Research ArticleMOLECULAR BIOLOGY

Structure and mechanisms of sodium-pumping KR2 rhodopsin

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Science Advances  10 Apr 2019:
Vol. 5, no. 4, eaav2671
DOI: 10.1126/sciadv.aav2671
  • Fig. 1 Architecture and cavities of KR2 protomers.

    (A) Overall view. Helices F and G are not shown. (B) Detailed view of the cytoplasmic part of KR2 protomer. Helix G is shown with 90% transparency. (C) Detailed view of the Schiff base region. Helix B is shown with 90% transparency, and helix A is not shown. (D) Detailed view of the extracellular part of KR2 protomer. The hydrophobic membrane core boundaries were calculated using the PPM server (48) and are shown as solid horizontal lines. The cavities were calculated using HOLLOW (49) and are pink colored.

  • Fig. 2 Overall architecture of KR2 pentamer and structure of the oligomerization interface.

    (A and B) View from the cytoplasmic and extracellular sides, respectively. Only contacts between chains A (yellow) and E (green) are shown. (C) Cytoplasmic side of the oligomerization interface. (D and E) Extracellular side of the oligomerization interface.

  • Fig. 3 Comparison of Schiff base region of different KR2 structures.

    (A) Chain A of pentameric Na+-pumping form (expanded conformation, pH 8.0) is shown in yellow. (B) Chain E of 4XTN model (compact conformation, pH 4.9) is shown in salmon. (C) The 4XTL model (one of two closely related to compact conformations pH 4.3) is shown in light blue. (D) The 3X3C model (closely related to compact conformation, soaked at pH 8.0 to 9.0) is shown in green. The red dashed ellipse shows double conformation of the Asp116 side chain. Red contoured arrows show the important displacement of the Asn112-Leu74 pair (colored teal). Helix A′ of nearby protomer and fragments of lipid molecules are shown in orange for pentameric and monomeric models, respectively. The cavities are colored pink. The cartoon representation of helix A is hidden for clarity. A prosthetic group retinal is colored dark green.

  • Fig. 4 Schiff base region structures in pentameric KR2 at different pH.

    (A) Detailed view of the RSB region of KR2 at pH 8.0 (yellow). Hydrogen bonds, stabilizing the expanded conformation, are shown as black dashed lines. (B) Detailed view of the RSB region of KR2 at pH 5.0 (magenta). The two parts show expanded and compact conformations, which coexist in KR2 at pH 5.0. Arrows show the important displacement of the Asn112-Leu74 pair. Helix A′ of nearby protomer is shown in orange. Helices A and B are not shown. The cavities are colored pink. Hydrogen bonds, stabilizing the compact conformation, are shown as black dashed lines. (C) 2Fo-Fc electron density maps of the double conformation of the Asn112-Leu74 pair in the KR2 model at pH 5.0. The maps are contoured at the level of 1.0σ.

  • Fig. 5 Schiff base cavity in K+-pumping mutants of KR2.

    (A) Monomeric blue form of G263F. (B) Pentameric red form of G263F. (C) Monomeric blue form of S254A. (D) Pentameric red form of S254A. The cavities are colored pink. A prosthetic group retinal is colored dark green.

  • Fig. 6 Effects of mutations of KR2 at oligomerization interface.

    (A) SEC profiles of wild-type (WT) KR2 at pH 4.3, 6.0, and 8.0. a.u, arbitrary units. (B) SEC profiles of wild-type KR2 and its mutants at pH 8.0. Protein with an initial concentration of 70 mg/ml was dissolved in buffer solution with 0.1% n-dodecyl β-d-maltoside (DDM) to a final concentration of 1 to 2 mg/ml and incubated for 72 hours. (C) Na+-pumping activity of KR2 and its mutants measured in E. coli cell suspension. The solutions contain 100 mM NaCl (black and dashed) and 100 mM NaCl and 30 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (black and solid). The cells were illuminated for 300 s (yellow area on the plots). (D) Ribbon representation of the structure alignment of H30K (green) and Y154F (orange) mutants and wild type (yellow) of KR2. Side chains at positions 30 and 154 are shown as sticks. The hydrophobic membrane core boundaries are shown as solid horizontal lines. (E and F) Y154F and H30K mutation region aligned with the wild-type protein, respectively. 2Fo-Fc electron density maps are shown for the mutant structures and are contoured at the level of 1.5σ (Y154F) and 1.0σ (H30K).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaav2671/DC1

    Fig. S1. KR2 ion uptake pore.

    Fig. S2. Comparison of orientations and cavities inside different KR2 structures.

    Fig. S3. Comparison of extracellular regions of different KR2 structures.

    Fig. S4. Activity tests of KR2 potassium-pumping mutants.

    Fig. S5. Structural alignment of potassium-pumping KR2 mutants with the model of wild-type protein.

    Fig. S6. Structures of potassium-pumping KR2 mutants.

    Fig. S7. KR2 crystal soaking.

    Fig. S8. KR2 dry and wet forms.

    Fig. S9. Activity tests of KR2 at different pH.

    Fig. S10. Electron density maps of KR2 structures.

    Table S1. Summary information of crystal structures obtained.

    Table S2. Data collection and refinement statistics of the wild-type KR2.

    Table S3. Data collection and refinement statistics of KR2 mutants.

    Table S4. Distance between Asp116 oxygen and RSB nitrogen atoms in KR2 models.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. KR2 ion uptake pore.
    • Fig. S2. Comparison of orientations and cavities inside different KR2 structures.
    • Fig. S3. Comparison of extracellular regions of different KR2 structures.
    • Fig. S4. Activity tests of KR2 potassium-pumping mutants.
    • Fig. S5. Structural alignment of potassium-pumping KR2 mutants with the model of wild-type protein.
    • Fig. S6. Structures of potassium-pumping KR2 mutants.
    • Fig. S7. KR2 crystal soaking.
    • Fig. S8. KR2 dry and wet forms.
    • Fig. S9. Activity tests of KR2 at different pH.
    • Fig. S10. Electron density maps of KR2 structures.
    • Table S1. Summary information of crystal structures obtained.
    • Table S2. Data collection and refinement statistics of the wild-type KR2.
    • Table S3. Data collection and refinement statistics of KR2 mutants.
    • Table S4. Distance between Asp116 oxygen and RSB nitrogen atoms in KR2 models.

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