Research ArticleSTRUCTURAL BIOLOGY

Pumping mechanism of NM-R3, a light-driven bacterial chloride importer in the rhodopsin family

See allHide authors and affiliations

Science Advances  07 Feb 2020:
Vol. 6, no. 6, eaay2042
DOI: 10.1126/sciadv.aay2042
  • Fig. 1 Structural characterization of NM-R3 retinal chromophores after light excitation.

    (A) Phylogenetic tree showing relationships among rhodopsins. (B) Difference absorption spectra showing the increasing red shift of the visible band near 600 nm with laser power. Spectra were obtained from the difference between the absorption spectra for the dark and light states, shown in fig. S1. (C) Difference electron density maps showing changes triggered near the retinal. |Fobs|light − |Fobs|dark difference Fourier electron density maps contoured at ±3.5σ. Positive and negative values are shown in blue and red, respectively.

  • Fig. 2 Atomic movements induced by photoexcitation.

    |Fobs|light – |Fobs|dark difference Fourier electron density map contoured at ±3.5σ in (A) CW95K-ND3, (B) CW95K-ND10, and (C) CW95K-ND30. For each map, three panels show different views of the retinal, and the central panel shows unexpected density peaks that appear consistently in each map. Red arrows suggest the movement of chloride ions following photoexcitation. Positive and negative values are shown in blue and red, respectively. The stick model represents the dark state of NM-R3. Additional views of the electron density maps are available in movies S1 and S2.

  • Fig. 3 Chloride ion conduction path from the extracellular to cytoplasmic face.

    |Fobs|light − |Fobs|dark difference Fourier electron density maps contoured at ±3.2σ covering a ribbon diagram of the entire protein model. Residues surrounding each chloride ion are represented by stick models. Positive and negative values are shown in blue and red, respectively. Two chloride ions (Cl-1 and Cl-2) are present in the refined dark model, and four additional ions (Cl-3, Cl-4, Cl-5, and Cl-6) were identified from the difference density maps. The Cl-1 ion moves slightly to site Cl-1B. A chloride ion at the extracellular face, Cl-3, was observed only in CW95K-ND3.

  • Fig. 4 Mutational analyses of functional residues in the chloride ion conduction pathway.

    (A) The anion-pumping activity of wild-type (WT) and mutant NM-R3 (C105A, C105F, S60F, Q224W, and F90W). Light-induced pH changes were measured using Escherichia coli cells expressing NM-R3 suspended in a solution containing 100 mM NaCl in the absence (gray solid lines) or presence (black solid lines) of the protonophore CCCP (30 μM) or in the presence of both 30 μM CCCP and 50 mM TPP+ (black broken lines). The broken red line in each panel indicates the maximum activity level achieved by WT protein with photo-stimulation. div., division. (B) Positive difference electron density of CW95K-ND30 contoured at ±3.5σ (top) and CW95K-ND3 contoured at ±3.2σ (bottom), showing the path of chloride ions through the protein.

  • Table 1 Summary of the intensity quantification of the difference Fourier electron density map for key water molecules, chloride ions, and residues.

    Values given in units of σ (equal to the root mean square electron density of the unit cell). Values below ±3.0σ were set to zero, as being of too low significance. Cells are colored according to the σ value, being darker green when the magnitude of the measured difference is stronger. Residue nomenclature: SC, side chain; BB, back bone; (+), positive density feature; (−), negative density feature.

    DatasetsCW95K-ND3CW95K-ND10CW95K-ND30Pulse-95KPulse-140K
    S/D (e.A3/s)0.0060.0120.0160.0090.016
    Retinal
      C14/C15 (+)5.47.66.99.36.1
      C14/C15 (−)−5.9−7.0−5.2−9.2−4.3
      C20 (+)4.33.94.44.94.8
      C20 (−)−3.5−4.5−5.4−5.8−4.2
    Lys235
      Nz/CE (+)11.09.88.510.94.9
      Nz/CE (−)−13.4−16.8−11.4−15.3−7.2
      CD (+)3.53.60.04.30.0
      CD (−)−7.1−7.5−3.8−7.6−3.8
      CG (+)0.03.83.154.70.0
      CG (−)0.0−6.00.0−6.60.0
      BB (+)3.34.50.04.60.0
      BB (−)−4.1−4.7−3.4−4.5−4.5
    Ser234
      SC (+)3.07.17.13.53.6
      SC (−)−4.2−6.6−6.0−4.1−3.6
      BB (+)3.74.53.44.83.0
      BB (−)−4.4−4.8−3.5−6.20.0
    Asp231
      SC (+)4.36.27.08.15.0
      SC (−)−9.1−11.4−9.8−13.0−10.4
      BB (+)0.00.00.03.93.5
      BB (−)0.0−4.6−4.2−5.1−4.4
    Arg95
      SC (+)0.00.04.94.14.1
      SC (−)−3.8−3.9−5.4−4.6−4.3
    Cys105
      SC (+)3.24.65.54.44.5
      SC (−)−5.2−5.8−6.4−5.1−5.6
    His29
      SC (+)3.44.96.15.46.4
      SC (−)0.0−4.8−5.3−5.9−7.0
      BB (+)0.00.04.90.03.5
      BB (−)0.00.0−3.20.00.0
    Met58
      SC (+)3.34.84.74.93.6
      SC (−)−5.5−9.2−9.0−9.8−7.3
    Trp99
      SC (+)0.03.53.80.00.0
      SC (−)0.0−3.8−4.20.00.0
    Trp201
      SC (+)3.63.26.14.1.4.0
      SC (−)−3.9−4.5−6.1−3.9−5.4
    Asn168
      SC (+)3.33.24.43.23.5
      SC (−)−4.4−3.4−5.1−4.2−5.5
    Water
      W4 (+)0.00.05.60.00.0
      W4 (−)0.0−3.9−5.10.00.0
      W7 (+)0.05.03.04.10.0
      W7 (−)−4.1−5.0−5.8−4.7−4.7
    Cl ion
      Cl-1 (−)−9.8−9.6−11.3−9.8−8.7
      Cl-1B (+)4.95.15.14.14.7
      Cl-3 (+)3.63.40.00.00.0
      Cl-4 (+)0.03.87.60.04.9
      Cl-5 (+)3.84.54.64.23.8
      Cl-6 (+)4.04.66.75.33.4

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/6/eaay2042/DC1

    Fig. S1. Absorption spectra of an NM-R3 crystal before and after laser irradiation.

    Fig. S2. Comparison of retinal conformations before and after laser irradiation.

    Fig. S3. Entire difference Fourier electron density map and average temperature factors of NM-R3 crystal structure in the dark and light state under different laser irradiation conditions.

    Fig. S4. Stereoscopic views of the difference electron density maps near the retinal.

    Fig. S5. Pathway for chloride ion transfer from extracellular to cytoplasmic face.

    Fig. S6. Difference distance matrix plots showing differences between the dark and light states of NM-R3.

    Fig. S7. Comparison of the NM-R3, HsBR, and HsHR retinal-binding pockets.

    Fig. S8. Mutation analysis of Ser234, an important residue for NM-R3 function.

    Table S1. Data collection and refinement statistics.

    Movie S1. Detailed view of changes around the chromophore of NM-R3.

    Movie S2. Difference electron density map showing changes at the chromophore of NM-R3.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. Absorption spectra of an NM-R3 crystal before and after laser irradiation.
    • Fig. S2. Comparison of retinal conformations before and after laser irradiation.
    • Fig. S3. Entire difference Fourier electron density map and average temperature factors of NM-R3 crystal structure in the dark and light state under different laser irradiation conditions.
    • Fig. S4. Stereoscopic views of the difference electron density maps near the retinal.
    • Fig. S5. Pathway for chloride ion transfer from extracellular to cytoplasmic face.
    • Fig. S6. Difference distance matrix plots showing differences between the dark and light states of NM-R3.
    • Fig. S7. Comparison of the NM-R3, HsBR, and HsHR retinal-binding pockets.
    • Fig. S8. Mutation analysis of Ser234, an important residue for NM-R3 function.
    • Table S1. Data collection and refinement statistics.
    • Legends for movies S1 and S2

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Detailed view of changes around the chromophore of NM-R3.
    • Movie S2 (.mp4 format). Difference electron density map showing changes at the chromophore of NM-R3.

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article