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. 2018 Sep;561(7723):343-348.
doi: 10.1038/s41586-018-0511-6. Epub 2018 Aug 29.

Crystal Structure of the Natural Anion-Conducting Channelrhodopsin GtACR1

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Free PMC article

Crystal Structure of the Natural Anion-Conducting Channelrhodopsin GtACR1

Yoon Seok Kim et al. Nature. .
Free PMC article

Abstract

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overall structure of GtACR1.
Crystal structure of the GtACR1 dimer, viewed parallel to the membrane (left) and from the extracellular side (right). Disulfide bonds are shown using a stick model (green), and ATR (orange) is depicted by a sphere model.
Fig. 2
Fig. 2. Structural comparison of GtACR1 with C1C2.
a, b, Side (left) and extracellular (right) view of GtACR1 (blue) superimposed onto C1C2 (green) (a), and CrChR2 (yellow) (b). Red arrows mark the differences between the structures. c, Magnified view of N termini of GtACR1, C1C2 and CrChR2 as delimited by orange boxes in a and b. Green sticks denote disulfide bonds; note intramolecular disulfides bonds in GtACR1 (C219-to-C21) compared to the exclusively intermolecular disulfide bonds in C1C2 (at C73, C75, and C66) and CrChR2 (at C34 and C36).
Fig. 3
Fig. 3. RBP of GtACR1.
a, b, RBP of GtACR1 (a) and C1C2 (b). c, Effects of mutations (on residues comprising the GtACR1 RBP) on off-kinetics (top, fast closing; bottom, slow closing). Colour codes summarize the role of each residue in setting kinetics, wavelength or both. Data are mean and s.e.m; n = 10 for wild type (WT), 7 for C102A, 4 for M105A, M105I and C133R, and 5 for the rest. *P < 0.05, **P = 0.0021, ****P < 0.0001, Kruskal–Wallis with Dunn’s test. d, Absorption spectra of wild-type GtACR1 and the C237A mutant. Spectra were measured in one experiment. e, Traces of the wild-type GtACR1 and four kinetics-shifted mutants. Scale bar denoted by corresponding colour.
Fig. 4
Fig. 4. The protonated Schiff base region of GtACR1 and its counterions.
a, Structures of the Schiff base in GtACR1 (top), C1C2 (middle) and HsHR (bottom). Red spheres and dashed lines represent water molecules and hydrogen bonds, respectively; in GtACR1, D234 forms hydrogen bonds with the protonated Schiff base, Y72 and Y207, more similarly to HsHR than C1C2. b, Similar absorption spectra of wild-type GtACR1 and the D234N mutant, suggesting D234 protonation in the dark (see also Extended Data Fig. 9a). c, Light-induced difference FTIR spectra at 77 K. Note disappearance of the 1,740(−)/1,732(+) cm−1 peak pair (assigned to C = O vibration of a protonated carboxylate,) in D234N. Findings in b and c hold from at least pH 5–9 (Extended Data Fig. 9c). d, Current densities of wild-type GtACR1and three mutants. Note D234N abolishes the photocurrent (surprising if protonated in the dark), and Y207 (but not Y72) is essential (consistent with the importance of the local hydrogen-bonded network). Data are mean and s.e.m.; n = 9 for WT, 8 for D234N, 5 for Y72F and 4 for Y207F. **P = 0.01, ***P = 0.0006, one-way ANOVA followed by Dunnett’s test. e, Light-induced difference FTIR spectra of wild type and D234N at 170 K and 200 K. Decreased intensity of negative bands at 1,640 and 1,655 cm−1 reveals smaller conformational change of transmembrane helices in D234N. All spectroscopy experiments were performed once.
Fig. 5
Fig. 5. Ion-conducting pathways of GtACR1 and C1C2.
a, b, Ion-conducting pathways of GtACR1 (a) and C1C2 (b). The surface is coloured by the electrostatic potential calculated using PDB accession 2PQR for both GtACR1 and C1C2. Green, purple and orange-dashed circles represent the extracellular constriction site (ECS), intracellular constriction site (ICS) and central constriction site (CCS), respectively. IV, intracellular vestibule.
Fig. 6
Fig. 6. Constriction sites of GtACR1.
a, The ECS separating EV1 and EV2. Hydrogen bonds are shown as dashed lines. b, Initial glimpse of a patent intracellular conduction pathway for a light-activated channel; architecture of the GtACR1 intracellular ion exit pore leading to the intracellular vestibule (IV). c, The CCS architecture: sole constriction site in the pore, which separates the extracellular and intracellular vestibules. d, Current densities of mutants in residues comprising the CCS. Note the importance of residues E68 and N239 for photocurrents. Data are mean and s.e.m. n = 9 for WT, 5 for Q46A, E68A, E68T and E239A, and 4 for the rest. *P < 0.05, **P < 0.01, one-way ANOVA followed by Dunnett’s test. e, Comparison of reversal potentials. Note the signature of increased cation flux (depolarized reversal potential), consistent with disrupted pore selectivity Data are mean and s.e.m. n = 10 for WT, 6 for Q46A and Q46C, 5 for E68A and 4 for the rest. *P = 0.014, one-way ANOVA followed by Dunnett’s test.
Extended Data Fig. 1
Extended Data Fig. 1. Crystallography.
a, Size exclusion chromatogram of the purified GtACR1 protein used for crystallography. Similar results were seen in more than 20 independent experiments. b, Electrophysiology of full-length GtACR1 (left) and the final crystallization construct (right); whole-cell voltage-clamp recordings in five cells held at −70 mV, with 513 nm light at 1.0 mW mm−2 irradiance delivered with timing as shown with green-coloured bars, while cells were held at resting potentials from −95 mV (lowest trace) to +5 mV (uppermost trace) in steps of 10 mV. Similar results were seen in 3–5 cells from each group, and no significant difference was seen in resting potential, input resistance, reversal potential or photocurrent magnitude. c, Confocal images of cultured hippocampal neurons expressing full-length GtACR1 (left) and the final crystallization construct (right). Similar results were seen in more than five cells from 3–5 coverslips. Note the markedly reduced aggregation of the truncated construct. d, Crystals of GtACR1. Similar crystals were generated in more than 200 experiments. e, Lattice packing of GtACR1 crystals, viewed parallel to the x axis (left) and the y axis (right). f, Different amino acid configurations at different chains within the asymmetric unit of GtACR1. g, C-terminal interactions among different chains within the asymmetric unit of GtACR1.
Extended Data Fig. 2
Extended Data Fig. 2. Structural analysis of GtACR1.
a, 2Fo − Fc maps (blue mesh, contoured at 1σ) for the retinal-binding pockets of chains A–D. b, 2Fo − Fc maps (blue mesh, contoured at 1σ) for the lipid molecules. c, 2Fo − Fc maps (blue mesh, contoured at 1σ) and Fo − Fc maps (green and red meshes, contoured at 3.0σ and 3.0σ, respectively) for the Schiff base region of chains A–D. Water molecules are shown as red spheres. d, Table describing data collection and refinement statistics of GtACR1. Dataset was collected from 80 crystals. Values in parentheses are for the highest-resolution shell.
Extended Data Fig. 3
Extended Data Fig. 3. Structure-based sequence alignment of microbial opsin genes.
The sequences are GtACR1 (GenBank accession AKN63094.1), GtACR2 (GenBank AKN63095.1), ZipACR (GenBank APZ76709.1), PsuACR1 (GenBank ID: KF992074.1), the chimaeric channelrhodopsin between CrChR1 and CrChR2 (C1C2, PDB code 3UG9), CrChR1 (GenBank 15811379), CrChR2 (GenBank 158280944), ChR1 from Volvox carteri (VcChR1, UniProtKB B4Y103), ChR1 from V. carteri (VcChR2, UniProtKB ID: B4Y105), Chrimson (GenBank ID: AHH02126.1), ChR from Tetraselmis striata (TsChR, GenBank ID: KF992089.1), HsBR (PDB code 1C3W), HsHR (PDB code 1E12), and Krokinobacter eikastus rhodopsin 2 (KR2, PDB code 3X3B). The sequence alignment was created using PROMALS3D and ESPript 3 servers. Secondary structure elements for GtACR1 are shown as coils and arrows. ‘TT’ represents turns. Cysteine residues forming intermolecular and intramolecular disulfide bridges are highlighted in green and yellow, respectively. The residues of retinal-binding pockets are coloured pink. The residues in the Schiff base region are coloured cyan. The residues forming the ECS2 and CCS are coloured orange and blue, respectively.
Extended Data Fig. 4
Extended Data Fig. 4. Structural comparison among GtACR1, HsBR, HsHR, C1C2 and CrChR2.
a, b, Side view and extracellular view of the superimposed transmembrane regions of GtACR1 (blue) and HsBR (cyan) (a), GtACR1 (blue) and HsHR (beige) (b), C1C2 (green) and CrChR2 (yellow) (c). The ATRs are shown as stick models, and are coloured orange (GtACR1), salmon (HsBR), light-yellow (HsHR), green (C1C2) and yellow (CrChR2).
Extended Data Fig. 5
Extended Data Fig. 5. Interactions between N- and C-terminal regions and the 7-TM domain.
a, Interactions between the C-terminal region and the 7-TM domain. Hydrogen bonds are shown by dashed lines. b, Fluorescent size-exclusion chromatography traces of the full-length GtACR1 (1–295), the crystallized construct (1–282), and the C-terminal truncated construct (∆C: 1–253), showing possible importance of the C terminus in proper folding and/or stability. Similar results were observed in three independent experiments. c, Interactions between the N-terminal region and the ECL1. Hydrogen bonds are shown by dashed lines. d, Fluorescent size-exclusion chromatography traces of wild-type and C-to-S mutants of GtACR1. Labels indicate estimated elution positions of the aggregate, GtACR1–eGFP, and free eGFP; C-to-S mutants show decreased (<1/3) expression compared to the wild type. Similar results were observed in three independent experiments. e, Stained SDS–PAGE gel image of wild-type and N-terminal 6-amino-acid-truncated GtACR1 in the presence and absence of reducing reagent (β-mercaptoethanol); the wild type runs as a mixer of monomer and dimer in β-mercaptoethanol,whereas N-terminal-truncated GtACR1 stays monomeric even in the absence of β-mercaptoethanol. This experiment was performed once, but similar experiments with different concentrations of β-mercaptoethanol were performed three times, all with similar results. fh, Dimer interfaces of GtACR1 (f), C1C2 (g) and CrChR2 (h) viewed at two angles from the side; note reduced interface area (outlined) for GtACR1. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 6
Extended Data Fig. 6. Conductances, reversal potentials, absorption spectra and kinetics of wild-type GtACR1 and mutants.
ac, Photocurrents (a), reversal potentials (b) and absorption spectra (c) of wild-type GtACR1 and ten mutants of the retinal-binding pocket. λmax values are listed in the table (c, bottom). Photocurrents are measured in whole-cell voltage-clamp recordings held at −70 mV, with 513 nm light at 1.0 mW mm−2 irradiance. Data are mean and s.e.m.; n = 9 for WT, 6 for E163Q, 5 for C102A, M105A, C133A, C133R, C153A, E163A and C237A, and 4 for the rest. *P < 0.05, **P < 0.01, one-way ANOVA followed by Dunnett’s test. Reversal potentials are measured with identical light stimulation while cells were held at resting potentials from −95 mV to +15 mV in steps of 10 mV. Data are mean and s.e.m. n = 10 for WT and C237A, 6 for E163A and E163Q, 5 for C102A, M105A, C133A and C153A, and 4 for the rest. **P = 0.0022, one-way ANOVA followed by Dunnett’s test. Spectra measurement was performed in two independent trials, with wild type as a positive control. d, Comparison of fast closing (left) and slow closing (right) coefficients of wild-type and Y72F mutant GtACR1. Data are mean and s.e.m. n = 10 for WT and 5 for Y72F. P = 0.7 for both graphs, two-tailed t-test.
Extended Data Fig. 7
Extended Data Fig. 7. Current–voltage (IV) relationships of wild-type GtACR1 and mutants.
The IV relationship between −95 mV and +15 mV was determined from the single current amplitude at the indicated potentials. Each measurement is normalized to the current amplitude measured at −25 mV. Data are mean and s.e.m. n = 10 for WT and C237A, 8 for E223A, 6 for Q46C, E163A and E163Q, 4 for E68S, E68T, C102S and M105I, and 5 for the rest.
Extended Data Fig. 8
Extended Data Fig. 8. Representative traces of the IV measurement of wild-type GtACR1 and mutants.
Voltage clamp traces corresponding to the IV relationships in Extended Data Fig. 7 between −95 mV and +15 mV.
Extended Data Fig. 9
Extended Data Fig. 9. Spectroscopic characterization of wild-type GtACR1 and the D234N mutant.
a, Absorption spectra of wild-type GtACR1 (top left) and the D234N mutant (top right) measured from pH 3.0 to 10.0. The λmax value at each pH is listed in the table (bottom). b, Difference FTIR spectra of wild-type GtACR1 and the D234N mutant measured at 77 K, 170 K and 200 K. c, Difference FTIR spectra of wild-type GtACR1 in the 1,690–1,770 cm−1 region measured at pH 5.0, 7.0 and 9.0. Forty identical recordings at 77 K and seven identical recordings at 170 K and 200 K were averaged.
Extended Data Fig. 10
Extended Data Fig. 10. Comparison of surface electrostatic potential of GtACR1 and C1C2.
a, b, Electrostatic potential surfaces of GtACR1 (a) and C1C2 (b) viewed from four angles. The surface is coloured on the basis of the electrostatic potential contoured from −15 kT (red) to +15 kT (blue). c, d, Representation of positively charged amino acids (lysine and arginine residues) in GtACR1 (c), and negatively charged amino acids (aspartate and glutamate residues) in C1C2 (d).

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