Crystal structure of a light-driven sodium pump

Abstract

Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

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Figure 1: Overall architecture of KR2.
Figure 2: Ion-translocation pathway in KR2.
Figure 3: Conformations of the Asn112 and Asp116 pair in different KR2 states.
Figure 4: Ion-uptake cavity in KR2.
Figure 5: Sodium ion in the KR2 structure.

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Acknowledgements

The work was supported by the CEA (Institut de Biologie Structurale)–Helmholtz-Gemeinschaft (Research Center Jülich) Special Topic of the Cooperation 5.1 Specific Agreement (I.G., V.S., V.P., K.K., A.A., E.R., V.B., T.B. and V.G.). Part of this work was supported by the German Ministry of Education and Research (PhoNa–Photonic Nanomaterials, 03152101F; T.B. and V.G.). This work was supported by the Russian Foundation for Basic Research (research project 13-04-91320; V.B., A.A. and K.K.) and the Helmholtz Association of German Research Centers (project HRJRG-401; V.G., I.G., V.S., V.P., E.R. and T.B.), the Russian Federal Target Program 'Research and Development' (contract 14.587.21.0004, RFMEFI58714X0004; V.B. and G.B.) and the 5top100 program of the Ministry for Science and Education of Russia and Governmental Task 120/2014 (K.K., A.A., E.R., V.B., G.B. and V.G.). Crystallization, X-ray data collection and treatment were supported by the Russian Scientific Foundation (research project 14-14-00995; I.G., V.P., K.K., A.A., V.B. and V.G.). The work used the platforms of the Grenoble Instruct Center (Integrated Structural Biology Grenoble (ISBG); UMS 3518 CNRS–CEA–Université Joseph Fourier–European Molecular Biology Laboratory) with support from the French Infrastructure for Integrated Structural Biology (FRISBI) (ANR-10-INSB-05-02) and the Grenoble Alliance for Integrated Structural and Cell Biology (GRAL) (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology. V.P. is deeply thankful to the Fondation Nanosciences for financial support. The diffraction experiments were performed on beamlines ID23-1 and ID29 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We are grateful to local contacts at ESRF for providing assistance with using these beamlines. We are also deeply thankful to G. Gotthard, A. Royant and D. von Stetten (ESRF Cryobench laboratory) for help with collection of the KR2 crystals' optical absorption spectra.

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V.S., K.K. and A.A. expressed and purified the protein and its mutants; T.B. oversaw the expression and purification, V.P. crystallized the protein and collected absorption spectra; E.R. helped with automated crystallization; I.G. collected the diffraction data and solved the structures; A.P. helped with data collection; V.B. and G.B. helped with structure analysis; K.K. conducted the E. coli activity measurements; I.G., V.S. and A.A. helped with E. coli activity measurements; C.F. and T.G. contributed to the functional studies of the wild-type protein; V.S. and C.B. conducted the BLM activity experiments; E.B. oversaw the BLM activity experiments and analysis of the data, and contributed to the analysis of the relevance of the protein and its mutants for optogenetics; D.W. helped with manuscript preparation, V.G. oversaw the project; I.G. and V.G. analyzed the results and prepared the manuscript with input from all the other authors.

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Correspondence to Valentin Gordeliy.

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Integrated supplementary information

Supplementary Figure 1 Normalized absorption spectra of KR2 in detergent and blue (type A) and red (types B and C) crystals and photographs of the crystals in the crystallization drops.

The absorption maxima are at 522, 566, 530 and 528 nm correspondingly. a, Comparison of the absorption spectra. The absorption is normalized to reach 1 at the maximum. b and c, Fit of the absorption by the types B and C red crystals with the combination of the absorption in detergent (deprotonated Asp116) and in the blue crystals (protonated Asp116). d-f, Photographs of the crystallization drops with the type A crystals (d), type B crystals (e) and type C crystals (f). The perceived color of the crystals may differ from the actual color due to the variations in the imaging conditions.

Supplementary Figure 2 Crystal packings of KR2 and examples of the electron density maps.

a-c, Crystal packing of KR2 in the type A (a), B (b) and C (c) crystals. Typically for in meso-grown crystals, in all the types of crystals the proteins are arranged in stacked membrane-like layers. Molecules of the same asymmetric unit are shown in the same color.d-e, f-g and h, Examples of the electron density maps. The presented 2Fo-Fc maps are contoured at the level of 1.5σ. d, Retinal binding pocket in the type A crystal. e, f-g and h, Electron density maps around the residues Leu74 and Asn112 in the type A (e), B (f-g) and C (h) crystals.

Supplementary Figure 3 Comparison of KR2 structure with structures of other microbial rhodopsins.

a, Comparison of the KR2 backbone structure in the type A crystals (yellow) with those of bacteriorhodopsin (BR, purple, PDB ID 1C3W) and halorhodopsin (HR, blue, PDB ID 1E12). Root mean square deviations of the KR2 transmembrane helix backbone N, C and Cα atoms relative to BR and HR are 2.5 and 2.3 Å correspondingly. b Comparison of the cytoplasmic sides. c, Comparison of the retinal positions. In KR2, the retinal is closer to the helices C and D, similar to blue proteorhodopsins (grey, PDB ID 4JQ6, salmon, PDB ID 4KLY) and opposite to BR, HR and Exiguobacterium sibiricum rhodopsin (green, PDB ID 4HYJ). d, Function-defining residues in KR2, BR and HR. While BR’s Asp212 is present in all the three proteins, the residues Asp85, Thr89 and Asp96 are replaced with Asn112, Asp116 and Gln123 in KR2 and other sodium pumps, earning them the designation NQ- or NDQ-rhodopsins.

Supplementary Figure 4 Comparison of the KR2 backbone structure in the type A (blue), type B (magenta) and type C (yellow) crystals.

a, Overall structure. Most of the variation is observed in the positions of the cytoplasmic part of the helix C, of the B-C loop and of the N-terminal helix. b, Cα-traces of the helices B and C. Twisting at the Pro118-induced break in the α-helical structure results in substantial displacements of Asn112 and other residues’ Cα positions. In the type A crystals the residues 111-114 are in the clearly α-helical conformation (the O111-N115 distance is 3.1 Å). In the molecule D of the type B crystals, where the helix C is twisted the most, the residues 111-114 are in 310-helix-like conformation (the O111-N114 distance is 3.0 Å, while the O111-N115 distance is 4.4 Å). c, Top view of the Asn112 region. Asn112 position is very variable in the type B crystals.

Supplementary Figure 5 Details of the KR2 ion-translocation pathway.

a, Gln123 coordination. b, Ion uptake cavity. Helix G is not shown for clarity. Several ordered lipid hydrophobic tail groups (green) are observed right underneath the cavity entrance. Putative hydrogen bonds are shown in black, hydrogen bonds to the helix G atoms are shown in red. c and d, Ion release regions in the monomeric KR2 in the type A crystals (c) and in the pentameric KR2 in the type B crystals (d). Putative hydrogen bonds are shown by the dashes. Conformations of the ion release regions in the ten molecules of the Type B crystals are identical. e, Val67 and Leu120 region of KR2.

Supplementary Figure 6 E. coli activity tests of KR2 and its mutants.

a, pH changes upon illumination in the media containing KR2-expressing E. coli cells. The solutions contain 100 mM NaCl (blue), 100 mM NaCl and 30 µM CCCP (green), 100 mM KCl (red), 100 mM KCl and 30 µM CCCP (magenta). The cells were illuminated for 300 s (light area on the plots). b, Pump activities of KR2 and its mutants expressed in E. coli normalized by characteristic protein absorption. The solutions contain 100 mM NaCl (blue), 100 mM NaCl and 30 µM CCCP (green), 100 mM KCl (red), 100 mM KCl and 30 µM CCCP (magenta). The activities marked with asterisk (*) are below the variation in the background. c, Tested mutations in the KR2 ion pathway. The mutants N61M, G263F,L; E11A, E160A and R243A,Q are tested in this work, the mutants R109A, N112A,D, D116A,E,N, Q123A,E and D251A,E,N have been tested by Inoue et al.

Supplementary Figure 7 Analysis of the KR2 pentamer and its possible carotenoid-binding site.

a, View at the KR2 pentamer from the cytoplasmic side. One of the protomers is shown in green for clarity. The ordered lipid hydrophobic chains observed in the structure are shown in magenta. b, Comparison of the KR2 pentameric arrangement (yellow and green) with that of HOT75 proteorhodopsin (magenta). c, Residues at the oligomerization interface at the cytoplasmic side. d, Residues at the oligomerization interface at the extracellular side. The Na+ ion bound to Tyr25’ and Asp102 may be seen in the background. Putative hydrogen bonds are shown in black, hydrogen bonds to the backbone atoms are shown in red. e, Comparison of the xanthorhodopsin (XR) carotenoid-binding site with the homologous site of KR2. KR2 is shown in yellow, XR and the bound carotenoid salinixanthin are shown in blue. The structure alignment was done locally to highlight the similarity. One of the main prerequisites for the carotenoid binding is direct access of its 4-keto ring to the retinal’s β-ionone ring. In XR and Gloeobacter violaceus rhodopsin (GR), which have been shown to employ the light-harvesting carotenoid antennas salinixanthin and echinenone, this is possible due to the absence of the side chain of the retinal’s proximal glycine amino acid (Gly156 in XR). Similarly to XR and GR, KR2 has a glycine residue at the position 171. Krokinobacter eikastus membranes were also reported to contain a carotenoid. Other residues, involved in the carotenoid binding, are also largely conserved in KR2. The most significant differences between the surfaces are KR2’s A199, I213 and T216 instead of R184, A198 and G201 in XR. However, alanine and leucine are also observed in GR at the positions corresponding to A199 and I213, and GR is capable of carotenoid binding. Thus, these substitutions are not crucial for the carotenoid binding and it is highly probable that KR2 also binds a carotenoid antenna in its native membrane. The effect of G201XR → T216KR2 substitution is not clear. f, Possibility of xanthorhodopsin-like carotenoid binding in the pentameric arrangement of KR2. One KR2 monomer is shown in yellow, the other four are shown in the surface representation in beige and light yellow, XR and the bound carotenoid salinixanthin are shown in blue. Salinixanthin and KR2 retinal are shown in the space filling representation.

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Gushchin, I., Shevchenko, V., Polovinkin, V. et al. Crystal structure of a light-driven sodium pump. Nat Struct Mol Biol 22, 390–395 (2015). https://doi.org/10.1038/nsmb.3002

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