Advertisement

The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants

Open AccessPublished:July 23, 2020DOI:https://doi.org/10.1074/jbc.RA120.014118
      Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.
      One of the foremost challenges in understanding functional diversity of microbial rhodopsins is dissecting their complex structure–function relationships (
      • Ernst O.P.
      • Lodowski D.T.
      • Elstner M.
      • Hegemann P.
      • Brown L.S.
      • Kandori H.
      Microbial and animal rhodopsins: structures, functions, and molecular mechanisms.
      ,
      • Kurihara M.
      • Sudo Y.
      Microbial rhodopsins: wide distribution, rich diversity and great potential.
      ,
      • Govorunova E.G.
      • Sineshchekov O.A.
      • Li H.
      • Spudich J.L.
      Microbial rhodopsins: diversity, mechanisms, and optogenetic applications.
      ,
      • Gushchin I.
      • Gordeliy V.
      Microbial rhodopsins.
      ,
      • Klare J.P.
      • Chizhov I.
      • Engelhard M.
      Microbial rhodopsins: scaffolds for ion pumps, channels, and sensors.
      ). Despite the distinct functional roles and different amino acid sequences, microbial rhodopsins have surprisingly similar structures, especially in their transmembrane domains frugally recycled by nature. It turns out that the functional differences among microbial rhodopsins are regulated by relatively small variations in the side chains, internal water molecules, and bound ions, which necessitate the acquisition of high-resolution structural data to probe structure–function relationships. This is particularly true for ion-pumping rhodopsins, a structurally uniform group with various ion specificities, whereas greater structural diversity has been seen in sensory rhodopsins, enzymerhodopsins, and channelrhodopsins (
      • Govorunova E.G.
      • Sineshchekov O.A.
      • Li H.
      • Spudich J.L.
      Microbial rhodopsins: diversity, mechanisms, and optogenetic applications.
      ,
      • Mukherjee S.
      • Hegemann P.
      • Broser M.
      Enzymerhodopsins: novel photoregulated catalysts for optogenetics.
      ).
      Ion-pumping microbial rhodopsins are widespread among Eukarya, Bacteria, and Archaea (
      • Kandori H.
      Ion-pumping microbial rhodopsins.
      ,
      • Pinhassi J.
      • DeLong E.F.
      • Béjà O.
      • González J.M.
      • Pedrós-Alió C.
      Marine bacterial and archaeal ion-pumping rhodopsins: genetic diversity, physiology, and ecology.
      ,
      • Brown L.S.
      • Jung K.H.
      Bacteriorhodopsin-like proteins of eubacteria and fungi: the extent of conservation of the haloarchaeal proton-pumping mechanism.
      ). These microbial rhodopsins share a similar structural template consisting of seven tightly bundled transmembrane α-helices with the N and C termini located outside and inside the cell, respectively (Fig. 1). The all-trans-retinal chromophore is covalently bound through a protonated retinal Schiff base (PRSB) linkage to the ε-amino group of a lysine residue in the middle of the seventh helix. For ion pumps, the absorption of a photon triggers the retinal chromophore to isomerize from all-trans- to 13-cis-configuration, which induces a photocycle involving protein structural changes to pump an ion through the membrane against a concentration gradient. Despite their similar overall architecture, ion specificities and transport vectorialities of these rhodopsins can be adjusted by fine-tuning their structures (
      • Muroda K.
      • Nakashima K.
      • Shibata M.
      • Demura M.
      • Kandori H.
      Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps.
      ,
      • Inoue K.
      • Nomura Y.
      • Kandori H.
      Asymmetric functional conversion of eubacterial light-driven ion pumps.
      ), yielding proteins able to pump H+ outwards or inwards, Na+ outwards, or Cl (and some other anions) inwards.
      Figure thumbnail gr1
      Figure 1Microbial rhodopsin ion pumps with function-determining motif. Shown is a seven-transmembrane helix (denoted A–G) structure of microbial rhodopsin with all-trans-retinal chromophore bound covalently to a conserved lysine residue (cyan) on helix G. Three amino acids on helix C serve as a functional indicator on whether the protein pumps H+ or Na+ outward (orange and yellow arrows, respectively) or Cl inward (green arrow). The motif derives from key residues in the BR proton-pumping photocycle, with Asp-85 (acceptor of Schiff base proton), Thr-89, and Asp-96 (proton donor for Schiff base) forming the DTD motif. For pumping of H+, Na+, and Cl, different motifs are employed as indicated. Cyanobacterial chloride pump MastR possesses a TSD motif, which was mutated to DSD in this study to convert the MastR chloride pump into a proton pump.
      Lately, a motif of three conserved residues on the third transmembrane helix (helix C) has been adopted as a function determinant for ion-pumping rhodopsins (
      • Inoue K.
      • Kato Y.
      • Kandori H.
      Light-driven ion-translocating rhodopsins in marine bacteria.
      ,
      • Béjà O.
      • Lanyi J.K.
      Nature's toolkit for microbial rhodopsin ion pumps.
      ). Archaeal (bacteriorhodopsins or BRs) and fungal outward H+ pumps usually have an Asp-Thr-Asp (DTD) motif, whereas bacterial outward H+ pumps more commonly have either a DTE, DTK, or DTG signature representing the proton acceptor, the acceptor hydrogen-bonding partner, and the cytoplasmic proton donor (or its nonprotonatable homolog), respectively. Light-driven retinal-binding Na+ pumps possess an NDQ motif, in which the three residues play critical roles in binding transported Na+ and in the accompanying critical H+ transfers (
      • Inoue K.
      • Konno M.
      • Abe-Yoshizumi R.
      • Kandori H.
      The role of the NDQ motif in sodium-pumping rhodopsins.
      ).
      Unlike the above-mentioned subfamilies of cation pumps, anion transporters do not have a common function-determining motif and are represented by at least three divergent groups. Long-known archaeal Cl pumps (halorhodopsins or HRs) have a TSA motif, whereas recently discovered eubacterial chloride-pumping rhodopsins (ClRs or NTQ rhodopsins) possess an NTQ motif; however, in both groups the first two amino acids of the function-determining motif bind a chloride anion in the dark (
      • Engelhard C.
      • Chizhov I.
      • Siebert F.
      • Engelhard M.
      Microbial halorhodopsins: light-driven chloride pumps.
      ,
      • Kouyama T.
      • Kanada S.
      • Takeguchi Y.
      • Narusawa A.
      • Murakami M.
      • Ihara K.
      Crystal structure of the light-driven chloride pump halorhodopsin from Natronomonas pharaonis.
      ,
      • Schreiner M.
      • Schlesinger R.
      • Heberle J.
      • Niemann H.H.
      Structure of halorhodopsin from Halobacterium salinarum in a new crystal form that imposes little restraint on the E–F loop.
      ,
      • 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.
      ,
      • Hosaka T.
      • Yoshizawa S.
      • Nakajima Y.
      • Ohsawa N.
      • Hato M.
      • DeLong E.F.
      • Kogure K.
      • Yokoyama S.
      • Kimura-Someya T.
      • Iwasaki W.
      • Shirouzu M.
      Structural mechanism for light-driven transport by a new type of chloride ion pump, Nonlabens marinus rhodopsin-3.
      ). Additionally, there is a novel distinct anion-pumping cyanobacterial rhodopsin group containing both chloride and sulfate transporters, which have sequence similarity both to HRs and BRs (
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ,
      • Niho A.
      • Yoshizawa S.
      • Tsukamoto T.
      • Kurihara M.
      • Tahara S.
      • Nakajima Y.
      • Mizuno M.
      • Kuramochi H.
      • Tahara T.
      • Mizutani Y.
      • Sudo Y.
      Demonstration of a light-driven SO4(2-) transporter and its spectroscopic characteristics.
      ,
      • Nakajima Y.
      • Tsukamoto T.
      • Kumagai Y.
      • Ogura Y.
      • Hayashi T.
      • Song J.
      • Kikukawa T.
      • Demura M.
      • Kogure K.
      • Sudo Y.
      • Yoshizawa S.
      Presence of a haloarchaeal halorhodopsin-like Cl pump in marine bacteria.
      ,
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ). Similar to HRs, this group possess threonine and serine as the first motif-forming residues, but it has aspartate, valine, leucine, or isoleucine as its third member (TSD/TSV/TSL/TSI), along with a large number of unique polar residues elsewhere. The first characterized member of this group is Mastigocladopsis repens rhodopsin (MastR or MrHR), which pumps chloride ions inwards through a function-determining TSD motif (
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ,
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ).
      Although high-resolution structures of representative HRs (
      • Kouyama T.
      • Kanada S.
      • Takeguchi Y.
      • Narusawa A.
      • Murakami M.
      • Ihara K.
      Crystal structure of the light-driven chloride pump halorhodopsin from Natronomonas pharaonis.
      ,
      • Schreiner M.
      • Schlesinger R.
      • Heberle J.
      • Niemann H.H.
      Structure of halorhodopsin from Halobacterium salinarum in a new crystal form that imposes little restraint on the E–F loop.
      ) and ClRs (
      • 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.
      ,
      • Hosaka T.
      • Yoshizawa S.
      • Nakajima Y.
      • Ohsawa N.
      • Hato M.
      • DeLong E.F.
      • Kogure K.
      • Yokoyama S.
      • Kimura-Someya T.
      • Iwasaki W.
      • Shirouzu M.
      Structural mechanism for light-driven transport by a new type of chloride ion pump, Nonlabens marinus rhodopsin-3.
      ) are available, the third (cyanobacterial) group of chloride-pumping rhodopsins has not been characterized structurally. Similar to proton-pumping rhodopsins, comparison of structures of divergent chloride-pumping rhodopsins with the same biological function (i.e. the same ion specificity) could help in defining common structural determinants of this function and its group-specific fine-tuning elements. Toward this goal, here we provide the high-resolution crystal structure of the chloride-pumping MastR, which reveals the unique fine structural elements of this novel anion transporter along with the common HR-like chloride-binding motif in the context of a predominantly BR-like structural template.
      An alternative, elegant approach to understanding key structural determinants of the functions of microbial rhodopsin ion pumps is their functional conversion, in which a WT rhodopsin of chosen specificity is mutated to obtain a different functionality. Functional conversion of ion pumps has been achieved with varying success through site-directed mutagenesis, usually targeting the conserved three amino acids at the function-determining motif (
      • Muroda K.
      • Nakashima K.
      • Shibata M.
      • Demura M.
      • Kandori H.
      Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps.
      ,
      • Inoue K.
      • Nomura Y.
      • Kandori H.
      Asymmetric functional conversion of eubacterial light-driven ion pumps.
      ,
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ,
      • Sasaki J.
      • Brown L.S.
      • Chon Y.S.
      • Kandori H.
      • Maeda A.
      • Needleman R.
      • Lanyi J.K.
      Conversion of bacteriorhodopsin into a chloride ion pump.
      ).
      In this paper, we compare the X-ray crystallographic structures of chloride-pumping MastR and its proton-pumping T74D mutant (Figs. S1S3) in the context of other chloride and proton pumps with known high-resolution structures. The structures suggest that the hydrogen bonds of the PRSB and adjacent water were both strong for MastR and MastR-T74D, supporting the hypothesis that outward proton transport requires both a strongly hydrogen-bonded water and a strongly hydrogen-bonded Schiff base. It appears that the BR-like structure of MastR, which ensures proper positioning of the water molecule interacting with the Schiff base, allows for its easy functional conversion to a proton pump, in contrast to HRs in which this is not possible, thereby revealing important structural prerequisites for outward proton pumping by microbial rhodopsins.

      Results and discussion

      Crystal structure of MastR

      We purified MastR and the mutant MastR-T74D after overexpression in Escherichia coli (Fig. S4) and crystallized the proteins using the bicelle crystallization method (
      • Faham S.
      • Bowie J.U.
      Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure.
      ). We obtained hexagonal crystals (Fig. S5, A and B) in which trimers of three parallel monomers arrange in a hexagonal pattern (Fig. S6, C and G) to yield layers that stack directly on top of each other (Fig. S6, D and H). Within each layer, MastR packs as parallel trimers (Fig. S6C), and MastR-T74D packs as antiparallel trimers (Fig. S6G), respectively. In the stacking pattern, MastR forms stacked alternating layers (Fig. S6D), whereas MastR-T74D forms stacked parallel layers (Fig. S6H). This difference in packing results in MastR possessing an asymmetric unit with two rhodopsins connected via their extracellular loops, whereas MastR-T74D contains only a single protein in the asymmetric unit (Fig. 2A). We determined the cryogenic X-ray crystal structures of MastR and MastR-T74D at resolutions of 2.33 and 2.50 Å, respectively (Fig. 2, Fig. S2, and Table S1).
      Figure thumbnail gr2
      Figure 2Structure of MastR and MastR-T74D. A, the asymmetric unit of MastR (pink, contains two proteins) is overlaid with the asymmetric unit of MastR-T74D (green, contains a single protein). MastR has seven transmembrane α-helices and a short β-sheet on the B–C loop (red). MastR and MastR-T74D share a nearly identical structural fold (RMSD 0.26 Å). MastR possesses two binding sites for Cl ions (green spheres) adjacent to the retinal Schiff base and the B–C loop, whereas the mutant does not possess any Cl ions. B and C, electron density of key elements in the MastR (B) and MastR-T74D (C) retinal-binding pocket. Key residues have been labeled with the mutated residue highlighted in red. In MastR, a Cl ion (green sphere) is part of an extended hydrogen-bonding network including Thr-74, Ser-78, a single water molecule (red sphere), and the PRSB. In the proton-pumping T74D mutant, the Cl ion is lacking, which rearranges the hydrogen-bonding network. 2FcFo electron density maps are contoured at 0.8 σ. The direction of ion transport is indicated.
      Like other microbial rhodopsins, MastR has seven transmembrane helices (helices A–G), connected by three intracellular and three extracellular loops (Fig. 2A). The intracellular loop connecting helices B and C (B–C loop) forms a short antiparallel β-sheet. The structure contains 45 water molecules, two chloride ions, a glucose moiety from octylglucoside detergent, and the retinal chromophore. The electron density of the chromophore revealed retinal in the all-trans-configuration (Fig. 2B), covalently bound to the ε-amino group of Lys-204 on helix G via a PRSB, in agreement with prior Raman spectroscopy experiments (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ). The MastR and MastR-T74D structures are almost identical with an RMSD of 0.26 Å and differ mainly in the PRSB region around the site of the mutation and the presence or absence of Cl ions for the WT and mutant, respectively (Fig. 2, B and C).

      Chloride ion–binding sites

      The MastR structure further shows two chloride ion-binding sites: one adjacent the PRSB and the other forming a crystal contact between the B-C loops of two stacked proteins. The primary Cl ion adjacent the PRSB is well-ordered between a single water molecule and Thr-74 and Ser-78 of the TSD motif as shown by the electron density in Fig. 2B. The Cl ion is the counterion of the positively charged PRSB and connected to it via a bridging water molecule. This chloride ion-binding site can also be occupied by bromide and iodide but not fluoride, as shown by UV-visible absorption (Fig. S7A) (
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ). The T74A mutation was previously shown to strongly reduce chloride affinity (from KD of 2 to 85 mm) but not impede Cl ion transport, whereas the S78A mutation decreased the pKa and stability of the PRSB (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ), and the S78T mutation decreased chloride affinity dramatically (almost 200-fold) (
      • Hasemi T.
      • Kikukawa T.
      • Watanabe Y.
      • Aizawa T.
      • Miyauchi S.
      • Kamo N.
      • Demura M.
      Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin.
      ). This agrees with the structure, which confirms that both Ser-78 and Thr-74 are involved in Cl ion binding, similar to HR (
      • Schreiner M.
      • Schlesinger R.
      • Heberle J.
      • Niemann H.H.
      Structure of halorhodopsin from Halobacterium salinarum in a new crystal form that imposes little restraint on the E–F loop.
      ).
      The second chloride ion-binding site is formed by the extracellular sides of two MastR protomers (Fig. 2A). A Cl ion is found to bridge two B-C loop β-sheets by interacting with the side-chain nitrogen of Asn-60 and backbone nitrogen of Val-61 of both proteins. This well-ordered Cl ion appears to be a crystal-stabilizing contact and may not be a functionally relevant Cl ion as concluded from bromide-replacement experiments (Fig. S8) and the MastR-T74D structure. Because MastR-T74D packs as stacked parallel layers, the B–C loops do not get into contact, and the lack of electron density for a Cl ion near the B–C loop indicates low chloride affinity when no crystal contact between B–C loops occurs (Fig. S6H).

      Oligomerization of MastR

      Trimers of MastR are formed by interaction of helix B of one protomer with helices D′ and E′ of another protomer (Fig. 3A). The interface is stabilized by three polar contacts: (i) the backbone oxygen of Leu-44 with the hydroxyl group of Tyr-106, (ii) a water-bridged hydrogen bond between Thr-37 and Ser-99, and (iii) a water-bridged hydrogen bond between Glu-30 and the backbone nitrogen of Leu-94. Main hydrophobic interactions at the intermonomer interface are provided by Phe-41 on helix B, Tyr-106 on helix D′, and Trp-125 on helix E′ (Fig. 3A).
      Figure thumbnail gr3
      Figure 3Oligomeric assemblies of MastR and KR2. A, MastR assembles as trimers via a B–D′/E′ interface shown in the inset. B, sodium pump KR2 (PDB code 6rew) assembles like homologous FR (chloride pump) and GR (proton pump) as pentamer with characteristic orientation of the extracellular B–C loop (red) interacting with the neighboring protomer. Retinal is highlighted in cyan, and chloride ions are shown as green spheres.
      The oligomeric structure of microbial rhodopsins in solution can also be determined by visible CD spectroscopy because the exciton coupling of the retinal chromophores gives rise to distinct visible CD curves (
      • Shibata M.
      • Inoue K.
      • Ikeda K.
      • Konno M.
      • Singh M.
      • Kataoka C.
      • Abe-Yoshizumi R.
      • Kandori H.
      • Uchihashi T.
      Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy.
      ). Monomeric rhodopsins do not possess exciton coupling and only display a single positive peak. Trimeric rhodopsins exhibit bilobed shape spectra with a positive peak at the short-wavelength side of the λmax and a negative peak on the long-wavelength side of λmax. In pentameric rhodopsins, the different mutual orientation of the retinals gives rise to an inverted bilobe shape, with the negative and positive peak on the short- and long-wavelength sides of λmax, respectively. We measured the visible CD spectra of detergent-solubilized MastR, which showed a positive peak at ∼510 nm and a negative peak at ∼560 nm relative to its λmax of 540 nm, indicating that trimers also form in n-dodecyl β-d-maltopyranoside (DDM) micelles (Fig. S9).
      The trimeric assembly found in the MastR crystal is commonly observed in crystals and purple membranes of archaeal rhodopsins such as BR (
      • Luecke H.
      • Schobert B.
      • Richter H.T.
      • Cartailler J.P.
      • Lanyi J.K.
      Structure of bacteriorhodopsin at 1.55 A resolution.
      ,
      • Henderson R.
      The structure of the purple membrane from Halobacterium hallobium: analysis of the X-ray diffraction pattern.
      ), HR (
      • Kolbe M.
      • Besir H.
      • Essen L.O.
      • Oesterhelt D.
      Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution.
      ), and archaerhodopsin (
      • Yoshimura K.
      • Kouyama T.
      Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2.
      ). A BR-like trimer was also found for Anabaena sensory rhodopsin (ASR) by solid-state NMR (
      • Wang S.
      • Munro R.A.
      • Shi L.
      • Kawamura I.
      • Okitsu T.
      • Wada A.
      • Kim S.Y.
      • Jung K.H.
      • Brown L.S.
      • Ladizhansky V.
      Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein.
      ). Interestingly, the interactions at the MastR intermonomer interface appear to be very strong, like for the ASR trimer (
      • Wang S.
      • Munro R.A.
      • Shi L.
      • Kawamura I.
      • Okitsu T.
      • Wada A.
      • Kim S.Y.
      • Jung K.H.
      • Brown L.S.
      • Ladizhansky V.
      Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein.
      ) and are stronger than for BR because ASR and MastR remain as a trimer in detergent (Fig. S9), whereas BR dissociates into monomers (
      • Dencher N.A.
      • Heyn M.P.
      Formation and properties of bacteriorhodopsin monomers in the non-ionic detergents octyl-β-d-glucoside and Triton X-100.
      ). This might explain why in similar bicelle crystallization conditions MastR formed a trimeric assembly, whereas BR crystallized as monomer (
      • Faham S.
      • Bowie J.U.
      Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure.
      ).
      The BR-like quaternary structure of MastR is in contrast to pentameric assemblies found for eubacterial ClRs such as chloride-pumping Fulvimarina rhodopsin (FR) (
      • Inoue K.
      • Koua F.H.
      • Kato Y.
      • Abe-Yoshizumi R.
      • Kandori H.
      Spectroscopic study of a light-driven chloride ion pump from marine bacteria.
      ), for which atomic force microscopy revealed a pentamer (
      • Shibata M.
      • Inoue K.
      • Ikeda K.
      • Konno M.
      • Singh M.
      • Kataoka C.
      • Abe-Yoshizumi R.
      • Kandori H.
      • Uchihashi T.
      Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy.
      ), as well as eubacterial sodium NDQ pumps (
      • Kwon S.K.
      • Kim B.K.
      • Song J.Y.
      • Kwak M.J.
      • Lee C.H.
      • Yoon J.H.
      • Oh T.K.
      • Kim J.F.
      Genomic makeup of the marine flavobacterium Nonlabens (Donghaeana) dokdonensis and identification of a novel class of rhodopsins.
      ) such as Krokinobacter eikastus rhodopsin 2 (KR2) (
      • Inoue K.
      • Ono H.
      • Abe-Yoshizumi R.
      • Yoshizawa S.
      • Ito H.
      • Kogure K.
      • Kandori H.
      A light-driven sodium ion pump in marine bacteria.
      ) and eubacterial proton pumps Gloeobacter rhodopsin (GR) (
      • Morizumi T.
      • Ou W.L.
      • Van Eps N.
      • Inoue K.
      • Kandori H.
      • Brown L.S.
      • Ernst O.P.
      X-ray crystallographic structure and oligomerization of gloeobacter rhodopsin.
      ) and proteorhodopsin (PR) (
      • Ran T.
      • Ozorowski G.
      • Gao Y.
      • Sineshchekov O.A.
      • Wang W.
      • Spudich J.L.
      • Luecke H.
      Cross-protomer interaction with the photoactive site in oligomeric proteorhodopsin complexes.
      ). The crystal structures of the eubacterial Nonlabens marinus ClR (NmClR/NM-R3) (
      • 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.
      ,
      • Hosaka T.
      • Yoshizawa S.
      • Nakajima Y.
      • Ohsawa N.
      • Hato M.
      • DeLong E.F.
      • Kogure K.
      • Yokoyama S.
      • Kimura-Someya T.
      • Iwasaki W.
      • Shirouzu M.
      Structural mechanism for light-driven transport by a new type of chloride ion pump, Nonlabens marinus rhodopsin-3.
      ) and sodium pumps (KR2) (
      • Kato H.E.
      • Inoue K.
      • Abe-Yoshizumi R.
      • Kato Y.
      • Ono H.
      • Konno M.
      • Hososhima S.
      • Ishizuka T.
      • Hoque M.R.
      • Kunitomo H.
      • Ito J.
      • Yoshizawa S.
      • Yamashita K.
      • Takemoto M.
      • Nishizawa T.
      • et al.
      Structural basis for Na+ transport mechanism by a light-driven Na+ pump.
      ,
      • Kovalev K.
      • Polovinkin V.
      • Gushchin I.
      • Alekseev A.
      • Shevchenko V.
      • Borshchevskiy V.
      • Astashkin R.
      • Balandin T.
      • Bratanov D.
      • Vaganova S.
      • Popov A.
      • Chupin V.
      • Büldt G.
      • Bamberg E.
      • Gordeliy V.
      Structure and mechanisms of sodium-pumping KR2 rhodopsin.
      ) have as a common feature an extended helix B and a 3-omega motif, which result in a flip of the B–C loop in the direction of helices A and B (Fig. 3B). It has been suggested that the flipped orientation of the B–C loop supports the specific pentameric assembly in eubacterial pumps (
      • Morizumi T.
      • Ou W.L.
      • Van Eps N.
      • Inoue K.
      • Kandori H.
      • Brown L.S.
      • Ernst O.P.
      X-ray crystallographic structure and oligomerization of gloeobacter rhodopsin.
      ). MastR does not have these pentamer-inducing structural features and exists as a trimer accordingly.
      When the structures of known chloride pumps are compared with MastR and BR, major differences are noticeable at the extracellular side where the Cl ion entrance is located (Fig. 4). Although the transmembrane regions of BR, HRs, and MastR all look very similar (but quite different from ClR), the length of the B–C loops and size of the β-sheets varies largely, leading to different shapes of the chloride entrance. An ion entrance pore is developed best for MastR as indicated by the solvent-accessible inlet shown in Fig. 4. This is due to MastR having the shortest B–C loop, even shorter than the proton pump BR with which MastR shows best overall structural homology on the extracellular side. In contrast, HRs have long B–C loops that cover most of the extracellular surface, and only small solvent-accessible areas are found that serve as inlets for ions. Natronomonas pharaonis halorhodopsin (NpHR) has the longest B–C loop, which together with an additional N-terminal helix running parallel the membrane forms a hydrophobic cap that was proposed to prevent a rapid exchange of charged ions between the active center and the extracellular medium (
      • Kouyama T.
      • Kanada S.
      • Takeguchi Y.
      • Narusawa A.
      • Murakami M.
      • Ihara K.
      Crystal structure of the light-driven chloride pump halorhodopsin from Natronomonas pharaonis.
      ). A histidine residue in the middle of HsHR's long B–C loop is critical for the anion-pumping mechanism (
      • Otomo J.
      Anion selectivity and pumping mechanism of halorhodopsin.
      ). The B–C loop thus appears to be an element that chloride pumps use to regulate the entrance of anions.
      Figure thumbnail gr4
      Figure 4Topology and cavity comparison of MastR with various ion pumps. Overlay of MastR (pink) with BR (PDB code 1c3w; RMSD, 0.72 Å), HsHR (PDB code 5ahy; RMSD, 1.08 Å), NpHR (PDB code 3a7k; RMSD, 1.05 Å), and NmClR (PDB code 5g28; RMSD, 7.15 Å) reveals that MastR has the best overlap with proton-pumping BR. Cl ions (green spheres) are only shown for the labeled pump. Cavities (red) and the extracellular solvent-accessible inlet (yellow) are traced out with spheres calculated by Hollow (
      • Ho B.K.
      • Gruswitz F.
      HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures.
      ). MastR has short intracellular loops, especially the B–C loop (red for MastR and blue for others), which creates a solvent-accessible pore that permeates deep into the extracellular side. On the intracellular side, MastR has the fewest and smallest cavities of the proteins compared.

      Putative chloride ion transport pathway

      The putative chloride pathway is shown in Fig. 5. In the ground state MastR structure, there is an open tunnel leading into a water-filled cavity. The tunnel is located between the B–C and F-G loops and is likely to serve as the chloride entrance. This tunnel is pinched by Glu-192 and Glu-182, which may act as a regulatory gateway, similar to the proton release pathway regulated by the homologous Glu-204 and Glu-194 in BR (
      • Garczarek F.
      • Brown L.S.
      • Lanyi J.K.
      • Gerwert K.
      Proton binding within a membrane protein by a protonated water cluster.
      ). Similar to Glu-194 of BR, Glu-182 of MastR seems to be electrostatically coupled to the Schiff base region, because its replacement strongly affects chloride affinity (
      • Hasemi T.
      • Kikukawa T.
      • Watanabe Y.
      • Aizawa T.
      • Miyauchi S.
      • Kamo N.
      • Demura M.
      Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin.
      ). Its transient protonation in the photocycle may be required for chloride uptake. Additionally, a mutation of Glu-192 was shown to shift the N/O state equilibrium toward the N-state and slows down the photocycle (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ), which suggests slower chloride uptake from the extracellular environment. This suggested entrance is in accord with the T193A and T197A mutant phenotypes (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ), which show no photocycle changes, because these residues are not near the entrance cavity and are located between helices A and G.
      Figure thumbnail gr5
      Figure 5Putative Cl ion transport pathway. Shown is the structure of MastR displaying key residues, retinal bound to Lys-204 (cyan), Cl ions (green spheres), water molecules (red spheres), internal cavities (gray surfaces) containing a water molecule, and the extracellular pore (yellow surface) containing water molecules. The seven transmembrane α-helices and a short β-sheet on the B–C loop are shown in pink. The TSD motif (Thr-74, Ser-78, Asp-85) is displayed in black. Thr-74 and Ser-78 stabilize the Schiff base Cl ion. Asp-85, Asn-39, and Ser-211 connect helices C, B, and G, respectively, through interhelical hydrogen bonding. Residues surrounding the cavities (calculated using Hollow (
      • Ho B.K.
      • Gruswitz F.
      HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures.
      )) are shown in gray for nonpolar side chains and pink for polar side chains. The Schiff base cavity possesses a Cl and water, whereas both cytoplasmic cavities possess only a single water molecule.
      In the dark, the Cl ion, stabilized by residues Thr-74 and Ser-78, together with a single water molecule, fills a small cavity close to the PRSB. After retinal photoisomerization, this Cl ion should move toward the cytoplasmic side, which has two small cavities, with only a single water molecule in each. We propose that the chloride is more likely to move toward the slightly larger cytoplasmic cavity, which is surrounded by polar residues His-166, Ser-203, and Trp-170, which are good candidates for binding chloride. This is in contrast with the other smaller cavity, which only possesses Cys-43 and Asn-39 as potential chloride interaction partners. It was previously shown that the H166A mutation results in much slower decay of the L2 and N states, which slows down the overall photocycle turnover by a factor of >10 (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ). Thus, His-166 is potentially part of the chloride release pathway, similar to the homologous conserved threonine of HRs (
      • Rüdiger M.
      • Oesterhelt D.
      Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.
      ). The His-166 forms a stacking interaction with Trp-170, which may also contribute to slower dynamics in the H166A mutant, because the homologous Trp residue is known to play a key role in isomerization-induced conformational changes in other microbial rhodopsins (
      • Lanyi J.K.
      • Schobert B.
      Local-global conformational coupling in a heptahelical membrane protein: transport mechanism from crystal structures of the nine states in the bacteriorhodopsin photocycle.
      ).
      Next to the other cytoplasmic cavity, we observe a hydrogen-bonding network comprising Asp-85, Asn-39, and Ser-211, which connect helix C, B, and G, respectively. A particularly strong interhelical hydrogen bond is observed between Asp-85 and Asn-39 (2.7 Å distance between the heavy atoms), which is consistent with the interaction of these residues suggested by FTIR results obtained earlier (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ). In the same work, it was observed that Asp-85 deprotonates in response to the chloride translocation and must be reprotonated in the last step for the photocycle to complete. The requirement for Asp-85 reprotonation suggests that it may be used to prevent the backflow of chloride. From the structure, it seems likely that the protonation state of Asp-85 is regulated by its interaction with Asn-39 and Ser-211, which may change as a result of changing interhelical distances originating from light-induced conformational changes (such as helical tilts characteristic for rhodopsins).
      Interestingly, FTIR revealed that there is an accompanying long-living perturbation of a buried cysteine (hydrogen bond weakening), which was not assigned conclusively. There are three cysteines in MastR: Cys-43, Cys-77, and Cys-130 (Fig. S10).
      Cys-77 is located close to the function-determining motif forming Thr-74 and Ser-78 and may form a weak intrahelical bond with the carbonyl oxygen of Val-73 (distance between the heavy atoms is 3.6 Å). This bond may be further weakened upon structural perturbations accompanying Cl ion translocation toward the cytoplasmic side. Cys-130 is located next to the β-ionone ring of retinal and does not appear to be involved in hydrogen bonding. In view of the discussed transient deprotonation of the neighboring Asp-85–Asn-39 complex, it is also tempting to ascribe the cysteine perturbation found by FTIR to Cys-43, which shares the water cavity with Asn-39.
      Finally, we propose that the Cl ion is released through the pathway delineated by Ser-211 and Thr-214. Although these residues are not essential for transport, the photocycles of S211A and T214A show an accumulation of the O state and slightly faster photocycle turnover. Moreover, Ser-211 is conserved among cyanobacterial ion pumps but is nonpolar in BR and HRs (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ). Along the Ser-211 and Thr-214 trajectory are residues Thr-217 and Ser-155, which may assist in Cl ion transportation. A clear understanding of the chloride transport and pathway will, however, require structural information on the photocycle intermediates.

      Retinal-binding pocket of MastR and MastR-T74D

      The most significant structural differences between MastR and MastR-T74D occur adjacent to the retinal chromophore as shown in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6. In the MastR structure, a Cl ion is located between the side-chain hydroxyl groups of Thr-74 (dCl-O = 3.1 Å) and Ser-78 (dCl-O = 3.0 Å) of the TSD motif. This Cl ion is part of an extended hydrogen-bonding network in which the Cl ion is connected to the Thr-74 and Ser-78 side chains, as well as via an adjacent water molecule (dCl-O = 3.1 Å) to the PRSB (dPRSB-H2O = 2.8 Å) and the carboxyl group of Asp-200. The Asp-200 and Arg-71 side chains further form an electrostatic interaction (Fig. 6A).
      Figure thumbnail gr6
      Figure 6Comparison of the hydrogen-bonding network around the protonated retinal Schiff base. A, MastR (PDB code 6xl3). B, chloride pumps HsHR (PDB code 5ahy), NpHR (PDB code 3ak7), and NmClR (PDB code 5g28). C, proton pump MastR-T74D mutant (PDB code 6wp8). D, proton pumps BR (PDB code 1c3w), Exiguobacterium sibiricum rhodopsin (PDB code 4hyj), and GR (PDB code 6nwd). Retinal and residues of the hydrogen-bonded PRSB network are shown as a stick model. Water is shown as red spheres, and chloride is shown as green spheres. Hydrogen bonding distances are indicated in angstrom (Å), with distances ≤3 Å shown in blue and those >3 Å shown in pink.
      The MastR-T74D structure, in contrast, contains no Cl ion in the retinal-binding pocket. The carboxylate side chain of Asp-74 fills the space where the Cl ion is located in MastR and takes over the function of the Cl ion in the extended hydrogen-bonding network. The electrostatic interaction between Asp-200 and Arg-71, however, is weakened, because the Arg-71 guanidinium group flipped away from Asp-200 (Fig. 6C). This is in line with the observed positions for the homologous Arg side chains in other proton (Fig. 6D) and chloride pumps (Fig. 6B), in which it points toward the extracellular surface for the former and toward retinal for the latter. The different electrostatic environments of the PRSB in WT and mutant result in different absorption spectra with maxima of 522 nm for MastR-T74D and 540 nm for MastR, respectively (Fig. S4C) (
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ).

      Functional conversion of chloride pumps

      A distinctive feature of MastR is that a change of the TSD motif to DSD (T74D mutation) converts the chloride pump into proton pump (
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ). However, functional conversion of archaeal HsHR and NpHR chloride pumps into proton pumps by altering their TSA motif failed. The introduction of single or multiple mutations to generate various putative proton pumps with an aspartate as proton acceptor yielding DSA, DSD, or DTD motifs were unsuccessful (
      • Muroda K.
      • Nakashima K.
      • Shibata M.
      • Demura M.
      • Kandori H.
      Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps.
      ,
      • Havelka W.A.
      • Henderson R.
      • Oesterhelt D.
      Three-dimensional structure of halorhodopsin at 7 A resolution.
      ,
      • Váró G.
      • Brown L.S.
      • Needleman R.
      • Lanyi J.K.
      Proton transport by halorhodopsin.
      ), although the DTD motif is characteristic for BR proton pump. On the other hand, conversion of BR in the opposite direction, i.e. H+ to Cl ion pump, by changing the motif from DTD to TSD (D85T mutant) was successful, even though the obtained chloride pump was fairly inefficient (
      • Sasaki J.
      • Brown L.S.
      • Chon Y.S.
      • Kandori H.
      • Maeda A.
      • Needleman R.
      • Lanyi J.K.
      Conversion of bacteriorhodopsin into a chloride ion pump.
      ,
      • Tittor J.
      • Haupts U.
      • Haupts C.
      • Oesterhelt D.
      • Becker A.
      • Bamberg E.
      Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein.
      ). Similarly, for eubacterial pumps, conversion was only possible in one direction. For FR, an NTQ-motif chloride pump, mutation of NTQ to DTE converted FR to a proton pump, but opposite conversion of GR, a DTE-motif proton pump, into a chloride pump by mutation of DTE to NTQ failed (
      • Inoue K.
      • Nomura Y.
      • Kandori H.
      Asymmetric functional conversion of eubacterial light-driven ion pumps.
      ). The possibility of functional conversion implied that the pumps have a common fundamental transport mechanism and shed light on the evolutionary conserved residues of proton and chloride pumps across all domains of life (
      • Brown L.S.
      • Jung K.H.
      Bacteriorhodopsin-like proteins of eubacteria and fungi: the extent of conservation of the haloarchaeal proton-pumping mechanism.
      ,
      • Ihara K.
      • Umemura T.
      • Katagiri I.
      • Kitajima-Ihara T.
      • Sugiyama Y.
      • Kimura Y.
      • Mukohata Y.
      Evolution of the archaeal rhodopsins: evolution rate changes by gene duplication and functional differentiation.
      ,
      • Sharma A.K.
      • Spudich J.L.
      • Doolittle W.F.
      Microbial rhodopsins: functional versatility and genetic mobility.
      ).
      A structural comparison of proton and chloride pumps can provide insights into why some conversions are possible but others are not (Fig. 6). The extended hydrogen-bonding network linked to the PRSB is at the heart of the pump, because upon retinal isomerization, the N–H dipole of the PRSB will flip in chloride pumps, and for proton-pumping rhodopsins the PRSB proton will even dissociate for proton transfer to the proton acceptor. The geometry of the extended hydrogen-bonding network defines the starting point in the pumping cycle and is thus a determinant of pump function.
      When we compare the hydrogen-bonding networks of proton pumps (Fig. 6, C and D), we find that in all cases, a central water molecule connects the PRSB and the carboxyl side chains of the proton acceptor (Asp-85 in BR) and a conserved aspartate (Asp-212 in BR (
      • Kandori H.
      Ion-pumping microbial rhodopsins.
      )). Characteristic is that these hydrogen bonds are strong, having distances between the heavy atoms shorter than or equal to 2.9 Å. In some proton pumps the hydrogen-bonding network is extended by additional water molecule(s) and polar side chains including the second amino acid of the function-determining motif. For the chloride pumps the extended hydrogen-bonding network is similar but in addition contains a Cl ion between the first two amino acids of the function-determining motif, which is bridged via a water molecule to the PRSB (Fig. 6, A and B). It is striking that the hydrogen bonds are weaker, especially the bond between the central water and PRSB, which is ∼3.5 Å for archaeal HRs and eubacterial NmClR. For MastR the hydrogen-bonding network is tighter, more similar to proton pumps, which might (together with the overall structural similarity between MastR and the proton pump BR) be an explanation for why the Cl to H+ ion pump conversion was successful. Strong hydrogen bonding of the PRSB water was concluded from FTIR studies to be a prerequisite for proton pumping and is consistent with the structural data on the PRSB hydrogen-bonding networks in Fig. 6 (
      • Muroda K.
      • Nakashima K.
      • Shibata M.
      • Demura M.
      • Kandori H.
      Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps.
      ).
      The position of the PRSB water in the hydrogen-bonding network seems to be crucial for a proton pump, because it constrains proper geometry between the aspartate proton acceptor and the PRSB, which affects the pKa of the acceptor group. A proper pKa is important for a proton transfer event in later stages of the photocycle. By comparing the structures, we find that Ala-53 in BR is an important determinant to achieve the correct geometry of the PRSB hydrogen-bonding network. This residue is a serine in the non-outward proton transporting families including HRs, Xenorhodopsins (such as ASR) (
      • Inoue K.
      • Ito S.
      • Kato Y.
      • Nomura Y.
      • Shibata M.
      • Uchihashi T.
      • Tsunoda S.P.
      • Kandori H.
      A natural light-driven inward proton pump.
      ), and Schizorhodopsins (
      • Inoue K.
      • Tsunoda S.P.
      • Singh M.
      • Tomida S.
      • Hososhima S.
      • Konno M.
      • Nakamura R.
      • Watanabe H.
      • Bulzu P.A.
      • Banciu H.L.
      • Andrei A.S.
      • Uchihashi T.
      • Ghai R.
      • Béjà O.
      • Kandori H.
      Schizorhodopsins: a family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps.
      ) (Fig. S3). 25 years ago, it was proposed that the short side chain of Ala-53 is crucial to allow changes of the PRSB–Counterion complex geometry during the photocycle to enable proton transfer to the acceptor (
      • Brown L.S.
      • Gat Y.
      • Sheves M.
      • Yamazaki Y.
      • Maeda A.
      • Needleman R.
      • Lanyi J.K.
      The retinal Schiff base-counterion complex of bacteriorhodopsin: changed geometry during the photocycle is a cause of proton transfer to aspartate 85.
      ). Indeed, decreasing the alignment of PRSB and Asp-85 in BR by the A53V mutation almost abolished formation of the M state and resulted in disappearance of the hydroxyl stretch band of the strongly bound water in the L state. The A53S mutation completely abolished the M state.
      L. S. Brown, R. Needleman, and J. K. Lanyi, unpublished observations.
      The available structures of various proton and chloride pumps and our newly added MastR structures confirm the importance of the geometry in the PRSB hydrogen-bonding network. It appears that the serine side chain in HRs may displace the Schiff base water away from the Schiff base, preventing their successful conversion to proton pumps.
      It is important to note that there must be additional determinants for the successful conversion of pump function. In an attempt to convert NpHR to a proton pump, Muroda et al. (
      • Muroda K.
      • Nakashima K.
      • Shibata M.
      • Demura M.
      • Kandori H.
      Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps.
      ) generated two mutants with six or ten replacements to yield sequence conservation patterns including BR's DTD motif and the Ala-53 equivalent. A functional proton pump, however, could not be generated, pointing at the additional fine-tuning required to achieve proper conformational changes and proton affinities in the ion-transporting photocycle.
      In an extensive pump interconversion study comprising proton, chloride, and sodium pumps (
      • Inoue K.
      • Nomura Y.
      • Kandori H.
      Asymmetric functional conversion of eubacterial light-driven ion pumps.
      ), the authors describe several examples of asymmetric functional conversion in which conversion was only possible when mutation reversed the evolutionary amino acid changes in the function-determining motif. In case of converting the FR chloride pump into a proton pump and increasing the proton pump characteristic accumulation of the M state, the S255F mutation outside the PRSB hydrogen-bonding network was required in addition. It has been proposed that MastR evolved from a proton pump (
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ,
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ). The high structural similarity of MastR to BR and the conservation of key functional residues (Fig. S3) explains why additional mutations were not necessary for the generation of an effective proton pump.

      Conclusion

      The crystal structures of MastR and MastR-T74D gave insight into the ion transport pathway and the mechanism of this new group of chloride ion pumps with TSD motif. MastR resembles and most likely evolved from an archaeal proton pump, explaining why the T74D mutation enabled functional conversion from Cl to H+ pump. The distinct hydrogen-bonding network including a strongly bound water molecule between the PRSB and Asp-74 is a prerequisite for the successful conversion. Proton transfer requires the correct pKa of proton acceptor Asp-74, which is achieved by a suitable geometry of the hydrogen-bonding network. In the future, it will be interesting to study MastR T74D photocycle intermediates to determine the proton acceptor pKa and understand details of the ion transport steps. In addition, because of MastR's slow photocycle (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ) (Fig. S11) and crystallization in bicelles, this chloride pump should be suitable for time-resolved serial synchrotron X-ray crystallography using fixed-target crystal delivery systems (
      • Schulz E.C.
      • Mehrabi P.
      • Müller-Werkmeister H.M.
      • Tellkamp F.
      • Jha A.
      • Stuart W.
      • Persch E.
      • De Gasparo R.
      • Diederich F.
      • Pai E.F.
      • Miller R.J.D.
      The hit-and-return system enables efficient time-resolved serial synchrotron crystallography.
      ,
      • Wierman J.L.
      • Paré-Labrosse O.
      • Sarracini A.
      • Besaw J.E.
      • Cook M.J.
      • Oghbaey S.
      • Daoud H.
      • Mehrabi P.
      • Kriksunov I.
      • Kuo A.
      • Schuller D.J.
      • Smith S.
      • Ernst O.P.
      • Szebenyi D.M.E.
      • Gruner S.M.
      • et al.
      Fixed-target serial oscillation crystallography at room temperature.
      ).

      Experimental procedures

      Gene construction, protein expression, and purification

      The plasmid for expression of MastR was described previously (
      • Harris A.
      • Saita M.
      • Resler T.
      • Hughes-Visentin A.
      • Maia R.
      • Pranga-Sellnau F.
      • Bondar A.N.
      • Heberle J.
      • Brown L.S.
      Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
      ). The gene encoding MastR was cloned into pET21a(+) vector via NdeI–XhoI restriction sites, which added a C-terminal hexahistidine tag. The MastR-T74D plasmid was prepared from the MastR plasmid using a QuikChange Lightning site-directed mutagenesis kit (Agilent). The mutation was confirmed by DNA sequencing (ACGT Corp, Toronto, Canada).
      For protein expression, chemically competent E. coli OverExpressTM C43(DE3) cells (Lucigen) were transformed with MastR or MastR-T74D plasmid. A single colony starter culture was used to inoculate several 1-liter cultures (Miller LB broth, 100 μg ml−1 ampicillin, 4-liter nonbaffled conical flask). The cultures were incubated (2 h, 37 °C, 220 rpm) and induced at an A600 nm between 0.6 and 0.8 by adding 0.5 mm isopropyl-β-d-thiogalactopyranoside (BioShop), then supplemented with 5 μm all-trans-retinal (Sigma–Aldrich), and further incubated (37 °C, 220 rpm). After 5 h the cells were harvested through centrifugation (Beckman rotor JLA-8.1, 30 min, 4 °C, 5,000 × g) yielding 3.2 ± 0.2 g of pellet per liter of cell culture. Four pellets were resuspended in 100 ml of buffer A (50 mm MES, pH 6.5, 300 mm NaCl) and then combined with a SIGMAFASTTM protease inhibitor mixture tablet (Sigma–Aldrich). The cells were lysed using an Emulsiflex C3 (Avestin, Ottawa, Canada) homogenizer (three passages, 4 °C, 15,000 p.s.i.), and the lysate was cleared by centrifugation (Eppendorf 5810R, 30 min, 4 °C, 4,600 × g). The cloudy suspension was carefully decanted, whereas the pellet was discarded. The crude membranes were prepared by ultracentrifugation (Beckman rotor 45Ti, 1 h, 4 °C, 125,000 × g). The pellet was resuspended in 10 ml of buffer A using a chilled glass/Teflon Potter–Elvehjem homogenizer. MastR protein was solubilized with 2% (w/v) DDM (Glycon, Luckenwalde, Germany) using a rotator (4 °C, 30 rpm, overnight). Nonsolubilized material was removed by ultracentrifugation (Beckman rotor 45Ti, 1 h, 4 °C, 125,000 × g).
      For purification, the bright red MastR-containing solution was filtered (0.22 μm, Millipore), equilibrated with 20 mm of imidazole, and loaded onto a HisTrapTM 1-ml nickel–nitrilotriacetic acid column (GE Healthcare). The column was washed with 10 column volumes (CV) of equilibration buffer (0.1% (w/v) DDM, 20 mm imidazole, buffer A). The DDM detergent was reduced to 0.05% (10 CV of 0.05% DDM, 20 mm imidazole, buffer A), and then exchanged to n-octyl β-d-glucopyranoside (OG) (20 CV of 2% OG, 20 mm imidazole, buffer A). The protein was eluted with 30 CV of a linear imidazole gradient (20–750 mm imidazole, buffer A, 1% OG). Elution was monitored at 280 and 537 nm (or 522 nm for MastR-T74D). Consecutive fractions of MastR in OG with sufficient purity (A537nm/A280nm > 0.6, eluted at ∼400–450 mm imidazole) were combined and concentrated to 10 mg/ml using a 30-kDa concentrator (Amicon). Gel filtration was performed on 1 ml of concentrated protein loaded on a SuperdexTM 200 10/300 GL column (GE Healthcare) using buffer B (10 mm MES, pH 6.5, 300 mm NaCl, 0.8% OG) at a rate of 0.3 ml min−1 with 0.4-ml fractions collected. Consecutive fractions with A537nm/A280nm > 0.6 were combined and prepared for crystallization. The obtained protein had a purity of >95% as analyzed by Coomassie-stained SDS-PAGE (Fig. S4, A and B).

      Crystallization and harvesting

      24% (w/v) bicelles composed of 2.8:1 1,2-dimyristoyl-sn-glycero-3-phosphocholine:CHAPSO were prepared in advance as previously described (
      • Agah S.
      • Faham S.
      Crystallization of membrane proteins in bicelles.
      ) and were stored at –20°C. Prior to crystallization, the bicelles were thawed at room temperature and then kept on ice. To crystallize MastR, the protein was first concentrated to ∼15 mg/ml assuming ε537 = 40,000 m−1 cm−1 (
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ). The protein and bicelles were combined in a 2:1 ratio (resulting in 10 mg/ml protein and 8% (w/v) bicelle) and incubated on ice for 2 h. The precipitant solution was prepared (pH 4.6, 3.6 m sodium phosphate monobasic monohydrate (Hampton Research), 180 mm 1,6-hexanediol, 3.5% triethylene glycol). Hanging-drop vapor-diffusion crystallization experiments were set up on standard pregreased 24-well crystallization trays. The 6 μl of hanging drop (4 μl of MastR-bicelle mixture, 1.5 μl of precipitant solution, 0.5 μl of 1% OG) was mixed 10 times on a thick siliconized cover slide and held over 0.5 ml of precipitant solution in the reservoir. Crystal trays were stored at 34 °C and left undisturbed for 3 days. The crystals appeared after 2–3 days and reached a maximum size of 50–150 μm in 4–6 days. The crystallization of MastR-T74D is nearly identical, with the only difference being the pH of the crystallization buffer (pH 4.0).
      To prevent dehydration while harvesting crystals, 6 μl of the precipitant solution was immediately pipetted onto the sample, followed by 6 μl of precipitant solution containing 12.5% ethylene glycol as cryoprotectant. The glass slide containing the hanging drop was placed on an ice pack to make the drop more fluid. After 90 s of incubation, single crystals were harvested using MicroLoop LDTM 50–200 μm (MiTeGen) and then flash frozen in liquid nitrogen.

      Bromide soaking experiment

      After MastR crystals had grown, the glass cover was removed, and 6 μl of 0.1 m sodium bromide in precipitant solution was added to the hanging drop. The cover was quickly resealed and then stored (10 min, 20 °C). The crystals were then soaked in cryoprotectant (5 min, 6 μl of precipitant solution with 12.5% ethylene glycol) and then flash frozen with liquid nitrogen.

      Data collection and analysis

      X-ray diffraction experiments were carried out on Beamline 23-ID-B of the Advanced Photon Source (APS) at Argonne National Laboratory (Lemont, IL, USA). The data were collected at 100 K using a 1.0332 Å, 20 × 20-μm X-ray beam that was attenuated by a factor of 5. Reflections were collected every 0.2° (0.4 s) for a total of 60° using a Dectris Eiger X 16M detector operated in continuous, shutterless data collection mode at a distance of 250 mm. For MastR-T74D, data from a single crystal was employed. For MastR, a single, large, hexagonal crystal was measured in three separate spots, and the resulting data were merged in data processing. Diffraction data were processed using XDS (
      • Kabsch W.
      XDS.
      ) and scaled using AIMLESS (
      • Evans P.R.
      • Murshudov G.N.
      How good are my data and what is the resolution?.
      ) from the CCP4i program package (
      • Winn M.D.
      • Ballard C.C.
      • Cowtan K.D.
      • Dodson E.J.
      • Emsley P.
      • Evans P.R.
      • Keegan R.M.
      • Krissinel E.B.
      • Leslie A.G.
      • McCoy A.
      • McNicholas S.J.
      • Murshudov G.N.
      • Pannu N.S.
      • Potterton E.A.
      • Powell H.R.
      • et al.
      Overview of the CCP4 suite and current developments.
      ). The MastR structure was solved by molecular replacement with Balbes (
      • Long F.
      • Vagin A.A.
      • Young P.
      • Murshudov G.N.
      BALBES: a molecular-replacement pipeline.
      ) using archaerhodopsin-1 (
      • Enami N.
      • Yoshimura K.
      • Murakami M.
      • Okumura H.
      • Ihara K.
      • Kouyama T.
      Crystal structures of archaerhodopsin-1 and -2: common structural motif in archaeal light-driven proton pumps.
      ) as a search model (PDB entry 1UAZ). The MastR-T74D structure was solved by molecular replacement using MastR as the starting model. The model was manually built in Coot (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ), and iterative refinement was done with the phenix.refine routine of Phenix (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). Refinement statistics are summarized in Table S1. Figures with three-dimensional structures were prepared using PyMOL (The Pymol Molecular Graphics System, Version 1.8.0.5, Schrödinger, LLC).

      CD spectroscopy

      CD spectra were recorded as described previously (
      • Ward M.E.
      • Wang S.
      • Munro R.
      • Ritz E.
      • Hung I.
      • Gor'kov P.L.
      • Jiang Y.
      • Liang H.
      • Brown L.S.
      • Ladizhansky V.
      In situ structural studies of Anabaena sensory rhodopsin in the E. coli membrane.
      ). In brief, the spectra were collected at 20 °C with a 10-mm-path-length quartz microcell on a model no. J-810 spectropolarimeter (JASCO, Easton, MD, USA). A spectral range of 700–400 nm with a scanning speed of 50 nm/min were used. Four independent accumulations were averaged.

      Flash photolysis

      Flash-photolysis spectroscopy was performed as described previously, using a custom-built single-wavelength spectrometer (
      • Waschuk S.A.
      • Bezerra Jr., A.G.
      • Shi L.
      • Brown L.S.
      Leptosphaeria rhodopsin: bacteriorhodopsin-like proton pump from a eukaryote.
      ). Briefly, 7-ns pulses of the second harmonic of an Nd-YAG laser at 532 nm (Continuum Minilite II) initiated the photocycle. Absorption changes of the monochromatic light (Oriel QTH source and two monochromators) were recorded with an Oriel photomultiplier, an amplifier with a 350 MHz bandwidth, and a Gage AD converter (CompuScope 12100-64M).

      Root-mean square deviation calculation

      The root-mean-square deviation (RMSD) was calculated using PyMOL. A sequence alignment was performed, and the Cα of aligned residues were superimposed. Five rounds of refinement were implemented, and structural outliers were discarded.

      Data availability

      The refined models have been deposited in the Protein Data Bank under codes 6xl3 (MastR) and 6wp8 (MastR-T74D). All other data that support the findings of this study are available from the corresponding author upon reasonable request.

      Acknowledgments

      We thank Emil F. Pai for discussions and helpful suggestions about determination of the MastR structure. This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office of Science User Facility operated for the Department of Energy Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357. The Eiger 16M detector at GM/CA-XSD was funded by NIH grant S10 OD012289. We specifically thank the staff at the GM/CA Beamlines 23-ID.

      Supplementary Material

      Author Profile

      References

        • Ernst O.P.
        • Lodowski D.T.
        • Elstner M.
        • Hegemann P.
        • Brown L.S.
        • Kandori H.
        Microbial and animal rhodopsins: structures, functions, and molecular mechanisms.
        Chem. Rev. 2014; 114 (24364740): 126-163
        • Kurihara M.
        • Sudo Y.
        Microbial rhodopsins: wide distribution, rich diversity and great potential.
        Biophys. Physicobiol. 2015; 12 (27493861): 121-129
        • Govorunova E.G.
        • Sineshchekov O.A.
        • Li H.
        • Spudich J.L.
        Microbial rhodopsins: diversity, mechanisms, and optogenetic applications.
        Annu. Rev. Biochem. 2017; 86 (28301742): 845-872
        • Gushchin I.
        • Gordeliy V.
        Microbial rhodopsins.
        Subcell. Biochem. 2018; 87 (29464556): 19-56
        • Klare J.P.
        • Chizhov I.
        • Engelhard M.
        Microbial rhodopsins: scaffolds for ion pumps, channels, and sensors.
        Results Probl. Cell Differ. 2008; 45 (17898961): 73-122
        • Mukherjee S.
        • Hegemann P.
        • Broser M.
        Enzymerhodopsins: novel photoregulated catalysts for optogenetics.
        Curr. Opin. Struct. Biol. 2019; 57 (30954887): 118-126
        • Kandori H.
        Ion-pumping microbial rhodopsins.
        Front. Mol. Biosci. 2015; 2 (26442282): 52
        • Pinhassi J.
        • DeLong E.F.
        • Béjà O.
        • González J.M.
        • Pedrós-Alió C.
        Marine bacterial and archaeal ion-pumping rhodopsins: genetic diversity, physiology, and ecology.
        Microbiol. Mol. Biol. Rev. 2016; 80 (27630250): 929-954
        • Brown L.S.
        • Jung K.H.
        Bacteriorhodopsin-like proteins of eubacteria and fungi: the extent of conservation of the haloarchaeal proton-pumping mechanism.
        Photochem. Photobiol. Sci. 2006; 5 (16761082): 538-546
        • Muroda K.
        • Nakashima K.
        • Shibata M.
        • Demura M.
        • Kandori H.
        Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps.
        Biochemistry. 2012; 51 (22583333): 4677-4684
        • Inoue K.
        • Nomura Y.
        • Kandori H.
        Asymmetric functional conversion of eubacterial light-driven ion pumps.
        J. Biol. Chem. 2016; 291 (26929409): 9883-9893
        • Inoue K.
        • Kato Y.
        • Kandori H.
        Light-driven ion-translocating rhodopsins in marine bacteria.
        Trends Microbiol. 2015; 23 (25432080): 91-98
        • Béjà O.
        • Lanyi J.K.
        Nature's toolkit for microbial rhodopsin ion pumps.
        Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (24737891): 6538-6539
        • Inoue K.
        • Konno M.
        • Abe-Yoshizumi R.
        • Kandori H.
        The role of the NDQ motif in sodium-pumping rhodopsins.
        Angew. Chem. Int. Ed. Engl. 2015; 54 (26215709): 11536-11539
        • Engelhard C.
        • Chizhov I.
        • Siebert F.
        • Engelhard M.
        Microbial halorhodopsins: light-driven chloride pumps.
        Chem. Rev. 2018; 118 (29882660): 10629-10645
        • Kouyama T.
        • Kanada S.
        • Takeguchi Y.
        • Narusawa A.
        • Murakami M.
        • Ihara K.
        Crystal structure of the light-driven chloride pump halorhodopsin from Natronomonas pharaonis.
        J. Mol. Biol. 2010; 396 (19961859): 564-579
        • Schreiner M.
        • Schlesinger R.
        • Heberle J.
        • Niemann H.H.
        Structure of halorhodopsin from Halobacterium salinarum in a new crystal form that imposes little restraint on the E–F loop.
        J. Struct. Biol. 2015; 190 (25916754): 373-378
        • 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.
        Nat. Commun. 2016; 7 (27554809): 12677
        • Hosaka T.
        • Yoshizawa S.
        • Nakajima Y.
        • Ohsawa N.
        • Hato M.
        • DeLong E.F.
        • Kogure K.
        • Yokoyama S.
        • Kimura-Someya T.
        • Iwasaki W.
        • Shirouzu M.
        Structural mechanism for light-driven transport by a new type of chloride ion pump, Nonlabens marinus rhodopsin-3.
        J. Biol. Chem. 2016; 291 (27365396): 17488-17495
        • Hasemi T.
        • Kikukawa T.
        • Kamo N.
        • Demura M.
        Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
        J. Biol. Chem. 2016; 291 (26578511): 355-362
        • Niho A.
        • Yoshizawa S.
        • Tsukamoto T.
        • Kurihara M.
        • Tahara S.
        • Nakajima Y.
        • Mizuno M.
        • Kuramochi H.
        • Tahara T.
        • Mizutani Y.
        • Sudo Y.
        Demonstration of a light-driven SO4(2-) transporter and its spectroscopic characteristics.
        J. Am. Chem. Soc. 2017; 139 (28257611): 4376-4389
        • Nakajima Y.
        • Tsukamoto T.
        • Kumagai Y.
        • Ogura Y.
        • Hayashi T.
        • Song J.
        • Kikukawa T.
        • Demura M.
        • Kogure K.
        • Sudo Y.
        • Yoshizawa S.
        Presence of a haloarchaeal halorhodopsin-like Cl pump in marine bacteria.
        Microbes Environ. 2018; 33 (29553064): 89-97
        • Harris A.
        • Saita M.
        • Resler T.
        • Hughes-Visentin A.
        • Maia R.
        • Pranga-Sellnau F.
        • Bondar A.N.
        • Heberle J.
        • Brown L.S.
        Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.
        Phys. Chem. Chem. Phys. 2018; 20 (29057415): 3184-3199
        • Sasaki J.
        • Brown L.S.
        • Chon Y.S.
        • Kandori H.
        • Maeda A.
        • Needleman R.
        • Lanyi J.K.
        Conversion of bacteriorhodopsin into a chloride ion pump.
        Science. 1995; 269 (7604281): 73-75
        • Faham S.
        • Bowie J.U.
        Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure.
        J. Mol. Biol. 2002; 316 (11829498): 1-6
        • Hasemi T.
        • Kikukawa T.
        • Watanabe Y.
        • Aizawa T.
        • Miyauchi S.
        • Kamo N.
        • Demura M.
        Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin.
        Biochim. Biophys. Acta Bioenerg. 2019; 1860 (30529327): 136-146
        • Shibata M.
        • Inoue K.
        • Ikeda K.
        • Konno M.
        • Singh M.
        • Kataoka C.
        • Abe-Yoshizumi R.
        • Kandori H.
        • Uchihashi T.
        Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy.
        Sci. Rep. 2018; 8 (29844455): 8262
        • Luecke H.
        • Schobert B.
        • Richter H.T.
        • Cartailler J.P.
        • Lanyi J.K.
        Structure of bacteriorhodopsin at 1.55 A resolution.
        J. Mol. Biol. 1999; 291 (10452895): 899-911
        • Henderson R.
        The structure of the purple membrane from Halobacterium hallobium: analysis of the X-ray diffraction pattern.
        J. Mol. Biol. 1975; 93 (239244): 123-138
        • Kolbe M.
        • Besir H.
        • Essen L.O.
        • Oesterhelt D.
        Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution.
        Science. 2000; 288 (10827943): 1390-1396
        • Yoshimura K.
        • Kouyama T.
        Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2.
        J. Mol. Biol. 2008; 375 (18082767): 1267-1281
        • Wang S.
        • Munro R.A.
        • Shi L.
        • Kawamura I.
        • Okitsu T.
        • Wada A.
        • Kim S.Y.
        • Jung K.H.
        • Brown L.S.
        • Ladizhansky V.
        Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein.
        Nat. Methods. 2013; 10 (24013819): 1007-1012
        • Dencher N.A.
        • Heyn M.P.
        Formation and properties of bacteriorhodopsin monomers in the non-ionic detergents octyl-β-d-glucoside and Triton X-100.
        FEBS Lett. 1978; 96 (32075): 322-326
        • Inoue K.
        • Koua F.H.
        • Kato Y.
        • Abe-Yoshizumi R.
        • Kandori H.
        Spectroscopic study of a light-driven chloride ion pump from marine bacteria.
        J. Phys. Chem. B. 2014; 118 (25166488): 11190-11199
        • Kwon S.K.
        • Kim B.K.
        • Song J.Y.
        • Kwak M.J.
        • Lee C.H.
        • Yoon J.H.
        • Oh T.K.
        • Kim J.F.
        Genomic makeup of the marine flavobacterium Nonlabens (Donghaeana) dokdonensis and identification of a novel class of rhodopsins.
        Genome Biol. Evol. 2013; 5 (23292138): 187-199
        • Inoue K.
        • Ono H.
        • Abe-Yoshizumi R.
        • Yoshizawa S.
        • Ito H.
        • Kogure K.
        • Kandori H.
        A light-driven sodium ion pump in marine bacteria.
        Nat. Commun. 2013; 4 (23575682): 1678
        • Morizumi T.
        • Ou W.L.
        • Van Eps N.
        • Inoue K.
        • Kandori H.
        • Brown L.S.
        • Ernst O.P.
        X-ray crystallographic structure and oligomerization of gloeobacter rhodopsin.
        Sci. Rep. 2019; 9 (31375689): 11283
        • Ran T.
        • Ozorowski G.
        • Gao Y.
        • Sineshchekov O.A.
        • Wang W.
        • Spudich J.L.
        • Luecke H.
        Cross-protomer interaction with the photoactive site in oligomeric proteorhodopsin complexes.
        Acta Crystallogr. D Biol. Crystallogr. 2013; 69 (24100316): 1965-1980
        • Kato H.E.
        • Inoue K.
        • Abe-Yoshizumi R.
        • Kato Y.
        • Ono H.
        • Konno M.
        • Hososhima S.
        • Ishizuka T.
        • Hoque M.R.
        • Kunitomo H.
        • Ito J.
        • Yoshizawa S.
        • Yamashita K.
        • Takemoto M.
        • Nishizawa T.
        • et al.
        Structural basis for Na+ transport mechanism by a light-driven Na+ pump.
        Nature. 2015; 521 (25849775): 48-53
        • Kovalev K.
        • Polovinkin V.
        • Gushchin I.
        • Alekseev A.
        • Shevchenko V.
        • Borshchevskiy V.
        • Astashkin R.
        • Balandin T.
        • Bratanov D.
        • Vaganova S.
        • Popov A.
        • Chupin V.
        • Büldt G.
        • Bamberg E.
        • Gordeliy V.
        Structure and mechanisms of sodium-pumping KR2 rhodopsin.
        Sci. Adv. 2019; 5 (30989112): eaav2671
        • Otomo J.
        Anion selectivity and pumping mechanism of halorhodopsin.
        Biophys. Chem. 1995; 56 (7662863): 137-141
        • Garczarek F.
        • Brown L.S.
        • Lanyi J.K.
        • Gerwert K.
        Proton binding within a membrane protein by a protonated water cluster.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (15738416): 3633-3638
        • Rüdiger M.
        • Oesterhelt D.
        Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.
        EMBO J. 1997; 16 (9233791): 3813-3821
        • Lanyi J.K.
        • Schobert B.
        Local-global conformational coupling in a heptahelical membrane protein: transport mechanism from crystal structures of the nine states in the bacteriorhodopsin photocycle.
        Biochemistry. 2004; 43 (14705925): 3-8
        • Havelka W.A.
        • Henderson R.
        • Oesterhelt D.
        Three-dimensional structure of halorhodopsin at 7 A resolution.
        J. Mol. Biol. 1995; 247 (7723027): 726-738
        • Váró G.
        • Brown L.S.
        • Needleman R.
        • Lanyi J.K.
        Proton transport by halorhodopsin.
        Biochemistry. 1996; 35 (8639608): 6604-6611
        • Tittor J.
        • Haupts U.
        • Haupts C.
        • Oesterhelt D.
        • Becker A.
        • Bamberg E.
        Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein.
        J. Mol. Biol. 1997; 271: 405-416
        • Ihara K.
        • Umemura T.
        • Katagiri I.
        • Kitajima-Ihara T.
        • Sugiyama Y.
        • Kimura Y.
        • Mukohata Y.
        Evolution of the archaeal rhodopsins: evolution rate changes by gene duplication and functional differentiation.
        J. Mol. Biol. 1999; 285 (9878396): 163-174
        • Sharma A.K.
        • Spudich J.L.
        • Doolittle W.F.
        Microbial rhodopsins: functional versatility and genetic mobility.
        Trends Microbiol. 2006; 14 (17008099): 463-469
        • Inoue K.
        • Ito S.
        • Kato Y.
        • Nomura Y.
        • Shibata M.
        • Uchihashi T.
        • Tsunoda S.P.
        • Kandori H.
        A natural light-driven inward proton pump.
        Nat. Commun. 2016; 7 (27853152): 13415
        • Inoue K.
        • Tsunoda S.P.
        • Singh M.
        • Tomida S.
        • Hososhima S.
        • Konno M.
        • Nakamura R.
        • Watanabe H.
        • Bulzu P.A.
        • Banciu H.L.
        • Andrei A.S.
        • Uchihashi T.
        • Ghai R.
        • Béjà O.
        • Kandori H.
        Schizorhodopsins: a family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps.
        Sci. Adv. 2020; 6 (32300653): eaaz2441
        • Brown L.S.
        • Gat Y.
        • Sheves M.
        • Yamazaki Y.
        • Maeda A.
        • Needleman R.
        • Lanyi J.K.
        The retinal Schiff base-counterion complex of bacteriorhodopsin: changed geometry during the photocycle is a cause of proton transfer to aspartate 85.
        Biochemistry. 1994; 33: 12001-12011
        • Schulz E.C.
        • Mehrabi P.
        • Müller-Werkmeister H.M.
        • Tellkamp F.
        • Jha A.
        • Stuart W.
        • Persch E.
        • De Gasparo R.
        • Diederich F.
        • Pai E.F.
        • Miller R.J.D.
        The hit-and-return system enables efficient time-resolved serial synchrotron crystallography.
        Nat. Methods. 2018; 15 (30377366): 901-904
        • Wierman J.L.
        • Paré-Labrosse O.
        • Sarracini A.
        • Besaw J.E.
        • Cook M.J.
        • Oghbaey S.
        • Daoud H.
        • Mehrabi P.
        • Kriksunov I.
        • Kuo A.
        • Schuller D.J.
        • Smith S.
        • Ernst O.P.
        • Szebenyi D.M.E.
        • Gruner S.M.
        • et al.
        Fixed-target serial oscillation crystallography at room temperature.
        IUCrJ. 2019; 6 (30867928): 305-316
        • Agah S.
        • Faham S.
        Crystallization of membrane proteins in bicelles.
        Methods Mol. Biol. 2012; 914 (22976019): 3-16
        • Kabsch W.
        XDS.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20124692): 125-132
        • Evans P.R.
        • Murshudov G.N.
        How good are my data and what is the resolution?.
        Acta Crystallogr. D Biol. Crystallogr. 2013; 69 (23793146): 1204-1214
        • Winn M.D.
        • Ballard C.C.
        • Cowtan K.D.
        • Dodson E.J.
        • Emsley P.
        • Evans P.R.
        • Keegan R.M.
        • Krissinel E.B.
        • Leslie A.G.
        • McCoy A.
        • McNicholas S.J.
        • Murshudov G.N.
        • Pannu N.S.
        • Potterton E.A.
        • Powell H.R.
        • et al.
        Overview of the CCP4 suite and current developments.
        Acta Crystallogr. D Biol. Crystallogr. 2011; 67 (21460441): 235-242
        • Long F.
        • Vagin A.A.
        • Young P.
        • Murshudov G.N.
        BALBES: a molecular-replacement pipeline.
        Acta Crystallogr. D Biol. Crystallogr. 2008; 64 (18094476): 125-132
        • Enami N.
        • Yoshimura K.
        • Murakami M.
        • Okumura H.
        • Ihara K.
        • Kouyama T.
        Crystal structures of archaerhodopsin-1 and -2: common structural motif in archaeal light-driven proton pumps.
        J. Mol. Biol. 2006; 358 (16540121): 675-685
        • Emsley P.
        • Lohkamp B.
        • Scott W.G.
        • Cowtan K.
        Features and development of Coot.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20383002): 486-501
        • Adams P.D.
        • Afonine P.V.
        • Bunkóczi G.
        • Chen V.B.
        • Davis I.W.
        • Echols N.
        • Headd J.J.
        • Hung L.W.
        • Kapral G.J.
        • Grosse-Kunstleve R.W.
        • McCoy A.J.
        • Moriarty N.W.
        • Oeffner R.
        • Read R.J.
        • Richardson D.C.
        • et al.
        PHENIX: a comprehensive Python-based system for macromolecular structure solution.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20124702): 213-221
        • Ward M.E.
        • Wang S.
        • Munro R.
        • Ritz E.
        • Hung I.
        • Gor'kov P.L.
        • Jiang Y.
        • Liang H.
        • Brown L.S.
        • Ladizhansky V.
        In situ structural studies of Anabaena sensory rhodopsin in the E. coli membrane.
        Biophys. J. 2015; 108 (25863060): 1683-1696
        • Waschuk S.A.
        • Bezerra Jr., A.G.
        • Shi L.
        • Brown L.S.
        Leptosphaeria rhodopsin: bacteriorhodopsin-like proton pump from a eukaryote.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (15860584): 6879-6883
        • Ho B.K.
        • Gruswitz F.
        HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures.
        BMC Struct. Biol. 2008; 8 (19014592): 49

      Linked Article

      • Shining light on rhodopsin selectivity: How do proteins decide whether to transport H+ or Cl–?
        Journal of Biological ChemistryVol. 295Issue 44
        • Preview
          The versatile microbial rhodopsin family performs a variety of biological tasks using a highly conserved architecture, making it difficult to understand the mechanistic basis for different functions. Besaw et al. now report structures of a recently discovered cyanobacterial Cl-pumping rhodopsin and its functionally divergent mutant that reveal how these transmembrane proteins create a gradient of activity with subtle changes. These insights are paralleled by a second recent report, which in combination answers long-standing questions about rhodopsin selectivity and will facilitate future engineering efforts.
        • Full-Text
        • PDF
        Open Access