Crystal structure of the channelrhodopsin light-gated cation channel

Abstract

Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

“…8). 44 In an FT-IR study on the C1C2 chimera, a water molecule with the O-D stretch at 2378 cm −1 was assigned by its sensitivity to site-directed mutations to a water molecule H-bonded to D292 in the dark state, 45 a residue equivalent to D212 in BR. In the dark state of CaChR1, we estimate the O-H (O-D) frequency of the Hbonded O-H group of a dangling water at 3300 cm −1 (2450 cm −1 ), only slightly upshifted in respect to water W401 of BR and to the above discussed water molecule of the C1C2 chimera.…”

“…Structural model of CaChR1 derived by homology modeling and build by the SWISS-MODEL server 81 using the C1C2 structure as template (pdb: 3UG9). 44 The gray horizontal lines indicate the membrane part of the channel. The retinal chromophore is located in the middle of the membrane (yellow sticks) and bound via a Schiff base, SB (blue), linkage to the apoprotein.…”

“…42,43 CrChR2 absorbs maximally at ∼470 nm and shows a photocycle comprising a minimum of four intermediate states: 43 The only ChR whose 3D structure has been resolved to atomic resolution is a chimera construct from CrChR1 and CrChR2 (C1C2). 44 Recently, the first report on active internal water molecules on a ChR was published, performed on the C1C2 chimera. 45 Light-induced FT-IR spectroscopy revealed changes in water O-D vibrations upon illumination at 77 K, i.e., between the closed dark and the early P 1 500 state.…”

Achievement of protein micro-crystallography at SPring-8 beamline BL32XU

“…Several successful structure determinations from tiny lipidic cubic phase (LCP) crystals proved the efficiency of this system. In the case described in reference [5], helical data collection using a 1(H) x 10(V) m beam and 0.5 m step was conducted. The total absorbed dose was kept below 5 MGy for each of three MAD datasets by using this concept.…”

Alternative Formation of Red-Shifted Channelrhodopsins: Noncovalent Incorporation with Retinal-Based Enamine-Type Schiff Bases and Mutated Channelopsin

“…19) In addition, the X-ray crystal structure of C1C2 at 2.3 Å resolution is available, and it provides precise information for three-dimensional architecture of C1C2. 20) Based on the crystal structure, the cavity of the retinal binding pocket does not seem to be large enough to allow incorporation of our synthetic enamine-type Schiff bases, as the retinal binding lysine residue would interfere with the Schiff base. Thus, we constructed two C1C2 mutants to accommodate the larger chromophore: one is C1C2-K296A, in which the lysine at the retinal binding site, K296, was replaced by alanine, and the other is C1C2-K296G, in which lysine 296 was substituted with glycine.…”