Structure of the light-driven sodium pump KR2 and its implications for optogenetics

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1 STRUCTURAL SNAPSHOT Structure of the light-driven sodium pump KR2 and its implications for optogenetics Ivan Gushchin 1,2, Vitaly Shevchenko 1,2, Vitaly Polovinkin 1,2,3,4,5, Valentin Borshchevskiy 1,2, Pavel Buslaev 2, Ernst Bamberg 6 and Valentin Gordeliy 1,2,3,4,5 1 Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre J ulich, Germany 2 Moscow Institute of Physics and Technology, Dolgoprudniy, Russia 3 Institut de Biologie Structurale, Universite Grenoble Alpes, France 4 Institut de Biologie Structurale, Centre National de la Recherche Scientifique, Grenoble, France 5 Institut de Biologie Structurale, Direction des Sciences du Vivant, Commissariat a l Energie Atomique, Grenoble, France 6 Max Planck Institute of Biophysics, Frankfurt am Main, Germany Keywords crystal structure; light-driven; microbial rhodopsins; optogenetics tools; potassium pump; sodium pump Correspondence V. Gordeliy, Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre J ulich, J ulich, Germany Fax: Tel: valentin.gordeliy@ibs.fr (Received 8 September 2015, revised 22 October 2015, accepted 30 October 2015) doi: /febs A key and common process present in organisms from all domains of life is the maintenance of the ion gradient between the inside and the outside of the cell. The gradient is generated by various active transporters, among which are the light-driven ion pumps of the microbial rhodopsin family. Whereas the proton-pumping and anion-pumping rhodopsins have been known for a long time, the cation (sodium) pumps were described only recently. Following the discovery, high-resolution atomic structures of the pump KR2 were determined that revealed the complete ion translocation pathway, including the positions of the characteristic Asn- Asp-Gln (NDQ) triad, the unusual ion uptake cavity acting as a selectivity filter, the unique N-terminal a-helix, capping the ion release cavity, and unexpected flexibility of the retinal-binding pocket. The structures also revealed pentamerization of KR2 and binding of sodium ions at the interface. Finally, on the basis of the structures, potassium-pumping KR2 variants have been designed, making the findings even more important for optogenetic applications. In this Structural Snapshot, we analyse the implications of the structural findings for understanding the sodium translocation mechanism and application of the pump and its mutants in optogenetics. Introduction Microbial rhodopsins constitute a large family of sevenhelix transmembrane proteins that utilize light energy to perform their function [1]. The light is captured by the covalently bound cofactor retinal, and absorption of a photon launches a series of structural transformations called the photocycle. The result of these transformations may be active ion translocation, channel opening or closing, or generation of a signal. Since the discovery of bacteriorhodopsin in 1971 [2], the members of the family have served as model systems for various experimental approaches aimed at membrane proteins, and they have recently found an application in the rapidly growing field of optogenetics [3,4]. Whereas the light-driven proton and anion pumps have been known for a long time, the first light-driven cation (sodium) pump, KR2 from Dokdonia eikasta, was discovered only in 2013 [5]. Soon, similar proteins from Nonlabens marinus [6], Gillisia limnaea [7] and 1232 FEBS Journal 283 (2016) ª 2015 FEBS

2 I. Gushchin et al. Structure of light-driven sodium pump other organisms [8,9] were described. The proteins are distinguished by their characteristic NDQ triad of the active site residues (Asn112, Asp116 and Gln123 in KR2), instead of DTD (Asp85, Thr89 and Asp96) in the classic proton pump bacteriorhodopsin, or DTE/DTK in other proton pumps [10,11], and TSA in chloride pump halorhodopsins. The fold of the sodium pumps was expected to be the same as in other microbial rhodopsins; however, their mechanism of action was far from clear. Here, we describe the recently determined structures of KR2 [12,13] and experimental data that shed light on the details of sodium translocation. Structure of the light-driven sodium pumps Similarly to those of many other microbial rhodopsins, the crystals of KR2 were obtained by use of the in meso approach [14], and several non-illuminated state structures were determined [12,13]. The protein readily crystallizes at nonphysiological acidic ph, with Gushchin et al. and Kato et al. reporting highly similar structures of monomeric KR2 at resolutions of 1.45 and 2.3 A, respectively. The higher-resolution structure reveals additional water molecules, a bound sodium ion, and alternative conformations for several side chains [12]. However, at acidic ph, the optical properties of KR2 are altered, and sodium translocation is not observed. Therefore, determination of the physiological-state structures at more neutral ph would be much more beneficial for understanding the transport mechanism. This was accomplished in two different ways. Kato et al. elucidated the KR2 structure at ph by soaking the crystals, obtained at ph 4.0, in solutions with ph [13]. Gushchin et al. found that raising the ph value of the precipitant solution to 4.9 or 5.6, at which sodium translocation can already be observed, results in crystals in two novel space groups [12]. The new crystals have optical properties close to those in the physiological state, and show the pentameric assembly of KR2, different conformation of the active site residues, and, again, the sodium ions bound at the oligomerization interface [12]. General features of the KR2 structure The light-driven sodium pump has the classic microbial rhodopsin fold, whereby seven transmembrane helices, named A G, form a bundle with the cofactor retinal at the centre, covalently bound to Lys255 of helix G (Fig. 1A). The distinguishing feature of KR2 is the short N-terminal a-helix that caps the inside of the protein (Fig. 1A,B). Although an N-terminal a- helix has also been observed in Natronomonas pharaonis halorhodopsin [15], there is no evidence for its involvement in the ion translocation pathway. The transmembrane helices of the sodium pump are connected by three intracellular and three extracellular loops, of which the loop connecting the helices B and C is notably extended and forms a b-hairpin, which is also involved in b-sheet interactions with the short linker joining the N-terminal a-helix and helix A (Fig. 1B). Ion translocation pathway Atomic structures of KR2 reveal the complete ion translocation pathway, consisting of the three major checkpoints: the ion uptake cavity, the cavity close to the retinal Schiff base, and the putative ion release cavity (Fig. 1A). The ion uptake cavity is unexpectedly open into the bulk solvent, unlike in proton or anion pumps, mostly because of the absence of the side chain of Gly263 of helix G. This cavity primarily serves as the selectivity filter: obstruction of the cavity with mutations G263F and G263W gives the pump potassium translocation ability, whereas mutation G263L impacts severely on sodium pumping [12,13] (Fig. 1C). Consequently, besides the NDQ triad, Gly263 is another requisite residue for sodium translocation. The retinal-binding pocket contains the retinal in the all-trans conformation, together with several ordered water molecules in a cavity underneath the retinal Schiff base. The cavity is surrounded by the polar and ionizable residues Ser70, Asn112, Asp116, and Asp251, and, as in other pumps, is separated from the ion release region by an arginine (Arg109; Fig. 1A). The ion release cavity also contains several water molecules and a cluster of three ionizable ion residues, namely Glu11, Glu160, and Arg243. Neutralizing mutations of these residues do not abolish the transport activity of the sodium pump, supporting the notion that these residues mostly have a structural function [5,12,13]. In the initial report describing the discovery of the sodium pump, Inoue et al. showed that nonilluminated KR2 can bind sodium ions [5]. Somewhat perplexingly, experiments with KR2 mutants showed that this sodium binding is not required for sodium pumping, and vice versa [5]. The high-resolution structures of KR2 monomer and KR2 pentamer [12] provide the explanation for these observations: there are no sodium ions bound inside KR2; however, one ion binds on the surface of the protein, coordinated by the FEBS Journal 283 (2016) ª 2015 FEBS 1233

3 Structure of light-driven sodium pump I. Gushchin et al. Fig. 1. Structure of KR2. The N-terminal helix is shown in blue, and the B C loop is shown in orange. Water-accessible cavities are shown as red surfaces. (A) Side view. The ion path is indicated by arrows. Hydrophobic membrane core boundaries calculated with the PPM server [18] are shown as black lines. (B) View from the extracellular side. (C) KR2 ion uptake cavity. Hypothetical effects of the G263L, G263F and G263W mutations are shown by use of the conformations of the homologous residues Leu224 of bacteriorhodopsin (BR) [19], Leu250 of Halobacterium salinarum halorhodopsin (HR) [20], Phe233 of Exiguobacterium sibiricum rhodopsin (ESR) [21], and Trp221 of blue proteorhodopsin (PR) [16]. (D) Pentameric assembly of KR2. Sodium ions are shown in violet. residues of helices A and B. In pentamers, this ion is at the oligomerization interface, bridging Tyr25 (helix A), Thr83 and Phe86 (helix B) of one protomer with Asp102 (B C loop) of another (Fig. 1D). Oligomeric assembly of KR2 When crystallized at ph 4.9 or higher, KR2 forms symmetrical pentamers [12] (Fig. 1D). KR2 also behaves as a pentamer during size-exclusion chromatography at physiological ph values, upon solubilization in the detergent decyl b-d-maltopyranoside or the detergent dodecyl b-d-maltopyranoside. Finally, closely related proton pump proteorhodopsins form pentamers or hexamers via a similar interface in crystals [16] and in native-like membranes [17]. On the basis of these findings, we suggest that KR2 forms pentamers under physiological conditions. Variations in the observed conformations of KR2 Although all of the determined KR2 structures have similar general features, there are notable differences in the conformations of the active site residues. At acidic ph, KR2 in crystals has an absorption maximum at 566 nm [12]. Correspondingly, the structures show a protonated Schiff base counterion (Asp116) that is too far from the Schiff base nitrogen to form a hydrogen bond (Fig. 2A,B) [12,13]. In crystals soaked in the solution with physiological ph, the Asp116 side chain partially reorients towards the retinal, and forms a hydrogen bond with the Schiff base (Fig. 2C); no other changes are observed [13]. Pentamerization significantly affects the Schiff base environment. The crystals with pentameric KR2 absorb maximally at nm at a ph as low as 4.9, whereas the crystals with monomeric KR2 absorb maximally at 566 nm at a ph as high as 4.6 [12]. This signals the strong dependence of the Asp116 pk a on the protein s oligomeric state. Comparison of the monomeric and pentameric structures reveals the reason for this: upon pentamerization, the retinal-binding pocket is remodelled, with the Asp116 side chain, although in the same conformation, now within hydrogen-bonding distance of the Schiff base nitrogen (Fig. 2D,E). As KR2 is expected to be pentameric under physiological conditions, we believe that the latter structures are representative of the correct position of the retinal and surrounding amino acids in the ground state. Finally, in the pentameric assembly, the KR2 protomers show a large spectrum of conformations differing in the degree of helix C bulging (two extreme 1234 FEBS Journal 283 (2016) ª 2015 FEBS

4 I. Gushchin et al. Structure of light-driven sodium pump Fig. 2. Schiff base environment in different states of KR2 and halorhodopsin (helices C and G). (A) First conformation observed in monomeric KR2 at acidic ph at a resolution of 1.45 A [12]. (B) Second conformation observed in monomeric KR2 at acidic ph at a resolution of 1.45 A [12]. (C) The conformation observed in monomeric KR2 soaked at neutral ph [13]. Two observed rotameric conformations of Asp116 are marked r1 and r2. (D) Compact conformation of the Schiff base pocket, observed in pentameric KR2 (molecule I in type B crystals) [12]. (E) Expanded conformation of the Schiff base pocket, observed in pentameric KR2 (molecule D in type B crystals) [12]. (F) Comparison of compact (yellow) and expanded (orange) conformations observed in pentameric KR2 [12]. (G) Comparison of compact ionfree (yellow) and expanded ion-bound (orange) conformations of NpHR [15,22]. (H) Distances between the C a atoms of the residues homologous to Asn112 and Asp251 in KR2 (Asp85 and Asp212 in bacteriorhodopsin; Thr126 and Asp252 in NpHR) in the microbial rhodopsins of known structure. ar-1, archaerhodopsin-1 [23]; ar-2, archaerhodopsin-2 [24]; AR2, Acetabularia rhodopsin II [25]; ASR, Anabaena sensory rhodopsin [26]; BPR 1, blue proteorhodopsin from HOT75 [16]; BPR 2, blue proteorhodopsin from Med12 [16]; ChR, chimera of channelrhodopsins 1 and 2 [27]; cr-3, cruxrhodopsin-3 [28]; dr-3, deltarhodopsin-3 [29]; ESR, E. sibiricum rhodopsin [21]; HmBRI, Haloarcula marismortui bacteriorhodopsin I [30]; HsBR, H. salinarum bacteriorhodopsin [19]; HsHR, H. salinarum halorhodopsin [20]; NpHR, N. pharaonis halorhodopsin [15,22]; NpSRII, N. pharaonis sensory rhodopsin II [31]. conformations are shown in Fig. 2F) [12]. Similar changes in the conformation of helix C are observed upon anion binding or unbinding in halorhodopsin (Fig. 2G). In fact, such changes, and the possibility of the expanded conformation, whereby the distance between the residues homologous to KR2 s Asn112 and Asp251 is increased, can be considered to be specific features of ion pumps: of all microbial rhodopsins of known structure, only anion-bound halorhodopsins and pentameric KR2 have a conformation with this distance above 12 A (Fig. 2H). In all other groundstate structures, the distance is ~10 A. As Asn112 and Asp251 are on the sides of the Schiff base-proximal cavity, the distance between them is directly related to the amount of ion-accessible space in the Schiff base vicinity, and 12 A might be a minimal value for accommodation of an ion other than a proton. However, additional experiments are needed to check this proposition. The mechanism of active sodium translocation Unfortunately, no atomic-resolution data on the photocycle intermediates of KR2 are available at present. Such data are crucial for deciphering the key events leading to ion translocation. Nevertheless, the obtained structural information for the sodium pump and the wealth of the information about other light-driven ion pumps allow us to formulate a hypothesis for the mechanism of active sodium translocation. For that, we employ the structural features shown by KR2 in the physiological pentameric state, and depict the major conformational states, i.e. the ground state, the M state, and the O state, in Fig. 3. In the nonilluminated ground state, there are no sodium ions bound inside the protein, the retinal Schiff base is protonated, Asp116 is deprotonated, and the FEBS Journal 283 (2016) ª 2015 FEBS 1235

5 Structure of light-driven sodium pump I. Gushchin et al. sodium ion from re-entering the cytoplasm, thus ensuring directed translocation. Finally, following the exit of the sodium ion into the extracellular space, flipping of the Asn112 side chain back towards the Schiff base, and reisomerization of the retinal back into the all-trans conformation, the photocycle is complete (Fig. 3). Fig. 3. Proposed model of the structural changes during sodium translocation. The photocycle starts with absorption of a photon by KR2 in the ground state, which, in the early intermediates, leads to isomerization and deprotonation of all-trans retinal. In the M state, the partially electronegative deprotonated Schiff base attracts the sodium ion, which, in the M-to-O transition, translocates towards Asn112, Asp116, and Asp251. This transformation is accompanied by Asn112 flipping and expansion of the Schiff base cavity. Reprotonation of retinal prevents exit of the sodium ion into the cytoplasm, thus ensuring vectorial transport. Finally, in the O-toground transition, the sodium ion is released into the extracellular solution, while the backbone returns to the contracted state and Asn112 flips back towards retinal. The arrows denote the motions of the corresponding elements (blue for the Schiff base nitrogen, grey for the Schiff base hydrogen, magenta for sodium, and yellow for the Asn112 side chain). In the O state, there is enough space for two or three hydration water molecules around the sodium ion, indicated by magenta shading. The presented structural models are based on the compact and expanded pentameric KR2 states. Schiff base cavity is in the compact conformation [12,13]. The photocycle starts with absorption of a photon by retinal, which leads to excitation and consequent all-trans to 13-cis isomerization of it in the early photocycle intermediates. The isomerization is followed by deprotonation of the Schiff base, with the proton being displaced to Asp116 [5,7]. In this new intermediate, the M state, the now partially electronegative Schiff base attracts the sodium ion, which enters the ion uptake cavity from the cytoplasm, passes the retinal, and occupies the position close to Asp251. The entry of the sodium ion leads to flipping of the Asn112 side chain and expansion of the Schiff base cavity. Following sodium uptake and reprotonation of the Schiff base from Asp116, the pump is in the O state [5,7]. The protonated Schiff base prevents the Implications for optogenetics Experiments with the nematode Caenorhabditis elegans and cortical rat neural cells have shown that KR2 can be used as an optogenetics tool [13]. The obtained structures simplify the engineering of new sodium pumps with desirable properties. However, even more important is that the structures open a way to engineering new optogenetics tools. Indeed, immediately after determination of the crystal structures, potassium-translocating variants were designed [12,13], representing the first examples of outwardly directed light-driven potassium pumps. Such pumps are also ideal for silencing of neural cells and muscle cells, because the activity of the pump is increased by the electrical potential and by the potassium gradient across the plasma membrane upon depolarization, thereby mimicking the natural effect of outwardly directed potassium channels other than sodium or proton pumps. Acknowledgements The work was supported by the CEA(IBS) HGF (FZJ) STC 5.1 specific agreement, the 5top100- program of the Ministry for Science and Education of Russia, FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The work was performed in the framework of ERA.Net RUS Plus grant (ID 323, Russian Federal Target Program Research and Development contract , RFME- FI58715X0011). Author contributions VS expressed and purified the protein, VP crystallized the protein and collected absorption spectra, IG collected the diffraction data and solved the structures, VB and PB helped with structure analysis, EB wrote about the implications for optogenetics, VG supervised the project, IG and VG analyzed the results and prepared the manuscript with input from all the other authors FEBS Journal 283 (2016) ª 2015 FEBS

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