Corrections

BIOPHYSICS AND COMPUTATIONAL BIOLOGYCorrection for “Structural insights into the proton pumping byunusual proteorhodopsin from nonmarine bacteria,” by IvanGushchin, Pavel Chervakov, Pavel Kuzmichev, Alexander N.Popov, Ekaterina Round, Valentin Borshchevskiy, AndriiIshchenko, Lada Petrovskaya, Vladimir Chupin, Dmitry A. Dolgikh,Alexander A. Arseniev, Mikhail Kirpichnikov, and ValentinGordeliy, which appeared in issue 31, July 30, 2013, of ProcNatl Acad Sci USA (110:12631–12636; first published July 19, 2013;10.1073/pnas.1221629110).The authors note that the author name Alexander A. Arseniev

should instead appear as Alexander S. Arseniev. The correctedauthor line appears below. The online version has been corrected.

Ivan Gushchin, Pavel Chervakov, Pavel Kuzmichev,Alexander N. Popov, Ekaterina Round, ValentinBorshchevskiy, Andrii Ishchenko, Lada Petrovskaya,Vladimir Chupin, Dmitry A. Dolgikh, Alexander S.Arseniev, Mikhail Kirpichnikov, and Valentin Gordeliy

www.pnas.org/cgi/doi/10.1073/pnas.1314549110

PHYSIOLOGYCorrection for “Sulfatides are required for renal adaptation tochronic metabolic acidosis,” by Paula Stettner, Soline Bourgeois,Christian Marsching, Milena Traykova-Brauch, Stefan Porubsky,Viola Nordström, Carsten Hopf, Robert Kösters, Roger Sandhoff,Herbert Wiegandt, Carsten A. Wagner, Hermann-Josef Gröne, andRichard Jennemann, which appeared in issue 24, June 11, 2013,of Proc Natl Acad Sci USA (110:9998–10003; first publishedMay 28, 2013; 10.1073/pnas.1217775110).The authors note that the author name Robert Kösters should

instead appear as Robert Koesters. The corrected author lineappears below. The online version has been corrected.

Paula Stettner, Soline Bourgeois, Christian Marsching,Milena Traykova-Brauch, Stefan Porubsky,Viola Nordström, Carsten Hopf, Robert Koesters,Roger Sandhoff, Herbert Wiegandt, Carsten A. Wagner,Hermann-Josef Gröne, and Richard Jennemann

www.pnas.org/cgi/doi/10.1073/pnas.1314463110

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Structural insights into the proton pumping by unusualproteorhodopsin from nonmarine bacteriaIvan Gushchina,b,c,d,1, Pavel Chervakove,1, Pavel Kuzmichevf,1, Alexander N. Popovg, Ekaterina Rounda,b,c,Valentin Borshchevskiyd,e, Andrii Ishchenkoe,h, Lada Petrovskayaf,2, Vladimir Chupinf,2, Dmitry A. Dolgikhf,i,Alexander S. Arsenievf, Mikhail Kirpichnikovf,i, and Valentin Gordeliya,b,c,d,e,2

aInstitut de Biologie Structurale J.-P. Ebel, Université Grenoble Alpes, 38027 Grenoble, France; bInstitut de Biologie Structurale J.-P. Ebel, Centre National de laRecherche Scientifique, 38027 Grenoble, France; cInstitut de Biologie Structurale J.-P. Ebel, Direction des Sciences du Vivant, Commissariat à l’ÉnergieAtomique, 38027 Grenoble, France; dResearch-Educational Centre “Bionanophysics,” Moscow Institute of Physics and Technology, Dolgoprudniy 141700,Russia; eInstitute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Juelich, 52425 Juelich, Germany; fShemyakin and OvchinnikovInstitute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia; gEuropean Synchrotron Radiation Facility, 38027 Grenoble, France;hInstitute of Crystallography, University of Aachen (Rheinisch-Westfälische Technische Hochschule), 52056 Aachen, Germany;and iBiological Department, Lomonosov Moscow State University, Moscow 119991, Russia

Edited by Richard Henderson, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved June 13, 2013 (receivedfor review December 12, 2012)

Light-driven proton pumps are present in many organisms. Here,we present a high-resolution structure of a proteorhodopsin froma permafrost bacterium, Exiguobacterium sibiricum rhodopsin(ESR). Contrary to the proton pumps of known structure, ESR pos-sesses three unique features. First, ESR’s proton donor is a lysineside chain that is situated very close to the bulk solvent. Second,the α-helical structure in the middle of the helix F is replaced by310- and π-helix–like elements that are stabilized by the Trp-154and Asn-224 side chains. This feature is characteristic for the pro-teorhodopsin family of proteins. Third, the proton release region isconnected to the bulk solvent by a chain of water molecules al-ready in the ground state. Despite these peculiarities, the positionsof water molecule and amino acid side chains in the immediateSchiff base vicinity are very well conserved. These features makeESR a very unusual proton pump. The presented structure shedslight on the large family of proteorhodopsins, for which struc-tural information was not available previously.

retinylidene protein | bacteriorhodopsin | retinal | photocycle

Retinal-containing membrane proteins are present in alldomains of life. They use light energy for a wide range of

different functions such as ion transport, photosensing, andchannel activity (1). The proteins are used for development ofmany applications including the optogenetic control of cell andtissue (2). All of these proteins contain at least seven trans-membrane α-helices and a retinal molecule that is covalentlybound via the Schiff base to the side chain of a lysine amino acid(3). The most studied protein of this family is bacteriorhodopsin(BR) from a halophilic archaea Halobacterium salinarum, a pro-ton pump, which provides the first key universal step of trans-formation of the energy in the cells: generation of protonelectrochemical gradient across the cell membrane (4). Studiesof this protein have provided a basis for the fundamental hy-pothesis of transport mechanisms and motivated the developmentof new experimental technologies (3).A significant number of BR structural homologs were revealed

in diverse microorganisms (proteobacteria, actinomycetes, cya-nobacteria, fungi, and others) including the proteorhodopsin(PR), whose gene was first discovered in metagenomic DNA li-brary from Monterey Bay, California (5). A potential bacterialrhodopsin gene was also identified in the genome of a Gram-positive bacterium Exiguobacterium sibiricum, isolated fromSiberian permafrost soil (6). This extreme environment containsa unique microbial community adapted to long-term freezing,cumulative radiation, and high-water osmolarity (7, 8). E. sibiricum,one of the Gram-positive microorganisms widely present in per-mafrost samples, can withstand a wide range of growth conditions,including the temperature from −5 °C to 40 °C (9). The E. sibiricum

rhodopsin (ESR) expressed well in Escherichia coli in a func-tionally active form. The protein with a covalently linked all-trans retinal had maximum absorbance at 534 nm and was ableto pump a proton upon illumination in a broad pH range of4.5–8.5 (10, 11).

Results and DiscussionHere, we present a high-resolution crystallographic structure ofESR and show that it is very unusual compared with thestructures of BR and xanthorhodopsin (XR) (12), the closestESR homolog and the only proton-pumping bacterial rhodopsinfor which the crystallographic structure is known. We show thatthe ESR structure possesses a proteorhodopsin-specific featureand may serve as a model for the protein family. We do notcompare the ESR structure with the solution NMR structure ofproteorhodopsin (13) for the reasons described in Fig. S1 andits legend.

Crystallographic Structure of E. sibiricum Rhodopsin and Its CharacteristicFeatures. The ESR crystals were grown using the in meso ap-proach (14, 15) and diffracted to 2.3 Å (Table 1). There are twoESR monomers with an almost identical structure in the crys-tallographic asymmetric unit. The most extended B–C loop couldbe built in one chain but not in the other (Fig. S2). Comparisonof the structure with the known structures of retinylidene pro-teins reveals that the overall fold is very well conserved (Fig. 1A)as well as the Schiff base-proximal region (Fig. 2A). The helix Gπ-bulge distortion observed in the other proteins is also presentin ESR. However, ESR possesses another feature that is uniqueto the protein (Fig. 1B and Figs. S3–S8). Namely, the α-helicalstructure in the middle part of the helix F is severely disrupted(Fig. S3). First, there is a π-bulge–like distortion near the car-bonyl oxygen of residue 185 (hydrogen bonds O185–N190 andO186–N191). Then, next to it, are 310-helix–like hydrogen bondsO189–N192, O190–N193, and O191–N194. Due to this concomitant

Author contributions: L.P., V.C., and V.G. designed research; I.G., P.C., P.K., A.N.P., E.R.,V.B., and A.I. performed research; I.G., P.C., P.K., A.N.P., E.R., V.B., A.I., L.P., V.C., D.A.D.,A.S.A., M.K., and V.G. analyzed data; I.G., P.C., P.K., A.N.P., E.R., V.B., A.I., L.P., V.C., D.A.D.,A.S.A., M.K., and V.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 4HYJ).1I.G., P.C., and P.K. contributed equally to this work.2To whom correspondence may be addressed. E-mail: valentin.gordeliy@ibs.fr, lpetr65@yahoo.com, or vvchupin@gmail.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1221629110/-/DCSupplemental.

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occurrence of the π- and 310-like structures, the overall registerof the helix is unchanged. The structural element is stabilized byhydrogen bonds between the backbone and neighboring sidechains (O187–Trp-154 and O190–Asn-224). The residues Trp-154and Asn-224 are present in all of the proteorhodopsins but areabsent in other proteins (Fig. 1C). Based on this fact, we con-clude that helix F should adopt the same structure in proteo-rhodopsins and that this unusual structure discriminates themfrom the other retinylidene proteins, including XR. This notionis supported by the recently released crystallographic structure ofblue light-absorbing proteorhodopsin from Med12 (PDB IDcode 4JQ6; Figs. S9 and S10).The role of the disruption of the helix F α-helical structure in

proteorhodopsins is not clear at present. Both 310- and π-structureshave been implicated to possess functional roles other than simplystructural in other membrane and soluble proteins (16–18). Onepossibility is that the novel structure facilitates the “unlatching” ofthe cytoplasmic site, similarly to what was proposed in bacterio-rhodopsin photocycle (19). As for the stabilizer residues, the roleof the Trp-154 position has not been highlighted previously, butAsn-224, the residue preceding the retinal-binding lysine, is knownto be important for the function of retinylidene proteins. For ex-ample, its mutation, A215T (a singlemutation in the retinal bindingpocket; two other mutations are needed for efficient signal trans-ducer binding), is enough to convert the proton pump bacterio-rhodopsin into a photosensor (20).Another difference between ESR and the proteins of known

structure is the position of the loop connecting the helices B andC, which interacts excessively with the helix A in ESR and XRand interacts with the helices D and E in BR (Fig. S11).Photosynthetic organisms often increase the efficiency of light

harvesting by utilization of antennae. Archaeal proton pumps BR

and archaerhodopsin function without any such additional mech-anisms. However, it was shown recently that a light-harvestingantenna can function also in a retinal protein. Xanthorhodopsin,a retinylidene protein of the extreme halophilic bacteriumSalinibacter ruber isolated from salt-crystallizer ponds, containsa single energy-donor carotenoid, salinixanthin, as an additionalchromophore (12, 21). The salinixanthin binds to the outer sur-face of the helix F at the protein–lipid boundary. The keto-ring ofsalinixanthin interacts with the residues at the extracellular endsof the helices E and F and the β-ionone ring of the retinal. Thebinding pocket of the keto-ring is formed by Leu-148, Gly-156,Phe-157, Thr-160, Met-208, andMet-211 and the retinal β-iononering (12). An intriguing question is whether other retinal proteinsmight also have an antenna. As it follows from our data, the resi-dues that are characteristic for xanthorhodopsin and Gloeobacterrhodopsin, another carotenoid-binding proton pump, are not con-served in ESR. Many of the ESR residues have larger side chainsthan the corresponding residues of XR. As a consequence,binding of the carotenoid or some other molecule in the retinalproximity is impossible (Fig. S12).

Structure of the Retinal Binding Pocket, Proton Release, and ProtonUptake Groups. The retinal binding pocket of ESR is presented inFig. 2 and compared with those of other proteins in Fig. 3. Theretinal is covalently linked to lysine 225 via a Schiff base, simi-larly to XR and BR. The electron densities around the retinaldefinitely show that the chromophore is in all-trans conformation(Fig. S13). The protonated Schiff base of the retinal points to-ward the extracellular part of the protein and, as in BR and XR,donates a hydrogen bond to a key water molecule W402. In itsturn, W402 donates hydrogen bonds to two anionic residues,Asp-85 and Asp-221 (Asp-85 and Asp-212 in BR correspond-ingly) (22, 23) (Figs. 2 and 3). This arrangement stabilizes thepositive charge of the Schiff base and is conserved in all of thecurrently known proton pumps (and in most other retinylideneproteins). The carboxylate of Asp-85 in ESR is oriented ina similar way to Asp-85 of BR and unlike its homolog Asp-96 inxanthorhodopsin, which is considerably rotated (12). One morewater molecule, W406, is observed in the retinal binding pocketof ESR, where it occupies a similar position to W406 of BR(Figs. 2 and 3). Whereas, in BR, W406 forms a hydrogen bond toArg-82 (22, 23), in ESR, it is close to other water moleculesdirectly facing the bulk solvent. In the structure of xantho-rhodopsin, only the water molecule W402 was found (12).A remarkable difference of the bacterial proteins ESR and

XR from archaeal BR is the presence of the histidine residue notfar from the retinal (Figs. 2 and 3). Like its homolog His-62 inXR, ESR’s His-57 forms a tight hydrogen bond to the protonacceptor Asp-85. The observed bond lengths in the two mono-mers of 2.58 Å and 2.66 Å are somewhat larger than those ofxanthorhodopsin, 2.55 Å and 2.42 Å. Although in both casesthese distances may be indicative of an unusual bonding, forexample low-barrier hydrogen bond (24–26), higher resolutionstructures are needed to test this hypothesis.Orientations of Asp–His pairs are completely different in ESR

and XR (Figs. 2 and 3). In ESR, His-57 is rotated toward Arg-82and is immersed in a cavity of a size sufficient for a water mol-ecule from the bulk to come in contact with it. The electrondensities in the cavity are indicative of several partially orderedwater molecules (Fig. 2); however, their positions differ in thetwo ESR molecules. The Asp–His pair is the only suitable protonacceptor group in the retinal vicinity. The Asp–His couplingexplains the experimental observation that the pKa of the His–Asp group is by several units higher than pKa of Asp-85 in BR(11). Lower affinity to the proton may explain the longer timeneeded for acceptor protonation (11). At the same time, as His-57 is almost in direct contact with the bulk solvent, it itself ortogether with Asp-85 could serve as a proton release group. In

Table 1. Data collection and refinement statistics

Property Value

Data collectionSpace group P321Cell dimensions

a, b, c, Å 96.099, 96.099, 124.380α, β, γ, ° 90, 90, 120

Resolution, Å 69–2.3 (2.42–2.3)*Rmerge, % 5.4 (33.4)I/σI 12 (3.7)Completeness, % 98 (96.7)Redundancy 3.9 (3.8)

RefinementResolution, Å 69–2.3No. of reflections 29,408Rwork/Rfree, % 17.5/21.5No. of atoms

Protein 3,499Ligand/ion 343Water 33

B-factors, Å2

Protein 37.0Ligand/ion 53.6Water 41.5

rmsdBond lengths, Å 0.008Bond angles, ° 1.4

Ramachandran statisticsFavored 98.2%Allowed 1.8%Outliers 0%

*Highest resolution shell is shown in parentheses.

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this respect, XR differs from ESR: in the former the analogoushistidine 62 points away from Asp-236 (analog of Asp-212 of BR)and does not participate in the hydrogen bond connection of theSchiff base to the solvent. Arginine 93 of XR separates the Schiffbase from the bulk solvent similarly to BR. There is no histidineresidue in the vicinity of the retinal in BR, the arginine 82 sep-arates the Schiff base from the bulk solvent, and the proton re-lease group is believed to consist of glutamates 194 and 204 andthe adjacent water molecules (22, 23, 27).

ESR’s Arg-82 points away from the retinal (Fig. 2), similarly tothe bacteriorhodopsin’s M state (27), and occupies the outermostposition compared with the other proteins (Fig. S14). It is be-lieved that Arg-82 plays a key role in proton release in BR (22, 23,27). In the BR photocycle, protonation of Asp-85 from the Schiffbase upon L to M state transition results in a flip of Arg-82 fromthe retinal to extracellular direction (outward from the retinal).However, already in the ground state of ESR, Arg-82 points tothe extracellular part of the protein. There, it forms a hydrogenbond to Glu-130 (Fig. 2B) and is a part of a larger cluster ofionizable and polar side chains (Fig. S11). This conformation isexpected to remain unperturbed during the whole photocycle,and, therefore, the role of Arg-82 in the ESR’s proton releasemechanism can be not as important as it is in BR. The data de-scribed here provide an explanation of a recent observation thatAsp-85 and Arg-82 are not coupled in ESR (as the former doesnot influence pKa of the latter, unlike in BR) (11).The ESR’s proton uptake region is also unique among the

proton pumps of known structure from both archaea and bac-teria. There is the cationic amino acid lysine 96 at its core,contrary to the anionic amino acids Asp-96 and Glu-107 in BRand XR, respectively (Figs. 1C and 4). The same amino acid isfound in the proton uptake region of recently identified pro-teorhodopsins from marine uncultured MG-II Euryarchaeota(28) (Fig. 1C and sequence alignment in Dataset S1). The Lys-96side chain is to some extent mobile as its positions differ in thetwo observed ESR molecules, and the electron densities areweak in one of them (Fig. 4). This side chain flexibility might berequired for the structural rearrangements needed for thereprotonation. However, it could also be a result of the overall

Fig. 1. Comparison of ESR with the retinylidene proteins of known structure. (A) Overlay of the known retinylidene protein structures. ESR is shown in darkblue, XR in light blue, ChR in yellow, and the other proteins in green. Most variation occurs in the helix A position. ESR differs markedly from the otherproteins by disrupted α-helical structure in the middle part of the helix F. The helices A, E, F, and G are labeled. N-terminal residues of ChR are not shown. (B)π-bulge and 310-helix–like structures in the ESR’s helix F. The disruption of normal α-helical structure is stabilized by the hydrogen bonds between N224 andthe carbonyl oxygen of G190, and between W154 and the carbonyl oxygen of I187. Whereas the proline 195 is conserved in other proteins, disruption ofα-helical structure in the region of residues 187–191 is unique to ESR. (C) Phylogenetic tree showing the relations between the retinylidene proteins of knownstructure, ESR, and other proteorhodopsins. Residues W154 and N224 are unique to the proteorhodopsin family. Lysine at the proton donor position is alsoobserved in the clade B proteorhodopsins (genes pop and pop-1). The multiple sequence alignment and the phylogenetic tree were generated using ClustalX2.1 (39), and the tree was drawn using the TreeDyn web server (40). The proteins used for comparison are channelrhodopsin (ChR), PDB ID code 3UG9 (41);bacteriorhodopsin (BR), PDB ID code 1C3W (22); archaerhodopsins 1 and 2 (aR-1 and aR-2), PDB ID codes 1UAZ and 1VGO (42); H. salinarum halorhodopsin(HsHR), PDB ID code 1E12 (43); Natronomonas pharaonis halorhodopsin (NpHR), PDB ID code 3A7K (44); N. pharaonis sensory rhodopsin II (NpSRII), PDB IDcode 3QAP (45); Anabaena sensory rhodopsin (ASR), PDB ID code 1XIO (46); Acetabularia rhodopsin 2 (AR2), PDB ID code 3:00 AM6 (47); and xanthorhodopsin(XR), PDB ID code 3DDL (12).

Fig. 2. Structure of the ESR Schiff base-proximal region with electron densitymaps. (A) View along the retinal. (B) View perpendicular to the retinal.Aspartates 85 and 221 are traditionally close to the Schiff base, with a watermolecule (W402) in between. There is a histidine residue (H57) close to D85.There are also additional electron densities that can be treated as partiallyordered water molecules (W406 and W407). The arginine 82 side chain pointsaway from the Schiff base and forms hydrogen bonds with E130.

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lesser quality of the proton uptake part of one of the two mole-cules in the crystallographic asymmetric unit (Fig. S2). The cavityaround Lys-96 is mostly surrounded by hydrophobic amino acidsand may accommodate at least three water molecules. Residualelectron densities are seen in this region although they are notsufficient for a reliable placement of these water molecules. Next,the cavity is very close to the bulk solvent as it is separated from itonly by a polar side chain of Thr-43, which is a remarkable

difference from BR and XR, where there is a large gap betweenthe proton uptake residue and the bulk solvent (Figs. 4 and 5).The proximity of the bulk solvent can result in an easy directaccess of the protons from the cytoplasm to Lys-96 througha water “funnel” and might explain why proton uptake precedesproton release in ESR. The small hydrophobic gate between K96and the Schiff base, created by the leucine 93 side chain, issimilar to that of XR and BR. We suggest that, upon isomeri-zation of the retinal, Leu-93 may change its conformation andallow a faster Schiff base reprotonation.Why do ESRs and some other proteorhodopsins possess a cat-

ionic amino acid at the proton uptake? E. sibiricum inhabitspermafrost soil, which is characterized by a wide range of pH(5.6–7.8). Therefore, the function of ESR must be adapted to thisvariation. Indeed, light-induced proton transport by ESR-con-taining E. coli cells and proteoliposomes is observed in a broad pHrange of 4.5–8.5 (11). It was shown that the pair Asp-85/His-57allows the protein to stabilize Asp85 in the unprotonated state ina wide range of pH, which is necessary to keep the proton pumpfunctional (11). However, in proteorhodopsin, where the protonacceptor pair is the same, direction of the proton flow may bereversed at acidic pH (29). Therefore, a priori, Asp-85/His-57cannot be considered as a sufficient and universal stabilizer ofproton pumping in a wide range of pH. One could speculate thatLys-96 with its reduced pKa is a key element of stabilization ofproton pumping. However, it is not clear how to explain a recentdiscovery that proteorhodopsins corresponding to the genesrevealed in the metagenomic sequences of uncultured marinegroup II Euryarchaeota contain a lysine residue in the protondonor position, similarly to ESR (28). This question about theproton pumping by ESR requires further investigation.

Proposed Model of the Photocycle. Structural and spectroscopicstudies of the intermediate states are necessary for a completedescription of the mechanism of vectorial proton translocation inESR and explanation of the role of Lys-96 and the Asp-85/His-57couple. Nevertheless, the outline of the ESR photocycle has beenobtained recently by Balashov et al. (11) (Fig. 6), and, thus, usingthe structure, we can propose the major steps of the protonpumping by the protein. First, upon isomerization of the retinal,the Schiff base flips from the extracellular to the cytoplasmic partof the retinal pocket. There, the position of the charged group inthe mostly hydrophobic environment is energetically unfavorable,and the Schiff base must be deprotonated as a result. The onlyprimary proton acceptor candidate is the residue Asp-85 (11),which is coupled strongly to His-57 (Figs. 2 and 3). Next, the Schiffbase must be reprotonated. Indirect experimental evidence showsthat the reprotonation proceeds via Lys-96 as it is the only ioniz-able residue in that region and the M state decay is significantly

Fig. 3. Comparison of the ESR, XR, and BR retinal binding pockets in the ground state. Both ESR and XR differ from BR by presence of the histidine residuenot far from the retinal. ESR’s retinal pocket also possesses other differences. First and probably most important, its histidine is turned toward the arginine 82,which in its turn is removed from the retinal, similarly to the bacteriorhodopsin’s M state. Second, in ESR, the residue preceding the retinal-bound lysine isasparagine, as opposed to alanine in BR, XR, and most of other proton-pumping bacterial rhodopsins.

Fig. 4. Comparison of the ESR, XR, and BR proton uptake regions. Theconformations differ in the two observed ESR molecules. Contrary to BR, XR,and other light-driven proton pumps of known structure, ESR has a lysine(Lys-96) residue at the proton uptake, whose electron densities are shown atthe level of 1.4σ. The cavity around Lys-96 is mostly surrounded by hydro-phobic amino acids and may accommodate at least three water molecules.The only polar residue, Thr-43, separates the cavity from the bulk solvent. Onthe contrary, the proton uptake residues Glu-107 of XR and Asp-96 of BR arefar from the bulk solvent, being separated from it by Ser-48 and Tyr-45 in XRand Phe-42 in BR. Lys-96 side chain is ordered in one ESR molecule andpartially disordered in the other. Additional positive electron densitiesaround Lys-96 (not shown) are observed in the difference maps that areprobably related to mobile water molecules.

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slowed down in the K96A mutant (11). It can be expected thatLys-96 is deprotonated in the ground state, as it is immersed ina mostly hydrophobic cavity, and it was shown that, in such cases,the lysine side chain pKa may be as low as 5.3 (30). Therefore, Lys-96 must be reprotonated following the Schiff base deprotonation,and only then can it pass the proton to the Schiff base. Thereprotonation step is probably facilitated by the presence of thecontinuous connection to the bulk solvent (Fig. 5). Finally, fol-lowing the experimental observations (11), release of the proton tothe bulk solvent occurs after the Schiff base reprotonation.

Materials and MethodsProtein Purification. ESR with a C-terminal hexahistidine tag was expressed inE. coli strain Rosetta2(DE3)pLysS and purified as described (10). ESR wasgrowing in a fermenter for 3 d at 30 °C in 2× ZYM5052 autoinduction me-dium (31) with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. Afterobtaining a membrane fraction, ESR was extracted by incubation in 50 mMTris, 1% n-dodecyl-β-D-maltopyranoside (DDM), 10 mM imidazole, pH 8.0overnight at +4 °C. The solubilized membrane fraction was purified on anNi-Sepharose 6 Fast Flow (GE Healthcare) column, washed with buffer 1 (50mM sodium phosphate, 500 mM NaCl, 2 M urea, 0.1% DDM, 10 mM imid-azole, pH 8.0), and buffer 2 (50 mM sodium phosphate, 200 mM NaCl, 0.1%

DDM, 20 mM imidazole, pH 8.0) and eluted with 50 mM sodium phosphate,200 mM NaCl, 0.1% DDM, 0.01% Na azide, 300 mM imidazole, pH 7.4. Afterremoving imidazole, the subsequent sample concentration was performedon an Amicon ultrafiltration device with a regenerated cellulose membrane(10 kDa molecular weight cut-off). The final concentration of ESR was up to2 mg/mL.

Crystallization. The crystals were grown using the in meso approach (14, 15).The solubilized protein in the crystallization buffer was added to the mon-ooleoyl-formed lipidic phase (Nu-Chek Prep). The best crystals were obtainedusing the protein concentration of 35 mg/mL and the 0.1 M Sodium acetate,0.2 M Sodium malonate, 12% (wt/vol) PEG 3350, pH 4.6, precipitant solution(Qiagen). The crystals were grown at 22 °C.

X-Ray Data Acquisition and Treatment. X-ray diffraction data (wavelengths0.934 Å and 0.976 Å) were collected at the beamlines ID14-1 and ID23-1 of theEuropean Synchrotron Radiation Facility (ESRF), Grenoble, France, usinga PILATUS 6M detector. Diffraction patterns were processed using theMOSFLM(32) and SCALA software from the CCP4 program suite (33). The crystals weretwinned, with the twin fraction in the range 0–45%. The crystal with the bestdiffraction was not twinned. The data statistics are presented in Table 1.

Structure Determination and Refinement. The initial phases were obtained bya molecular replacement (MR) method using MOLREP (34). The MR solutionwas found in the spacegroup P321. There are two monomers in the asym-metric unit in this solution. Although the overall configuration of the proteinsin the crystal is close to the higher-order symmetry P6322, multiple analysesshow that the correct spacegroup is P321. Typical Rmerge values are 1.5 timeshigher if the data are integrated in P6322, and systematic absences, charac-teristic of P6322, are not observed. Also, the MR solution could not be found inP6322. In P321, similar solutions were found using either XR coordinates (PDBID code 3DDL) (12) or the homology model of ESR, based on PDB ID code3DDL. The homology model created using the SWISS-MODEL server (35) waseventually used that resulted in the R-factors of ∼45% after the MR step.

The initial MR model was iteratively refined using intensity-based twinrefinement in REFMAC5 (36) and Coot (37). During the starting stage of therefinement, the medium noncrystallographic symmetry (NCS) restraints wereapplied to the monomers. After most of the model was built, release of theNCS restraints led to the improvement of the R and Rfree factors.

The initial atomic model was obtained using the 2.9-Å data from thetwinned crystal (twin fraction ∼40%) and was later refined against the 2.3-Ådata from the crystal that was not twinned. Along with the protein struc-ture, positions of 33 water molecules and 30 lipid tail fragments were de-termined. The final refinement, including the translation/libration/screw (TLS)treatment of the B-factors, was conducted using the PHENIX software suite (38).

Fig. 5. The cavities on the putative proton path in ESR, XR, and BR that may be occupied by water molecules. The upmost and the lowest cavities on thefigure face the bulk solvent. The important ionizable and charged residues are shown explicitly. In the proton uptake region, ESR’s Lys-96 is separated only bythe Thr-43 side chain from the solvent whereas there is a large gap in XR and BR between the bulk and Glu-107 and Asp-96 correspondingly. There is enoughspace for at least three water molecules around Lys-96 in ESR. In ESR, the histidine residue of a putative proton release group Asp-221/His-57 is immersed ina cavity of a size sufficient for a water molecule from the bulk to come in contact with it. This continuous cavity contains the ordered water molecules 402 and406, and transitions into the bulk. In XR, the release group is shielded by Arg-93. In BR, the proton is released from the completely different group, a pair ofglutamates (Glu-194 and Glu-204) that are separated by the Ser-193 side chain from the bulk. The cavities are determined as a composition of the crystal-lographically recognizable water molecules and the space determined using the Hollow software (48) with a 1.4-Å probe and 0.2-Å grid spacing.

Fig. 6. The models of the BR and ESR photocycles (11, 27). Some of thetransitions are reversible. The timescales are very approximate as they de-pend strongly on the conditions such as pH, temperature, and other factors.

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ACKNOWLEDGMENTS. The diffraction experiments were performed onthe ID14-1 and ID23-1 beamlines at the European Synchrotron RadiationFacility (ESRF), Grenoble, France. We are grateful to local contacts atESRF for providing assistance in using these beamlines. This work wassupported by the program “Chaires d’excellence” edition 2008 ofAgence Nationale de la Recherche France, Commissariat à l’ÉnergieAtomique(Institut de Biologie Structurale)-Helmholtz Gemeinschaft(Re-search Centre Juelich) Special Topic of Cooperation 5.1 specific agree-ment, Marie Curie grant (Seventh Framework Programme-PEOPLE-2007-1-1-Initial Training Networks, project Structural Biology of Membrane

Proteins) and an European Commission Seventh Framework Programmegrant for the European Drug Initiative on Channels and Transportersconsortium (HEALTH-201924). Part of this work was supported by GermanMinistry of Education and Research (PhoNa-Photonic Nanomaterials). Thiswork was partially supported by the Federal Target Program “Scientificand Academic Research Cadres of Innovative Russia” for the years 2009–2013, by the “Molecular and Cellular Biology” program of the RussianAcademy of Sciences, and by Russian Foundation for Basic Research Re-search Project 13-04-01700. We greatly acknowledge support of this workby ONEXIM, Russia.

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