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Non-cryogenic structure of a chloride pump provides crucial clues to temperature-dependent channel transport efficiency

      Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.

      Introduction

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      The abbreviations used are: LCP
      lipidic cubic phase
      SFX
      serial femtosecond crystallography
      XFEL
      X-ray free electron laser
      ATR
      all-trans retinal
      PSB
      protonated Schiff base
      ClR
      chloride-pumping rhodopsin
      bR
      bacteriorhodopsin
      HR
      halorhodopsin
      CCCP
      carbonyl cyanide 3-chlorophenylhydrazone
      DDMP
      difference distance matrix plot
      DDM
      n-dodecyl-β-d-maltoside
      LCLS
      Linac Coherent Light Source.
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      ). In microbial light-driven ion-pumping rhodopsin, the chromophore all-trans retinal (ATR) of rhodopsin binds covalently to a lysine residue through a Schiff base linkage. Absorption of light induces a conformational change in the retinal from all-trans to 13-cis, leading to overall structural changes in the protein, thereby activating ion transport and a photocycle that traverses several key intermediate states coming back to the dark state (
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      ). The motif of the protonated Schiff base (PSB) changes according to the unique residue of the proton acceptor and proton donor in rhodopsin, which induces a difference in the ion transport pathway and pumping mechanism. Several studies have reported proton- and chloride-pumping rhodopsins (ClRs) with various PSB motifs, including bacteriorhodopsin (bR) with DTD motifs, thermophilic bacteria rhodopsin (TR) with DTE motifs, and halorhodopsin (HR) with TSA motifs (
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      Implications for the light-driven chloride ion transport mechanism of Nonlabens marinus rhodopsin 3 by its photochemical characteristics.
      ). Therefore, it is very important to clarify the three-dimensional structure and accurate ion transport pathway of ClR at room temperature using XFEL.
      Recently, a ClR with a novel PSB (NTQ) motif exhibiting unique ion-transporting activities and pathways has been reported in the marine bacterium Nonlabens marina S1-08(T) (
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      ). Growth of this strain is modulated by environmental temperature, with a growth temperature range of 10–30 °C; the optimal temperature for growth is 20–25 °C, and no growth occurs below 5 °C or above 37 °C (
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      ). In early studies investigating the photoelectric signal of bacteriorhodopsin, it had been suggested that the rate constant could be changed according to the pH or temperature change resulting in the modulation of rhodopsin’s functions (
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      Electric response of a back photoreaction in the bacteriorhodopsin photocycle.
      ). In the present study, we also found that the channel functions of ClR are modulated by temperature in a similar manner. Therefore, it would be of interest to know the correlation between temperature and function of ClR, and in this respect, ClR could serve as a model protein to obtain structural dependence on temperature.
      Here, we report the first room temperature crystal structure of ClR containing the NTQ motif in the chloride ion transfer pathway using XFEL. The structure of ClR at room temperature, determined at a resolution of 1.85 Å, reveals tremendous structural details. We analyzed structural differences by comparing two structures at both room temperature and the cryogenic temperature. Although the overall structure determined by XFEL was comparable to that determined by synchrotron X-ray crystallography, the chloride ion-binding sites (one at the active center of the pump and the other near the loop on the cytoplasmic side) were found to be in distinct positions showing different hydrogen bond networks. Interestingly, one additional water molecule forming a hydrogen bond network with a chloride ion (Cl-I) near PSB was clearly observed in the XFEL-derived structure, but not in the structure determined at the cryogenic temperature.
      Our findings will be essential in gaining a better understanding of the structural differences and mechanical properties attributed to temperature during data collection by both XFEL and synchrotron analyses, providing hints into the temperature dependence of hydrogen bonding in ClR and its correlation with chloride-pumping function. The room temperature structure of ClR thus sets the foundation for further time-resolved SFX studies on chloride ion transportation pathways and their underlying molecular mechanism.

      Results

       Hyperactivity and anion-binding affinity of CIR for anion transport at low temperatures

      The Gram-negative, orange-pigmented, and rod-shaped bacteria Nonlabens marina S1-08(T) were isolated from seawater of the western north Pacific Ocean (30°11′ N, 145°05′ E; depth 0 m). It is well-known that Nonlabens marina S1-08(T) ceases cell growth at temperatures below 10 °C, unlike other marine bacteria (
      • Park S.
      • Yoshizawa S.
      • Chiura H.X.
      • Muramatsu Y.
      • Nakagawa Y.
      • Kogure K.
      • Yokota A.
      Nonlabens marina sp. nov., a novel member of the Flavobacteriaceae isolated from the Pacific Ocean.
      ), and the optimal growth temperature is between 20 °C and 25 °C (Table S1). Thus, to determine how temperature affects the anion transport activity of the ClR derived from Nonlabens marina S1-08(T), we measured light-induced pH changes for different monovalent salts, sodium chloride, and sodium bromide at five different temperatures, −20 °C, −10 °C, 4 °C, 10 °C, and 25 °C. As the temperature gradually decreased, the chloride ion transport rate increased and was almost 2-fold faster at −20 °C than at 25 °C in the absence of a protonophore, and the same trend was not observed for bromide ions (Fig. 1, A and B). Likewise, in the presence of the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), the pH change was much more drastic during chloride and bromide ion transportation at lower temperatures. The enhanced activity observed in the presence of CCCP completely disappeared following the addition of tetraphenylphosphonium ion (TPP+), a hydrophobic cationic reagent that disrupts membrane potential. This implied that the physiological function of ClR in pumping activity can be sensitively modulated by temperature changes. In this regard, abnormal functions, such as ClR hyperactivity involved in phototransduction biochemical reactions, could also play a role in cell death or suspended growth of Nonlabens marina S1-08(T) by altering the electrochemical balance of the bacterial cell.
      Figure thumbnail gr1
      Figure 1The anion-pumping activity of ClR depending on different temperatures. A and B, light-induced pH changes in E. coli cell suspensions expressing ClR in solution containing (A) 100 mm NaCl and (B) 100 mm NaBr observed in the absence (gray solid lines) or presence (black solid lines) of the protonophore CCCP (30 μm), or in the presence of 30 μm CCCP and 50 mm tetraphenylphosphonium ion (TPP+) (black broken lines) at different temperatures from −20 to 25 °C. ClR-expressing E. coli cells were light-induced by a green-light laser-on (540 nm) for 5 min and were dark-adapted before and after green-light laser-on for 5 min each.
      The binding affinity of ClR for different anions was measured by UV-visible absorption spectrometry to determine whether the hyperactivity was caused by a change in the binding affinity with the anion because of the temperature difference. The maximum wavelength with and without 1 m sodium chloride was 533 nm and 555 nm, at both room temperature (25 °C) and 4 °C, respectively (Fig. 2, A and B). Likewise, the maximum wavelength with and without 1 m sodium bromide was 537 nm and 555 nm at room temperature (25 °C), and 533 nm and 555 nm at 4 °C, respectively (Fig. S1, A and B). A similar blue shift was observed with bromide and chloride ions in the maximum absorption peak upon titration at both 25 °C and 4 °C. Anion titration allowed for the determination of dissociation constants (Kd) from the absorbance changes at 581 nm (Fig. 2, C and D and Fig. S1, C and D). The apparent Kd values were estimated using the Hill equation and are summarized in Table S2. Surprisingly, the binding affinity of chloride and bromide ions were approximately twice as strong at 4 °C than at room temperature (Fig. 2E). These binding-affinity results were consistent with the anion transport activity results (Table S2).
      Figure thumbnail gr2
      Figure 2The UV-visible absorption spectroscopy and binding affinity of ClR at 25 °C and 4 °C. A and B, the UV-visible absorption spectra of ClR following the addition of NaCl up to 1 m at (A) 4 °C and (B) 25 °C. The maximum absorption wavelength values in the absence and presence of chloride ions at 4 °C and 25 °C were 555 and 533 nm, respectively. C and D, absorption changes at 581 nm in the difference spectra plotted against chloride ion concentrations at (C) 4 °C and (D) 25 °C. E, the data were fitted using the Hill equation (solid lines) to estimate the anion affinity at 4 °C and 25 °C. All fitting parameters were normalized as Δλmax and set to 1. Error bars represent S.D. from three independent experiments. The titration experiments were performed with chloride ions and bromide ions at 4 °C and 25 °C. See also and .

       XFEL- and synchrotron-based structures demonstrate subtle differences in two chloride positions and hydrogen-bonding networks

      The ClR structures were determined by XFEL and synchrotron analysis to investigate structural changes that can explain functional differences of ClR at different temperatures. Crystal structures were collected at a resolution of 1.85 Å and 1.75 Å at room temperature (294 K) by XFEL and at cryogenic temperature (93 K) by the synchrotron, respectively. Overall, the two structures showed good agreement with no significant changes in the overall topology. The root mean square deviation (RMSD) of the 253 Cα atoms of a single molecule in the asymmetric unit was calculated to be 0.128 Å with similar overall residual B-factors (Å2) except for some structural points (Fig. S2). Specifically, helices A and B and part of helix G showed lower B-factor values, especially, Lys235, which formed a covalent bond with retinal and exhibited the lowest B-factor value in the ClR structure at room temperature. Moreover, we also identified several important structural differences through a detailed comparison of these two structures.
      First, we investigated the near-retinal region, which is the main trigger for ion-transporting activity. Interior residues surrounding the retinal chromophore were revealed with high-quality electron density maps, especially with a clear retinal β-ionone ring (Fig. 3, A and B). The covalent bonding angle between the retinal C15 and Lys235 residue in the two structures differed by about 3° (Fig. 3, C and D). This difference could be related to the dynamic properties that cause structural changes in response to temperature changes. Moreover, the chloride ion (Cl-I), located in the active core of the XFEL-derived structure, moved toward the PSB region, resulting in a shorter distance between Cl-I and the PSB by ∼0.3 Å compared with that of the synchrotron-derived structure (Fig. 3, C and D). Ion pumps such as ClR stabilize chloride and water molecules around the PSB region by interacting with proton acceptors or donor residues for ion transport, and the distance between them significantly differed between the two structures (Fig. S3, A, B, D, and E). Disruption of hydrogen bonds is essentially a temperature-dependent process (
      • Mizan T.I.
      • Savage P.E.
      • Ziff R.M.
      Temperature dependence of hydrogen bonding in supercritical water.
      ). The rearrangement of water molecules and chloride ions leads to a change in the hydrogen network. Important information gained from the XFEL structure was the discovery of new hydrogen bonding networks with an additional water molecule (W614), having B-factor of 26.4 Å2 and near Cl-I (Fig. 3, A and B). ClR was previously thought to have unique hydrogen bond networks with two water molecules distinct from other chloride pumps such as HR, which have three well-resolved water molecules mediating the hydrogen bond network between Cl-I–PSB and Asp239-Arg108 (in HR from Halobacterium salinarum) (
      • Kim K.
      • Kwon S.K.
      • Jun S.H.
      • Cha J.S.
      • Kim H.
      • Lee W.
      • Kim J.F.
      • Cho H.S.
      Crystal structure and functional characterization of a light-driven chloride pump having an NTQ motif.
      ). However, the ClR structure derived from XFEL confirms that it also has three well-resolved water molecules, W507, W508, and W614, and forms similar hydrogen bonding networks between Cl-I–PSB and Asp231-Arg95, like other chloride pumps (Fig. S3A). The B-factors of W507 and Cl-I were ∼16.3 Å2 and 20.8 Å2 in the XFEL-derived ClR structure, compared with the corresponding values of 24.4 Å2 and 29.2 Å2 in the synchrotron-derived structure, indicating more stable dynamics in this region.
      Figure thumbnail gr3
      Figure 3The structural comparison at two chloride-binding sites near the PSB and cytoplasmic regions. A and B, the 2Fo–DFc electron density map, contoured at 1.5 σ, near the PSB region of the (A) XFEL- and (B) synchrotron-derived ClR structures. Selected side chains close to the ATR are shown as stick models. The chloride ions are shown as green spheres. C and D, the chloride (Cl-I)-binding site near (C) ATR linked covalently to Lys235, and the Cl-II–binding site near (D) cytoplasmic region from the XFEL- and synchrotron-derived structures are shown as a stick model colored cyan and green, respectively. The two chloride ions located near the PSB and cytoplasmic regions are depicted as cyan and green spheres in the XFEL- and synchrotron-derived structures, respectively. E and F, the 2Fo–DFc electron density map, contoured at 1.5 σ, of Cl-II and two water molecules (W514 and W515) near the cytoplasmic region of the (E) XFEL- and (F) synchrotron-derived ClR structures. The B-factor values of water molecules were reflected by the electron density map.
      As a result, the XFEL-derived ClR structure determined at room temperature showed much stronger water-mediated hydrogen bonds and considerably more stable dynamic properties near the PSB and Cl-I. In particular, chloride ions exhibited increased interactions with W507 and the PSB in the XFEL-derived structure. The hydrogen bond between Arg95 and the water molecule W508 was similar, whereas the hydrogen bond between Asp231 and the water molecule W507 was very different because of the presence of the additional water molecule W614 (Fig. S3, A, B, D, and E).
      Second, structural rearrangement was detected around the chloride ion (Cl-II) in the cytoplasmic region (Fig. 3D). The location of Cl-II changed by ∼0.5 Å, resulting in a longer distance between Cl-II and Lys46 amide nitrogen in the XFEL-derived structure by ∼0.2 Å compared with that in the synchrotron-derived structure. The B-factors of Cl-II in the two structures were quite comparable: Both are about 40 Å2.
      Additionally, one water molecule in the cytoplasmic region, W515, showed different positions and B-factor values between the two structures. W515 forms a hydrogen bond between Lys46 and Ile112 by 3.0 and 2.7 Å in the XFEL-derived structure, although it shows a much weaker hydrogen bond between W515 and Lys46 in the synchrotron-derived structure (Fig. S3, C and F). In the XFEL-derived structure, the position of W515 shifted by 1.4 Å toward the backbone carbonyl oxygen of Lys46 and the B-factor was increased from 27.8 Å2 to 47.1 Å2 (Fig. 3, E and F). The information of hydrogen bonding distance and B-factors from the structure determined at room temperature using the XFEL method provides a structural explanation for the abnormal activities of ClR at low temperature.

       Channel cavities of ClR

      For more in-depth structural comparisons, the volume sizes and side chain orientations of the participating residues were investigated for four core cavities buried in the seven transmembrane domains of ClRs. The volume of individual cavities showed quantitative dependence on the number of atoms (or residues) and water lining the cavity. The cavity volumes (Å3) in the two ClR structures from the XFEL and the synchrotron are marked in Fig. 4, A and B. Overall, cavities 1 and 3 in the XFEL-derived ClR structure were slightly larger (87.79 and 2.76 Å3 versus 86.05 and 2.63 Å3), whereas cavities 2 and 4 were smaller than that in the synchrotron-derived structure (11.02 and 0.17 Å3 versus 13.90 and 0.29 Å3).
      Figure thumbnail gr4
      Figure 4Channel cavities of the XFEL- and synchrotron-derived ClR structures. A and B, the cavities embedded in the XFEL- and synchrotron-derived ClRs with water molecules are shown as cavity 1, 2, 3, and 4 marked by red, cyan, orange, and violet meshes, respectively. The volume (Å3) of individual cavities in XFEL- and synchrotron-derived ClRs are labeled. C–F, superimposed structure of ClR derived from XFEL and synchrotron in (C) cavity 1, (D) cavity 2, (E) cavity 3, and (F) cavity 4 colored by slate and orange. Participating amino acid residues for each cavity are represented by a line model. Chloride ions and water molecules from the XFEL- and synchrotron-derived structures are depicted as blue, slate, red, and orange spheres, respectively.
      In addition, although the structures were generally similar, the hydrogen bonds between the residues forming the cavity and the surrounding water differed. In cavity 1, Cl-I and five water molecules form a hydrogen bond network, and the side chain orientation of participating residues were very similar in the two structures, with some notable variations in the distances between certain residues, as explained in the previous section (Fig. 4C and Fig. S3). In cavity 2, different side chain packing for Ile64, Lys65, Arg95, Asn98, Trp99, Thr102, Asp231, and Lys235 was observed in the hydrogen bond network, with Cl-I and water molecules depending on the presence of an additional water molecule, W614 (Fig. 4D).
      Interestingly, the water molecule W509 did not share the same position, and its B-factor value differed between the two resolved structures (34.2 versus 41.4 Å2). In cavity 3, Asn3 and Asn92 showed very different side chain orientations, and the position of one nearby water molecule, W501, was not observed in the XFEL-derived structure (Fig. 4E and Fig. S4, A and B). In cavity 4, Met58, Lys106, Gln109, and Lys235 showed slight differences with respect to side chain orientation (Fig. 4F). Thus, the high-resolution ClR structures measured by both XFEL and synchrotron allow accurate comparison of the cavity and side chain orientations.

       Temperature-dependent conformational change of ClR

      Because the overall conformational changes were quite subtle, we constructed a difference distance matrix plot (DDMP) to detect regional structural rearrangements that might be caused by the temperature differences (Fig. 5) (
      • Richards F.M.
      • Kundrot C.E.
      Identification of structural motifs from protein coordinate data: secondary structure and first level supersecondary structure.
      ). DDMP analysis demonstrates dramatic internal distance differences in several regions based on the distances among Cα atoms. According to the DDMP results, structural perturbation resulting from the cryo-cooling conditions occurred during data collection. Specifically, major structural perturbations occurred in the B-C loop, the C-D loop, a part of helix C in the extracellular side, a part of helices E and G in the intracellular side, and the C-terminal helix (Fig. 5, A and B). The poly-chains Val79-Leu85 and Gln86-Thr89, located in the B-C loop, moved in opposite directions, and this may have affected the π-stacking interaction that formed the 3-ω motif of ClR (Fig. 5C). The extracellular section of helix C moved toward the interior of the protein, with the same orientation as poly-chain Val79-Leu85 in the B-C loop (Fig. 5C, upper panel). The intracellular portion of helix E is another region that showed the widest conformational change (Fig. 5, A and B) (
      • Richards F.M.
      • Kundrot C.E.
      Identification of structural motifs from protein coordinate data: secondary structure and first level supersecondary structure.
      ).
      Figure thumbnail gr5
      Figure 5The overall differences between the XFEL- and synchrotron-derived ClR structures by the DDMP analysis. A, the DDMP according to Cα atom deviations between the XFEL- and synchrotron-derived ClR structures. Red and blue dots indicate the relative movement closer or further, respectively, with color saturation indicating a difference of 0.5 Å or more. The XFEL- and synchrotron-derived ClRs differed slightly, as shown by the regions highlighted with black dotted boxes. B, blue dotted arrows connect these blocks to a ribbon diagram, indicating the position of the highlighted regions within the structure such as helix C, helix E, helix G, the B-C loop, the C-D loop, and the C-terminal helix. C, the direction of the relative movement of the transmembrane domains based on DDMP. Opposite motional directions are indicated by red and blue arrows in the extracellular (upper) and cytoplasmic (lower) regions.
      Interestingly, C-terminal helix movement was clearly observed in the direction opposite to helix G movement (Fig. 5C, lower panel). These motions were reported to be important for maintaining the function and stability of ClR in previous studies (
      • Kim K.
      • Kwon S.K.
      • Jun S.H.
      • Cha J.S.
      • Kim H.
      • Lee W.
      • Kim J.F.
      • Cho H.S.
      Crystal structure and functional characterization of a light-driven chloride pump having an NTQ motif.
      ) and are similar to the related motion in the function of bR (
      • Nogly P.
      • Panneels V.
      • Nelson G.
      • Gati C.
      • Kimura T.
      • Milne C.
      • Milathianaki D.
      • Kubo M.
      • Wu W.
      • Conrad C.
      • Coe J.
      • Bean R.
      • Zhao Y.
      • Båth P.
      • Dods R.
      • et al.
      Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.
      ). As a result, the structural orientation of the C-D loop, a part of helix E, and C-terminal helix moved toward the exterior of a protein, whereas helix G moved in the opposite direction (Fig. 5C, lower panel).
      The detailed structural information of the XFEL-derived ClR provided the basis for revealing the ion channel mechanism of the light-induced photocycle of ClR at room temperature.

      Discussion

      A number of functionally important membrane protein structures have been solved using crystals grown with the LCP method. However, several problems remain unsolved in order for membrane protein crystallization to obtain crystals suitable for synchrotron X-ray diffractions. Recently, SFX using XFELs has been developed and has emerged as a powerful method for the structural determination of macromolecules, especially membrane proteins such as G protein–coupled receptors. The experimental environment for XFEL is different from that used for synchrotron-based crystallography which requires cryogenic cooling protection, and this difference could influence the interaction of ligands or ions that are related to membrane protein function. The structure and dynamics for some membrane proteins depend on temperature and pressure.
      Studies have indicated that XFEL-derived structures do not suffer from perturbations because of cryogenic cooling or radiation damage from synchrotrons (
      • Nogly P.
      • Panneels V.
      • Nelson G.
      • Gati C.
      • Kimura T.
      • Milne C.
      • Milathianaki D.
      • Kubo M.
      • Wu W.
      • Conrad C.
      • Coe J.
      • Bean R.
      • Zhao Y.
      • Båth P.
      • Dods R.
      • et al.
      Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.
      ,
      • Edlund P.
      • Takala H.
      • Claesson E.
      • Henry L.
      • Dods R.
      • Lehtivuori H.
      • Panman M.
      • Pande K.
      • White T.
      • Nakane T.
      • Berntsson O.
      • Gustavsson E.
      • Båth P.
      • Modi V.
      • Roy-Chowdhury S.
      • et al.
      The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography.
      ). Here, we directly compared structures of ClR at cryogenic and room temperatures and revealed subtle structural differences that could explain the hyperactivity of ClR at low temperatures. The DDMP analysis depicted regional conformation changes for temperature-sensitive residues. The high-resolution data (1.85 Å and 1.75 Å of the structures from the XFEL and synchrotron, respectively) allowed us to accurately compare hydrogen bonding networks mediated by chloride ions and water molecules through direct structure comparison. The overall unit cell volume of microcrystals in the XFEL experiment was larger (by about 3.2%) than that of the crystals used in the synchrotron crystallography experiment, indicating that the cryogenic cooling slightly dehydrates the crystals and increases the crystal packing. In a previous bR study, the electron density map around the C2–C4 atoms of the retinal β-ionone ring was found to be partially depleted in the XFEL structure, indicating the possibility of multiple conformers at ambient temperature (
      • Nogly P.
      • Panneels V.
      • Nelson G.
      • Gati C.
      • Kimura T.
      • Milne C.
      • Milathianaki D.
      • Kubo M.
      • Wu W.
      • Conrad C.
      • Coe J.
      • Bean R.
      • Zhao Y.
      • Båth P.
      • Dods R.
      • et al.
      Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.
      ). In the present study, the ClR structure derived from XFEL showed a very clear electron density map of the retinal β-ionone ring, indicating that the structure was thermodynamically stable at physiological temperature. These findings suggest that bR and ClR may have different dynamic motions of the retinal, leading to a cis-trans transition even at the same temperature conditions (Fig. S4, C and D).
      Here, we also identified the hydrogen bond networks mediated by the additional water molecule, W614, near the Cl-I and PSB regions. In general, the energy associated with hydrogen bonds is 6–30 kJ/mol, and even a slight change in the angle and distance between the relevant atoms causes a drastic change in the energy barrier (
      • Watson J.D.
      ). For this reason, hydrogen bonding is a key element in maintaining the binding strength and ion specificity in a transporter protein, which in turn affects the ion transport efficiency. These differences were also observed at the Cl-II position in the cytoplasmic region. The weak hydrogen-bonding network near Cl-I in ClR would eventually lead to rapid chloride ion diffusion into the cytoplasm direction at a reduced energy cost. In this respect, the non-cryogenic structure determined at ambient temperature is helpful to better understand protein functions at the optimum temperature of their activity.
      Considering the structural changes observed in the cavity and DDMP analyses, temperature-dependent structural movement of ClR can be inferred. At ambient temperatures, the ClR structure has a relatively wider intracellular portion and narrower extracellular portion compared with its structure at low temperatures. In this study, we concluded that the ClR structure derived from the XFEL method provided a structural foundation in understanding its chloride ion-pumping activity at physiological conditions.

      Experimental procedures

       Protein expression and purification

      The ClR gene (GI: 594833795) from Nocardioides marinus in the pET21b vector was transformed into Escherichia coli BL21-CodonPlus (DE3; Agilent Technologies, Santa Clara, CA), and the cells were grown in high-salt Luria-Bertani medium at 37 °C. When the optical density at 600 nm (A600) was over 1.0, 50 μm ATR (Sigma-Aldrich) and 0.5 mm isopropyl-β-d-thiogalactopyranoside (IPTG) were added to induce ClR expression for 6–8 h at 30 °C. Harvested cells were lysed by sonication in lysis buffer containing 50 mm Tris-HCl (pH 7.0) and 150 mm NaCl. The membrane fraction was isolated by ultracentrifugation (Beckman) at 370,000 × g for 40 min at 4 °C, resuspended in solubilization buffer containing 50 mm Tris-HCl (pH 7.0), 150 mm NaCl, 1% n-dodecyl-β-d-maltoside (DDM), and 0.2% cholesteryl hemisuccinate (CHS), and incubated for 2 h at 4 °C for solubilization. The solubilized protein was purified by TALON affinity chromatography and the eluate was applied to a Superdex 200 size-exclusion column (GE Healthcare) equilibrated with buffer containing 20 mm HEPES (pH 7.5), 150 mm NaCl, 0.05% DDM, and 0.01% CHS (Fig. S5A).

       Microcrystal preparation for XFEL and synchrotron analysis

      ClR microcrystals appeared in 30% PEG dimethyl ether 500, 0.1 m sodium chloride, and 0.1 m MES (pH 6.0) buffer at a final concentration of 50 mg/ml for conventional synchrotron experiments (
      • Kim K.
      • Kwon S.K.
      • Jun S.H.
      • Cha J.S.
      • Kim H.
      • Lee W.
      • Kim J.F.
      • Cho H.S.
      Crystal structure and functional characterization of a light-driven chloride pump having an NTQ motif.
      ). For the LCP-XFEL approach, a high density of microcrystals is required for efficient data collection. We followed optimized protocols (
      • Liu W.
      • Ishchenko A.
      • Cherezov V.
      Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography.
      ) to obtain samples for a complete dataset collection. The purified protein was mixed with monoolein (1-oleoyl-rac-glycerol) at a 1:1.5 molar ratio (w/w) using a syringe lipid mixer (Hamilton, Reno, NV). After formation of a clear lipidic cubic phase, ∼5 μl of the protein-laden LCP sample was injected using a 100-μl syringe filled with 55–60 μl of precipitant solution. After sealing the needle stopper, the syringe was wrapped in a moist tissue to maintain the humidity and placed in a 20 °C incubator for 1 week (Fig. S5B). Five additional syringes were prepared to produce a large volume of the LCP crystal sample. Microcrystals of ClR were grown in the syringes, and red-colored crystals were identified using a microscope. For the synchrotron experiment, ClR microcrystals were grown on LCP plates (Fig. S5C).

       Experimental setup at Linac Coherent Light Source, and data collection from XFEL and synchrotron

      The experiment was performed at the Coherent X-ray Imaging (CXI) end station (
      • Liang M.
      • Williams G.J.
      • Messerschmidt M.
      • Seibert M.M.
      • Montanez P.A.
      • Hayes M.
      • Milathianaki D.
      • Aquila A.
      • Hunter M.S.
      • Koglin J.E.
      • Schafer D.W.
      • Guillet S.
      • Busse A.
      • Bergan R.
      • Olson W.
      • et al.
      The coherent x-ray imaging instrument at the Linac Coherent Light Source.
      ) at the Linac Coherent Light Source (LCLS) of the SLAC National Accelerator Laboratory. Prior to LCP-XFEL data collection, ClR crystals grown in syringes were incubated at 25 °C for 1 h. Approximately 40 μl of the LCP sample was transferred into a new syringe after removing the precipitant solution, and ∼10 μl of 9.9 MAG was applied to the LCP sample and homogenized. The sample was transferred into an LCP injector through an LCP loading needle. Data were collected at 120 Hz using a photon energy of 9.5 keV (λ = 1.3 Å) and a pulse duration of ∼60 fs. Because of evaporative cooling, the sample temperature at the X-ray interaction region (∼100 μm downstream of the nozzle exit) was estimated to be slightly below room temperature. The samples were injected using an LCP injector with a 75-μm diameter nozzle at an average flow rate of 2 μl/min. A Cornell-SLAC Pixel Array Detector (CSPAD) was used to collect the diffraction data. The panels of the CSPAD were tiled to form a two-dimensional detector that was placed at 77.5 mm from the interaction plane, allowing for diffraction data collection up to a 1.33-Å resolution at the given wavelength. About 420,465 diffraction patterns were collected in ∼1 h from 120 μl of the sample. The Cheetah program (
      • Barty A.
      • Kirian R.A.
      • Maia F.R.
      • Hantke M.
      • Yoon C.H.
      • White T.A.
      • Chapman H.
      Cheetah: software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data.
      ) was used to preprocess and filter the raw data to select diffraction patterns (actual “hits” of crystals from a liquid background). A pattern was classified as a “hit” if it contained at least 20 peaks with a signal-to-noise ratio larger than 6.0. A total of 22,094 patterns were identified as hits for an overall hit rate of 5.26%. The CrystFEL software suite (
      • White T.A.
      • Mariani V.
      • Brehm W.
      • Yefanov O.
      • Barty A.
      • Beyerlein K.R.
      • Chervinskii F.
      • Galli L.
      • Gati C.
      • Nakane T.
      • Tolstikova A.
      • Yamashita K.
      • Yoon C.H.
      • Diederichs K.
      • Chapman H.N.
      Recent developments in CrystFEL.
      ) was used to index the diffraction patterns and merge the intensity to form the three-dimensional diffraction volume. After refinement of the experimental conditions, including detector geometry, 10,805 patterns were indexed using the indexamajig module of the CrystFEL suite, and the unit cell parameters were determined for data merging. Finally, all reflections, including those predicted based on the observed peaks of indexed patterns, were merged into a dataset with a Monte Carlo approach implemented in CrystFEL. For conventional synchrotron data, we collected diffraction data from ClR crystals on the BL17A beamline at the Photon Factory (Tsukuba, Japan) using a 0.03 × 0.01 mm microbeam. The diffraction data were processed with XDS software (
      • Ramachandraiah G.
      • Chandra N.R.
      • Surolia A.
      • Vijayan M.
      Re-refinement using reprocessed data to improve the quality of the structure: A case study involving garlic lectin.
      ). We determined the cryogenic structure at Photon factory as described in a previous report (
      • Kim K.
      • Kwon S.K.
      • Jun S.H.
      • Cha J.S.
      • Kim H.
      • Lee W.
      • Kim J.F.
      • Cho H.S.
      Crystal structure and functional characterization of a light-driven chloride pump having an NTQ motif.
      ). All parameters for data collection, model refinement, and statistical analysis of both the XFEL and synchrotron data are summarized in Table 1.
      Table 1Data collection and refinement statistics
      XFEL-derived ClRSynchrotron-derived ClR
      Data collection
       Space groupC2C2
       Cell dimensions
      a, b, c (Å)103.36, 50.09, 69.40102.76, 49.40, 69.33
      α, β, γ (°)90.00, 109.65, 90.0090.00, 109.85, 90.00
       No. of collected images420,465NA
       No. of hits22,094NA
       No. of indexed patterns10,805NA
       No. of merged patterns10,801NA
       Indexing rate (%)48.9NA
       No. of total reflections1,644,02032,667
       No. of unique reflections28,973NA
       Resolution (Å)18.52–1.85 (1.92–1.85)
      Values in parentheses are for highest-resolution shell.
      23.63–1.75 (1.81–1.75)
      Rmerge (%)NA8.6 (26.9)
      Rsplit (%)16.6 (72.7)NA
      Rpim (%)NANA
      I/σ(I)4.04 (1.61)24.47 (5.0)
       Completeness (%)100 (100)98.4 (97.1)
       Multiplicity56.7 (37.3)3.1 (2.5)
       CC1/2 (%)96.7 (38.6)NA
      Refinement
       Resolution (Å)18.52–1.8523.63–1.75
       No. of reflections/test set28,681/142032,667/1655
      Rwork/Rfree0.24/0.280.19/0.22
       No. of atoms
      Protein20452072
      Retinal2020
      Water12072
      Chloride22
      Lipid9899
       B-factor (Å2)
      Wilson B/overall B22.53/23.5920.92/23.12
       Root mean square deviations
      Bond lengths0.010.01
      Bond angles0.970.93
       Ramachandran plot (%)
      Favored9899
      Allowed21
      Disallowed00
      * Values in parentheses are for highest-resolution shell.

       Structure determination and refinement

      Crystals grew in space group C2, with a = 103.36 Å, b = 50.09 Å, c = 69.40 Å, α = 90.00°, β = 109.65°, and γ = 90.00° for the XFEL-derived structure, and a = 102.76 Å, b = 49.40 Å, c = 69.33 Å, α = 90.00°, β = 109.85°, and γ = 90.00° for the synchrotron-derived structure, with one molecule in the asymmetric unit. Initial models of the near-isomorphous crystal data were obtained by molecular replacement using PHASER (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ) with a previously solved structure as a search model (PDB ID: 5G28). The solutions of ClR were successfully obtained with a final translation function Z-score of 19.0. The models were refined with the simulated annealing protocol using bulk-solvent correction with data between a resolution of 50.0 Å and 3.0 Å. Electron density was interpreted and traced using COOT (
      • Emsley P.
      • Cowtan K.
      Coot: Model-building tools for molecular graphics.
      ), and the model was refined with PHENIX (
      • Adams P.D.
      • Grosse-Kunstleve R.W.
      • Hung L.W.
      • Ioerger T.R.
      • McCoy A.J.
      • Moriarty N.W.
      • Read R.J.
      • Sacchettini J.C.
      • Sauter N.K.
      • Terwilliger T.C.
      PHENIX: Building new software for automated crystallographic structure determination.
      ) and autoBUSTER (
      • Blanc E.
      • Roversi P.
      • Vonrhein C.
      • Flensburg C.
      • Lea S.M.
      • Bricogne G.
      Refinement of severely incomplete structures with maximum likelihood in BISTER-TNT.
      ). After several rounds of alternate refinement by autoBUSTER in the absence and presence of ATR, the structure of ClR containing ATR was further refined, resulting in Rwork/Rfree = 0.24/0.28 over a resolution range of 18.52–1.85 Å. Likewise, the synchrotron-derived structure was refined, resulting in Rwork/Rfree = 0.19/0.22 over a resolution range of 23.63–1.75 Å. Solvent molecules were placed at positions where spherical electron density peaks were found above 1.3 σ in the |2Fo−DFc| map and above 3.0 σ in the |Fo–Fc| map, and where stereochemically reasonable hydrogen bonds could form. Validation of the final model was carried out using PROCHECK (
      • Laskowski R.A.
      • MacArthur M.W.
      • Moss D.S.
      • Thornton J.M.
      PROCHECK: A program to check the stereochemical quality of protein structures.
      ). During structure refinement, we tried to reduce the resolution gap and the artifact by applying the same σ cutoff value between the XFEL- and synchrotron-derived structure, especially for selection of chloride ions and water molecules.

       Data availability

      Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5ZTL (XFEL-derived ClR structure) and 5ZTK (synchrotron-derived ClR structure). Remaining data are available from the corresponding authors on reasonable request.

       Difference distance matrix plot analysis

      The pairwise distance map was computed between all possible amino acid residue pairs in a three-dimensional structure and represented with a two-dimensional matrix. We used the DDMP (Center for Structural Biology, Yale University, New Haven, CT) program to analyze the protein contact map. In the difference distance map, the changes in distance between Cα-Cα atoms were reflected by the brightness of the colors, and color saturation levels were set to represent Cα shifts of 0.5 Å closer (red) and 0.5 Å away (blue) (
      • Richards F.M.
      • Kundrot C.E.
      Identification of structural motifs from protein coordinate data: secondary structure and first level supersecondary structure.
      ).

       Cavity calculation

      The PDB coordinates of ClR structures were submitted to the CASTp Web server (http://sts.bioe.uic.edu/castp/calculation.html)
      Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
      (
      • Tian W.
      • Chen C.
      • Lei X.
      • Zhao J.
      • Liang J.
      CASTp 3.0: Computed atlas of surface topography of proteins.
      ) for putative substrate-binding pocket and retinal cavity calculation, and identification as a probe radius of 1.4 Å. Each pocket was assigned a unique identification number, roughly corresponding in order of decreasing volume. After selection of the specific pocket, graphical representations of ClR were made using PyMol.

       Measurement of pumping activities of ClR

      E. coli BL21-CodonPlus (DE3) cells, expressing ClR, were incubated at 37 °C in YT media supplemented with 100 mg/ml ampicillin. When the A600 nm was greater than 1.0, the expression of ClR was induced by adding 1 mm isopropyl-β-d-thiogalactopyranoside and 50 μm ATR. After additional cultivation for 4 h at 37 °C, the cells were collected by centrifugation at 4000 × g for 5 min, washed three times with 100 mm NaCl or NaBr including 50% glycerol, and suspended in the desired solvents for measurement by adjusting the A600 nm value to 8.0. Cell suspensions were placed in the dark for more than 2 h, under control of desired temperature from −20 °C to 25 °C, and then, proton ion flux changes in the buffer were measured using a pH electrode F-72G (Horiba, Kyoto, Japan) at five different temperatures, −20 °C, −10 °C, 4 °C, 10 °C, and 25 °C, respectively. The light source used was a 200 milliwatt 520 nm Xeon lamp (Elpisbio, Seoul, South Korea).

       UV-visible spectroscopy

      ClR was purified and buffer exchange was conducted using 10 mm MOPS (pH 6.5) and 0.05% DDM on a Superdex 200 size-exclusion column (GE Healthcare). The absorption spectra were scanned for the protein samples using a V-650 spectrophotometer (Jasco, Easton, MD) at two different temperatures, 4 °C and 25 °C. Anion titration experiments were performed by adding chloride and bromide ions and standard deviations were calculated from triplicate experiments. Anion-binding affinities were calculated from absorption changes at a wavelength of 581 nm using the Hill equation. The binding affinities are listed in Table S2.

      Author contributions

      J.-H. Y., H. L., and Weontae Lee conceptualization; J.-H. Y., X. L., M. S. H., and R. G. S. data curation; J.-H. Y., J.-H. P., M. O., Z. J., and Wonbin Lee formal analysis; J.-H. Y., H.-S. C., H. L., and Weontae Lee funding acquisition; J.-H. Y., X. L., J.-H. P., M. O., S.-Y. P., and C. H. Y. validation; J.-H. Y. and H. L. investigation; J.-H. Y., M. O., H. L., and Weontae Lee writing-original draft; J.-H. Y., S.-Y. P., and H. L. writing-review and editing; X. L., J.-H. P., Y. W., S.-Y. P., N. Z., M. S. H., R. G. S., C. H. Y., and L. T. software; M. O., S.-Y. P., H. H., C. L., N. Z., M. S. H., R. G. S., J. K., C. H. Y., and U. W. methodology; H.-S. C. and U. W. resources.

      Acknowledgments

      We are grateful to the staff scientists at the Coherent X-ray Imaging (CXI) station of the Linac Coherent Light Source of the SLAC National Accelerator Laboratory, BL17A beamline of the Photon Factory. We also thank the staff scientists at the Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) for their technical support on the initial screening. This research was supported by a Tianhe-2JK computing time award at the Beijing Computational Research Center (CSRC). Portions of this research were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. This LCLS beam time was part of the Protein Crystal Screening (PCS) program. LCLS is an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. Parts of the sample delivery system used at LCLS for this research were funded by the National Institutes of Health Grant P41GM103393, formerly P41RR001209.

      Supplementary Material

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