Structural basis for Na+ transport mechanism by a light-driven Na+ pump

Abstract

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na+ pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na+ transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na+ transport. Together with the structure-based engineering of the first light-driven K+ pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

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Figure 1: Overall structure of KR2 and comparison with BR.
Figure 2: Structures and functions of the N-helix and ECL1.
Figure 3: The intracellular cavity and the retinal binding pocket
Figure 4: The Schiff base regions under acidic and neutral conditions.
Figure 5: Proposed model of Na+ transport.
Figure 6: Optogenetic applications and molecular engineering of KR2.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors for KR2 in acidic and neutral conditions have been deposited in the Protein Data Bank, under the accession codes 3X3B and 3X3C, respectively.

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Acknowledgements

We thank M. Hattori and H. Nishimasu for useful discussions and critical comments on the manuscript; A. Kurabayashi, N. Sahara, and J. Sasaki for technical assistance; and S. Doki, K. Hirata and the beam-line staff members at BL32XU of SPring-8 for assistance in data collection. The synchrotron radiation experiments were performed at BL32XU of SPring-8, with approval from RIKEN. This work was supported by the Platform for Drug Discovery, Informatics and Structural Life Science, of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), by JSPS KAKENHI (grant nos. 11J06643, 24115508, 24655009, 25104009, 24681003, 24227004, and 25291011), by the FIRST program, PRESTO, CREST, JST, and by a Grant-in-Aid for JSPS Fellows.

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Contributions

H.E.K. expressed, purified, and crystallized KR2, and collected the diffraction data and solved the structures. K.I., H.O., Y.K., R.Y. and M.K. performed the functional analyses of KR2 in E. coli. H.K. and Y.I. performed the behavioural analysis in C. elegans. S.H., T.I., M.R.H., J.I., A.D.M. and H.Y. performed the electrophysiology in mammalian cells. M.T. and N.T. helped to make KR2 constructs for expression in mammalian cells. R.T. helped to measure the expression levels of KR2 in mammalian cells. K.Y. assisted with structure solution. S.Y. and K.K. provided the KR2 gene and helped to organize the project. H.E.K., K.I., R.I., H.K. and O.N. wrote the manuscript. H.K. and O.N. directed and supervised all of the research.

Corresponding authors

Correspondence to Hideki Kandori or Osamu Nureki.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Ion transport mechanisms of light-driven proton pumps and chloride pumps.

a, The proton transport mechanism for light-driven proton pumping in BR3,4. The Schiff base proton works as the substrate, and avoids the electrostatic repulsion between itself and the substrate. b, The chloride transport mechanism for light-driven chloride pumping in HR3,4. The numbering corresponds to the residue numbers in HR from Halobacterium salinarum. c, The electrostatic repulsion problem has to be solved by the light-driven cation pumps, rather than the proton pumps. The substrate cation (A+) must avoid the electrostatic repulsion with the Schiff base proton.

Extended Data Figure 2 Crystallography.

a, Table describing data collection and refinement statistics of KR2 in acidic and neutral conditions. b, The crystal packing of KR2, viewed parallel to the membrane. c, Electron-density map for the retinal binding pocket of KR2. A stereo view of the 2Fo − Fc maps (blue mesh, contoured at 1.5σ) for the retinal binding pockets of KR2 under the acidic conditions is shown. Water molecules are depicted by red spheres.

Extended Data Figure 3 Structure-based sequence alignment of microbial rhodopsins.

The sequences are KR2, putative Na+-pump rhodopsin (NaR) from Dokdonia sp. PR095 (DsNaR, GenBank ID: AEX55013.1), putative NaR from Nonlabens dokdonensis DSW6 (NdNaR, GenBank ID: AGC76155.1), putative NaR from Truepera radiovictrix (TrNaR, NCBI reference sequence: YP_003706581.1), putative NaR from Gillisia limnaea (GlNaR, NCBI reference sequence: WP_006989277.1), putative NaR from Indibacter alkaliphilus (IaNaR, NCBI reference sequence: WP_009036080.1), xanthorhodopsin from Salinibacter ruber (Xanthorhodopsin, PDB ID: 3DDL)18, blue proteorhodopsin from Med12 (Blue_Proteorhodopsin, PDB ID: 4JQ6)43, bacteriorhodopsin from Halobacterium salinarium (Bacteriorhodopsin, PDB ID: 1IW6)44, halorhodopsin from Halobacterium salinarium (Halorhodopsin, PDB ID: 1E12)45, sensory rhodopsin II from Natronomonas pharaonis (Sensory_rhodopsinII, PDB ID: 3QAP)46, and the chimaeric channelrhodopsin between ChR1 from Chlamydomonas reinhardtii and ChR2 from Chlamydomonas reinhardtii (C1C2, PDB ID: 3UG9)47. The sequence alignment between KR2, xanthorhodopsin, blue proteorhodopsin, bacteriorhodopsin, halorhodopsin, sensory rhodopsin II and C1C2 was created using the T-Coffee server48. Secondary structure elements for KR2 are shown as coils and arrows. TT represents turns. Identical and conservatively substituted residues are highlighted in red. The N-helix residues and the residues tethering the N-helix to the protein core (Glu160 and Arg243 in KR2) are coloured green. Asp112 and Asp116 in the NDQ motif, and the Ser residue interacting with Asp116 (Ser70 in KR2) are coloured blue. Carboxylates on ECL1 are coloured yellow. The glutamine (Gln123 in KR2) that contributes to the putative intracellular Na+ binding site is coloured purple. The glycine and tyrosine residues that form the hydrophobic hole near the β-ionone ring of ATR are coloured orange. The asterisk under the alignment indicates the lysine that forms the Schiff base with ATR.

Extended Data Figure 4 Structural comparisons between KR2, BR and XR.

a, b, Side views (left) and extracellular views (right) of the superimposed structures of KR2 and BR (a) or KR2 and XR (b). The ATRs are shown as stick models. c, d, Structural comparison of the Schiff base orientations between KR2 and BR. c, Stereo view of the KR2 structure and its electron density in the Schiff base region. The 2Fo − Fc map (blue mesh, contoured at 2.0σ) is shown. d, Stereo view of the BR structure in the same region. The water molecule is depicted by a red sphere, and the hydrogen bond between the Schiff base and the water molecule is represented by the black dashed line.

Extended Data Figure 5 Functional characterization of wild-type KR2 and its mutants.

a, Na+ binding to wild-type (WT) KR2 and three ECL1 mutants. atrFTIR difference spectra of wild-type KR2 and three ECL1 mutants (E90Q/E91Q, D98N, and D102N) upon the exchange of 50 mM NaCl/KCl. Dotted lines are the duplicated difference spectra of wild type. bd, Photocycle of the KR2 D102N mutant. b, c, Transient absorption spectra of the KR2 D102N mutant (b) and time traces of the absorption changes (c) at specific probe wavelengths. d, Photocycle scheme for the Na+ pump, determined from the analysis of the results shown in b and c. The lifetime of O-decay of wild-type KR2 is only shown for the major component (90.6% of total O-accumulation), and the previously reported minor component (9.4%) with τ = 112 ms is not discussed here15. Values are means and s.d. calculated from the traces shown in c. e, Expression levels of wild-type KR2 and its mutants. Blue bars indicate the expression levels of wild-type KR2 and its nine mutants (mean ± s.d., n = 3). The expression of the Δ1–18 mutant was too low to be determined. f, Left, magnified view of the interactions between the N-helix and TM4, TM7 and ECL1. Right, stereo view illustrating the interactions between the N-helix and TM4, TM7 and ECL1. Hydrogen bonds are shown by dashed lines and water molecules are represented by red spheres, the residue on the β-sheet is represented as a green stick model. g, Time constants of the decay of detergent-solubilized wild-type KR2 and mutant proteins in thermostability assays, shown as means ± s.d. estimated by the least squares fitting technique with an exponential function. All mutant proteins were assayed in buffer containing 100 mM NaCl (or RbCl for wild-type KR2), 50 mM Tris-HCl (pH 8.0), and 0.1% DDM.

Extended Data Figure 6 The electron densities around Asp116 under neutral conditions.

ac, Stereo views of the structures and densities in the Schiff base region. The 2Fo − Fc maps (blue mesh, contoured at 1.0σ) and Fo − Fc maps (green and red meshes, contoured at 3.0 and −3.0σ, respectively) are shown. a, Structural refinement with Asp116 rotamer 1 resulted in a strong negative peak, suggesting its alternative conformation. b, The refinement with Asp116 omitted also suggested the presence of a mixture of Asp116 with different conformations. c, Asp116 with both rotamers 1 and 2 fits well in the electron density.

Extended Data Figure 7 Electrophysiological and behavioural analyses.

a, Membrane expression and localization of KR2 in mammalian cortical neurons (left, scale bar 20 μm) and C. elegans (right, scale bar 100 μm). b, KR2-dependent stable hyperpolarization. A current pulse was injected into a KR2-expressing neuron to evoke repetitive action potentials. The membrane potential was hyperpolarized for 10 s (top), 30 s (middle) and 60 s (bottom) when the neuron was illuminated by green light (5.4 mW per mm2, green bar). The resting potential was −74 mV throughout the experiment. c, The recovery of the locomotion speed in KR2-expressing C. elegans after cessation of illumination with green light. The green bar represents the light illumination.

Extended Data Figure 8 The retinal binding pockets and their surfaces in BR, XR and KR2.

a, Magnified views of the retinal binding pockets in BR (left panel), XR (middle panel) and KR2 (right panel). The residue that occludes the hydrophobic pore is indicated by the red dashed circle. b, Surface representations of the pocket, from the same viewpoint as in a. The blue and red dashed circles indicate the hydrophobic pores of XR and KR2, respectively. c, Surface representations of the pocket, from the same viewpoint as in a and b, showing the (putative) interaction between the second chromophore and the protein in the crystal structure of XR with salinixanthin (PDB ID: 3DDL) (left panel), and the putative binding site of the second chromophore, carotenoid (right panel).

Extended Data Figure 9 Putative water molecule between Asn112, Trp113 and Asp251.

Stereo view of the structures and densities in the Schiff base region, including the putative water molecule between Asn112, Trp113 and Asp251. The 2Fo − Fc maps (blue mesh, contoured at 1.5σ) are shown. The black dashed lines and the numbers are the possible hydrogen bonds and their distances (in Å) between the putative water molecule and the neighbouring residues.

Extended Data Figure 10 Calculated absorption spectra of wild-type KR2 in the O intermediate state.

The O absorption spectra of wild-type KR2 in the solution containing NaCl (red) or LiCl (yellow), calculated from the previously reported spectra15. The green dotted line represents the dark absorption spectrum of wild-type KR2 in the solution containing NaCl. The number above each spectrum is the peak wavelength (λmax).

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Kato, H., Inoue, K., Abe-Yoshizumi, R. et al. Structural basis for Na+ transport mechanism by a light-driven Na+ pump. Nature 521, 48–53 (2015). https://doi.org/10.1038/nature14322

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