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X-ray crystal structure of voltage-gated proton channel

Abstract

The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn2+. Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-Å resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge–transfer center. The extracellular region of each protomer coordinated a Zn2+, thus suggesting that Zn2+ stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.

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Figure 1: Electrophysiological properties and crystal structure of mHv1cc.
Figure 2: Zn2+-binding site and Zn2+ sensitivity of mHv1 mutants.
Figure 3: Comparison of S4 position between mHv1cc and other VSDs.
Figure 4: Double hydrophobic layers (HLex and HLin), cavity and inner vestibule.
Figure 5: Gating mechanism of Hv1.

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Acknowledgements

We wish to thank N. Nakamura, K. Nishiwaki, M. Kobayashi and W. Kumano for their support with experiments. We thank D.M. Standley for suggestions and comments on the manuscript. We are grateful to S. Ogasawara, S. Iwata and S. Yokoyama for advice on membrane-protein crystallization. We wish to thank T. Tsukihara and Y. Yoneda for encouragement throughout this project. We also thank all members of the Nakagawa and Okamura laboratories for their suggestions and comments. This work was supported by the Target Proteins Research Program (A.N. and Y. Okamura) and the Platform for Drug Discovery, Informatics, and Structural Life Science (A.N.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan, by Grants-in-Aid for basic research S (no. 25253016) and A (no. 21229003) (Y. Okamura and A.N.), a Grant-in-Aid for Scientific Research on Innovative Areas (no. 24111529) (Y. Okamura) from the Japan Society for the Promotion of Science, by the JAXA-GCF (Japan Aerospace Exploration Agency–Granada Crystallization Facility) project “High quality protein crystallization project on the protein structure and function analysis for application,” conducted from the Japan Aerospace Exploration Agency (A.N.), and by the National Project on Protein Structural and Functional Analyses from the MEXT (A.N.). Diffraction data were collected at the Osaka University beamline BL44XU at SPring-8 (Harima, Japan) under proposal numbers 2010A6500, 2010B6500, 2011A6500, 2011B6500, 2012A6500, 2012B6500 and 2013A6500. The detector, MX225HE, is financially supported by Academia Sinica and the National Synchrotron Radiation Research Center in Taiwan, Republic of China.

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Authors

Contributions

K.T. expressed, purified and crystallized, collected and processed X-ray data, refined and analyzed the structure and wrote the paper. E.Y. assisted with collection and processing of X-ray data. S.S. performed physiological experiments. Y.F. assisted with physiological and structural studies. T.K., Y. Okochi and A.K. assisted with physiological studies. M.M. and H.N. assisted with protein expression. Y. Okamura and A.N. designed the study and wrote the paper. All authors commented on the manuscript.

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Correspondence to Yasushi Okamura or Atsushi Nakagawa.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Crystallized chimera construct (mHv1cc)

(a) Sequence alignment between mHv1cc and other VSDs. These sequences (mHv1cc, Kv1.2–Kv2.1; shaker family voltage–gated potassium channel Kv1.2–Kv2.1 paddle chimera channel, NavAb; voltage–gated sodium channel from Acrobacter butzleri, Kv1.2; voltage–gated potassium channel subunit Kv1.2, KvAP; voltage–gated potassium channel from Aeropyrum pemix, NavRh; NaChBac orthologue from the marine alphaproteobacterium HIB114, and Ci–VSP; voltage sensor phosphatase from Ciona intestinalis were aligned using program Clustal W39. The red characters show the periodically basic residues on S4. The green characters show the hydrophobic residues highly conserved among VSDs. (b) The cartoon diagram of crystallized chimera constructs (mHv1cc). The S2–S3 half intracellular–side (Glu149–Phe171) and cytoplasmic coiled–coil (Val216–Asn269) of mouse Hv1 or VSOP were replaced with the intracellular portion of Ci–VSP (Asp164 – Leu188) and the leucine zipper transcriptional activator GCN4 from S. cereviece (Arg249–Arg281), respectively. The intracellular portion of Ci–VSP and GCN4 show red and green diagrams, respectively. (c, d) Thermal stability assay of mHv1cc with CPM. (c) A raw fluorescence signal plotted against increasing temperature. (d) Melting temperature (Tm) was determined as the first inflection point by calculating derivative of raw fluorescence shown in a against temperature. Tm of mHv1cc was estimated to be 70.6 °C. The red and blue show plots with (n = 4 technical replicates) and without mHv1cc (n = 2 technical replicates), respectively. (e, f) Proton current of mHv1cc recorded at the indicated two different pH environments from HEK293T cells. (g) Stoichiometry of mHv1cc in HEK293T cells as shown by cross–linking with DSS. The arrow shows a band corresponding to dimer of mHv1cc. (h) H+ flux assay using proteoliposome. Data from vesicles with and without mHv1cc were shown in red and black curves, respectively. Error bars depict means ± S.D. (n = 3 technical replicates).

Supplementary Figure 2 Zn2+ binding and S4 position

(a–d) Se–Met and Zn2+ anomalous Bijvoet signals. These blue mesh show the initial map phasing by Se–MAD at 4.3Å (contoured at 1.0σ level). All Bijvoet anomalous difference signals (green mesh, contoured at 3.0σ level) were calculated by Se–Met1(a), Se–Met2(b), and Se–Met3(c) data collections, respectively. The asterisk shows unknown weak Bijvoet anomalous signal. The red mesh shows the Bijvoet anomalous difference map of Zn2+ calculated by Cryst–Z1 data collection (contoured at 5.0σ level) (d). (e) Electron density map of mHv1cc and anomalous Bijvoet signal of Zn2+, shown in stereogram (wall-eyed viewing). σA–weighted 2mFo - DFc map of mHv1cc (blue mesh) contoured at 1.0σ with the final model shows stick model. The map was calculated using native crystal (Cryst–B, S76–mHv1cc) at 3.45Å resolution. The magenta mesh shows the Bijvoet anomalous difference map of Zn2+ contoured at 5.0σ (20.00–5.0Å). The Zn2+ anomalous map was calculated from the native crystal data (Cryst–Z1) collected at 1.275Å wavelength using the phase as the refined coordinate of mHv1cc. (f) Stereo drawing of long helix (S4 and coiled–coil) in mHv1cc. The S4 consists of 310 helix (Arg201–Arg204) and α helix (Arg204–Arg207). Three arginine residues (Arg201, Arg204, and Arg207), and Met217 were drawn by stick model. The blue mesh shows the experimental map with MAD phases contoured at 1.0σ. The green mesh shows the Bijvoet anomalous difference map of selenomethionine (Met217) contoured at 5.0σ. The anomalous map of selenomethionine was calculated from MAD phases of Se-Met1 (Table 1). Since cell dimensions of Cryst–A were slightly deviated from those of Se-Met1, the refined model was fitted onto the Se–MAD map of Se-Met1 by rigid-body refinement. (g–i) Representative current traces in the presence of distinct doses of zinc ions. Data sets in each mutant [E115S(g), D119S(h), and ΔNΔC(i)] were recorded from the same patches. ΔNΔC denotes a version of mHv1 in which C-terminus and N-terminus were truncated at position 216 and 77, respectively16. Currents were elicited by test pulses to 100 mV under pHout/pHin = 7.0/7.0. The holding potential was -60 mV (black curve; 0 μM, red curve; 1 μM, and blue curve; 10 μM). (j, k) Sequence alignment of S4 (j) and comparison of S4 position among VSDs [mHv1cc (this structure, 3WKV)], Kv1.2 [2A79 (ref. 2)], KvAP (1ORS39), and NavRh (4DXW36) (k).

Supplementary Figure 3 Sequence alignment between mHv1cc and other VSDs.

Sequence alignment of Hv1 orthologs from different species. The red characters show the positions of conserved, periodically aligned arginine residues of S4. The orange and blue layers show the hydrophobic residues in a lower hydrophobic layer (HLin) and an upper layer (HLex), respectively. The green layers show the Zn2+ binding residues. Hv1 sequences of Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), Danio rerio (Dr), Xenopus laevis (Xl), Ciona intestinalis (Ci), Coccolithus pelagicu (Cp), Strongylocentrotus purpuratus (Sp, purple sea urchin), are aligned with mHv1cc, using Clustal W39. In S2–S3 linker of Hv1cc, the length increased by one residue when the original sequence of mHv1 was replaced by the corresponding region of Ci–VSP (underlined). Pro184 in Ci–VSP corresponds to Pro159 of mHv1cc. In order to avoid confusion, Pro159 (shown by the red asterisk) of mHv1cc was re–assigned to Pro158a.

Supplementary Figure 4 Water-accessibility profile of mHv1cc.

Comparison of water accessibility profile of mHv1cc with previous findings on mHv1, CiHv1 and hHv1. The symbols “+” and “–” show the accessible and the inaccessible residues, respectively. The information of accessible or inaccessible sites were cited from several studies of the cysteine–scanning with accessibility to MTS or the histidine–scanning of zinc sensitivity by electrophysiology. State–dependent MTS or Zn2+ accessibility are shown with three colored characters (blue; site extracellularly accessible to MTS or Zn2+ in activated–state from CiHv1 or hHv1. red; site intracellularly accessible to MTS in resting–state from CiHv1. green; site intracellularly accessible to Zn2+in activated–state from hHv1). #cysteine scanning of accessibility to MTS reagent done by electrophysiology on CiHv1 (Ref. 19) histidine scanning of Zn2+ sensitivity done by electrophysiology on hHV1 (Ref. 20) §cysteine scanning of accessibility to MTS reagent done by electrophysiology on CiHv1 (Ref. 18) accessibility to AMS reagent44,45

Supplementary Figure 5 Proposed dimer model of mouse Hv1.

(a) The amino acid sequences were represented by the ‘abcdefg’ convention for the coiled–coil. The red characters show residues of dimer–interface (a and d). Dimer–interface of mHv1cc was predicted from dimer–interface of GCN4 leucine zipper and mouse Hv1 cytoplasmic coiled–coil (3VMX28). (b) The dimer model of mHv1cc. Superimposition of cytoplasmic coiled–coil region of mouse Hv1 (Ile224–Gly266) and mHv1cc (Ile225–Leu242) was performed by least–squares superposition of Cα atoms (RMSD = 0.670Å). The arrow shows a coiled–coil region. The trimer in crystal packing is shown in box.

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Takeshita, K., Sakata, S., Yamashita, E. et al. X-ray crystal structure of voltage-gated proton channel. Nat Struct Mol Biol 21, 352–357 (2014). https://doi.org/10.1038/nsmb.2783

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