no direct or water-mediated hydrogen bonding between an aspartate and a cysteine (D156-O…S-C128) that would define a direct-current gate in C1C2 was observed in our simulations. Finally, we show that a single hydrogen bond between a glutamic acid (E90) and an asparagine (N258) residue suffices to keep the gate of C1C2 closed and to disable free water and ion passage through the putative pore, in contrast to the double bond proposed earlier for ChR2. We anticipate that this work will provide context for studies of both the gating process and water and ion transport in C1C2, and will spark interest in further experimental studies on the chimera.

Retinal isomerization and water-pore formation in channelrhodopsin-2

“…S4 ). Similar results were reported for previous models of ChR2 ( 6 , 31 , 39 ), and in simulations of related opsins ( 16 ).…”

“…Given the high sequence similarity between the C1C2 chimera and ChR2, structural and spectroscopic differences between them are rather small or nonexistent ( 16 – 18 ). Therefore, the crystal structure of the dark-adapted C1C2 is a good approximation of the closed ChR2, and a starting point for models of the open channel.…”

QM/MM Photodynamics of Retinal in the Channelrhodopsin Chimera C1C2 with OM3/MRCI

“…All calculations were carried out with a recent version of the program package COBRAMM, developed in Bologna, Lyon and Dü sseldorf. [27,28] It provides interfaces to several QM-and MMprograms, enabling hybrid QM/MM calculations in a subtractive scheme with electrostatic embedding and the hydrogen link atom approach.…”

“…Furthermore, mutation of D292 to alanine resulted in a substantial decrease in photocurrents, while the E162A mutant showed moderately decreased photocurrents . The MD study of VanGordon et al . reveals hydrogen bonds between RPSB and both counterions, too, and the authors propose aspartate to be the primary proton acceptor, at the same time emphasizing that glutamate cannot be completely neglected in this sense.…”

Channelrhodopsin C1C2: Photocycle kinetics and interactions near the central gate

“…We followed the previously reported molecular dynamics simulation set-up. 22 The pK a values of the ionizable C1C2 chimera side chains were calculated with the PROPKA code and averaged over the last 20 ns of molecular dynamics simulations.…”