Research ArticleStructural Biology

Design of a light-gated proton channel based on the crystal structure of Coccomyxa rhodopsin

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Science Signaling  19 Mar 2019:
Vol. 12, Issue 573, eaav4203
DOI: 10.1126/scisignal.aav4203
  • Fig. 1 Structural architecture of CsR-WT.

    (A) Overall crystal structure of monomeric CsR. The transmembrane domain consists of seven α helices (TM1 to TM7). Membrane boundaries were calculated using the positioning of proteins in membrane-server (PPM) (74) and are shown as red and blue dashed lines. (B) Close-up view of the counterion complex displaying a conserved pentameric configuration of the counterions and three well-ordered water molecules. (C) Residues involved in forming a hydrophobic cage around Tyr14 (green). (D) Close-up views of the hydrogen-bond network connecting the RSB to the extracellular side, composed of the counterion complex and a highly entangled proton release complex. (E) Close-up view of the cytoplasmic half-channel. Important amino acid residues involved in forming the half-channel are depicted as sticks. The covalently linked chromophore all-trans-retinal (RET) is shown in red. Several ordered lipids are displayed as green sticks. Hydrogen bonds are depicted as dashes. Water molecules (W) are shown as purple spheres.

  • Fig. 2 Electrophysiological characterization of CsR-WT and CsR variants with different amino acids at position 14 in X. laevis oocytes.

    (A) Absolute photocurrent amplitudes of different CsR variants at three different holding potentials and pHo values. Number of measured oocytes at pH 5, pH 7.5, and pH 10: WT, n = 10, 34, 7; Y14F, n = 5, 6, 5; Y14Q, n = 5, 7, 5; Y14K, n = 6, 6, 6; Y14D, n = 4, 6, 3; and CySeR (Y14E), n = 4, 10, 4. Error bars represent mean SE. (B) Representative photocurrents for CsR-WT (top) and CySeR (bottom) at an external pHo of 7.5 and different holding potentials. Positive currents indicate outward-directed proton flux, whereas negative currents indicate inward-directed proton flux. (C) Reversal potential of CySeR at different pHo values. The black line represents the reversal potential of a pure proton channel according to the Nernst equation. Acidification of the protein environment shifts the reversal potential toward active transport.

  • Fig. 3 Structural comparison of short molecular dynamic simulations of CsR-WT and CySeR.

    (A) Molecular dynamic simulation of CsR-WT (blue ribbon representation) showing the environment of Tyr14. The side chain of Tyr14 (green sticks) is tight and cage-like embedded by hydrophobic amino acid side chains (gray sticks). The hydrogen bonding network of Tyr14-Arg83-Glu203 stabilizing this structural microenvironment, intramolecular interactions, and connection to the proton release complex are shown. (B) Superposition of CsR-WT (blue ribbons and light gray sticks, Tyr14 highlighted as green sticks) and CySeR (black ribbons and sticks, Y14E highlighted as magenta sticks), after short molecular dynamic simulations (1 ns). The simulated structure of CsR-Y14E suggests that the side chain of the substituted Glu14 is shifted, most likely by repulsion and delocalization of the negatively charged and hydrophilic side chain within this region. (C) Close-up view and surface representation of Tyr14. (D) Close-up view and surface representation of Glu14, showing the >5-Å distance between Arg83 and Glu14.

  • Fig. 4 Functional comparison of CsR-WT and CySeR.

    (A and B) Absorption spectra of purified CsR-WT (A) and CySeR (B) at pH 5 (green), pH 7.4 (red), and pH 9 (blue). Maximum absorption of the chromophore is displayed. (C and D) Averaged electrical recordings of CsR-WT (C; n = 6 cells, mean ± SE) and CySeR (D; n = 6 cells, mean ± SE) in HEK293 cells in 110 mM NaCl (pHo/i 7.2) under different holding potentials in single turnover experiments (top). Averaged laser flash photolysis data of CsR-WT (C; n = 15 measurements) and CySeR (D; n = 15 measurements) in 20 mM tris-HCl (pH 7.4), 150 mM NaCl, and 0.03% DDM (bottom). Distinguishable photocycle intermediates are marked with the nomenclature of analogous BR intermediates.

  • Fig. 5 Kinetic analysis of current components in CsR-WT and CySeR.

    (A) Charge transfer within the photocycle of CsR-WT for each of the calculated time constants averaged over n = 6 cells. Total transported charge represented by a black curve. (B) Voltage dependence of the determined time constants for CsR-WT. (C) Voltage dependence of the time constants (left) and amplitudes (right) for CySeR. Error bars represent the SD.

  • Table 1 Data collection and refinement statistics.

    PDB, Protein Data Bank; RMSD, root mean square deviation.

    CsR-WT (PDB entry 6GYH)
    Data collection
    (wavelength)
    ESRF, ID23-2
    λ = 0.8729 Å
      Space groupH3
    Cell dimensions
      a, b, c (Å)78.08, 78.08, 143.97
      α, β, γ (°)90.0, 90.0, 120.0
      Resolution (Å)32.91–2.00
    (2.05–2.00)*
      Rmerge0.110 (1.391)
      Rpim0.066 (0.901)
      <I/σ(I)>6.8 (0.8)
      CC1/20.997 (0.357)
      Completeness (%)100.0 (100.0)
      Redundancy4.4 (4.6)
    Refinement
      Resolution (Å)61.2–2.00
      No. of reflections20932
      Rwork/Rfree (%)19.2/22.5
      No. of atoms1715
      Protein118
      Water42
    B-factors
      Protein43.50
      Ligand/ion53.52
      Water51.90
    RMSD
      Bond length (Å)0.013
      Bond angle (°)1.77
    Ramachandran plot
      Favored (%)98.0
      Allowed (%)1.6
      Outlier (%)0.4

    *Highest resolution shell is shown in parentheses.

    †Ramachandran plot created by RAMPAGE using the Richardsons’ data.

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/12/573/eaav4203/DC1

      Fig. S1. Current/voltage (I/V) curve comparison of full-length and truncated CsR-WT in X. laevis oocytes.

      Fig. S2. Overall crystal packing of CsR-WT.

      Fig. S3. Quality of the electron density of the chromophore retinal, Arg83, and Tyr14 in CsR-WT.

      Fig. S4. Structural superposition of CsR-WT and BR.

      Fig. S5. Close-up views of the structural arrangement around the conserved Arg in various microbial rhodopsins.

      Fig. S6. pH titration of Asp86 in CsR-R83Q.

      Fig. S7. Single wavelength traces of photochemical Y14F and Y14E.

      Fig. S8. Current/voltage (I/V) curves of stationary photocurrents at different extracellular salt conditions.

      Fig. S9. Steady-state FTIR measurements of CsR-WT and CySeR.

      Fig. S10. Calculated absolute spectra of the photocycle intermediates of CsR-WT.

      Table S1. Time constants of photocycle transitions in CsR-WT and CySeR.

      Reference (75)

    • This PDF file includes:

      • Fig. S1. Current/voltage (I/V) curve comparison of full-length and truncated CsR-WT in X. laevis oocytes.
      • Fig. S2. Overall crystal packing of CsR-WT.
      • Fig. S3. Quality of the electron density of the chromophore retinal, Arg83, and Tyr14 in CsR-WT.
      • Fig. S4. Structural superposition of CsR-WT and BR.
      • Fig. S5. Close-up views of the structural arrangement around the conserved Arg in various microbial rhodopsins.
      • Fig. S6. pH titration of Asp86 in CsR-R83Q.
      • Fig. S7. Single wavelength traces of photochemical Y14F and Y14E.
      • Fig. S8. Current/voltage (I/V) curves of stationary photocurrents at different extracellular salt conditions.
      • Fig. S9. Steady-state FTIR measurements of CsR-WT and CySeR.
      • Fig. S10. Calculated absolute spectra of the photocycle intermediates of CsR-WT.
      • Table S1. Time constants of photocycle transitions in CsR-WT and CySeR.
      • Reference (75)

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