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Shining light on rhodopsin selectivity: How do proteins decide whether to transport H+ or Cl?

Open AccessPublished:October 30, 2020DOI:https://doi.org/10.1074/jbc.H120.016032
      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.
      Light serves as a source of energy and information across all domains of life. Miniscule photons can be used to initiate large changes in biomolecules, particularly in the microbial rhodopsins, where photon-induced isomerization of all-trans-retinal chromophore affects the biological functions of ion pumps, ions channels, phototactic sensors, enzymes, and so on. Microbial rhodopsins are part of a large family of photoreceptive membrane proteins with a conserved structure made up of seven-transmembrane α helices (
      • Ernst O.P.
      • Lodowski D.T.
      • Elstner M.
      • Hegemann P.
      • Brown L.S.
      • Kandori H.
      Microbial and animal rhodopsins: Structures, functions, and molecular mechanisms.
      ). Because rhodopsins can perform such a wide variety of functions using this common compact structure, they have provided ideal model systems to study structure–function relationships in proteins using spectroscopy, biochemistry, and structural biology. Moreover, ion pumps and ion channels have been widely adopted as tools in optogenetics to manipulate the firing pattern of animal nerves with light, and further elucidation of the mechanism of ion-transporting rhodopsins is increasingly required to construct new ion-transporting optogenetic tools.
      So far, four types of ion-pumping rhodopsins, including outward and inward H+ pumps, an inward Cl pump, and an outward Na+ pump, have been identified. Of these, the archeal outward H+ pump, called bacteriorhodopsin (BR), was the first to be discovered, in 1971. Then the archeal inward Cl pump halorhodopsin (HR) was reported in 1977 and functionally characterized in 1982. BR has an Asp-Thr-Asp (DTD) motif in the third helix that plays a critical role during H+ pumping; these three residues are highly conserved in closely related H+ pumps. HR, in contrast, has a TSA motif, which is similarly critical for its chloride-specific activity. Interestingly, the mutation of the first D of the DTD motif to T in BR confers an inward Cl-pumping function similar to HR (
      • 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.
      ), suggesting that this residue (Asp-85 in BR) might be responsible for determining whether an H+ or Cl is transported. However, the reverse mutation of HR, changing the T of the TSA motif to D, did not result in the conversion to an H+ pump (
      • 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.
      ). Hence, the determinants separating H+ and Cl pump rhodopsins were not yet completely clear.
      Recently, it was revealed that cyanobacteria have a new type of Cl-pumping microbial rhodopsins with a TSD motif. One of them, MastR (also known as MrHR) from Mastigocladopsis repens, functions as a Cl pump similar to the TSA-containing HR, but in this case, the Thr to Asp mutation (MastR T74D) was successful in creating an H+ pump (
      • Hasemi T.
      • Kikukawa T.
      • Kamo N.
      • Demura M.
      Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump.
      ), thus achieving complete functional conversion between an H+ pump (BR) and a Cl pump (MastR) by swapping only one residue. Moreover, another TSD-containing HR from Synechocystis sp. PCC 7509 called SyHR was shown to be able to pump a sulfate ion (SO42) that other HRs are unable to transport (
      • 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 SO42− transporter and its spectroscopic characteristics.
      ). These findings suggest that TSD-containing HRs have molecular mechanisms for ion transport that differ from the archeal TSA-containing HRs. To understand the mechanisms of these unique abilities, however, demands their three-dimensional structure at atomic level.
      Besaw et al. now answer this call, revealing the X-ray crystallographic structure of MastR and its H+ pumping mutant (
      • Besaw J.E.
      • Ou W.L.
      • Morizumi T.
      • Eger B.T.
      • Sanchez Vasquez J.D.
      • Chu J.H.Y.
      • Harris A.
      • Brown L.S.
      • Miller R.J.D.
      • Ernst O.P.
      The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants.
      ) in bicelles at 2.33 and 2.50 Å, respectively. In the structure of WT MastR, the substrate Cl is bound to Thr-74 and Ser-78, corresponding to the T and S in the motif, and to the Schiff base part of the retinal via a water molecule. In MastR T74D, the Asp occupies the position adopted by Cl in the WT protein, and strong hydrogen bonds similar to those found in BR are formed between the Asp, water molecule, and retinal (Fig. 1). This result supports the idea that a strong hydrogen-bonding network is essential for the H+ pump, in agreement with previous spectroscopic and theoretical studies (
      • Ernst O.P.
      • Lodowski D.T.
      • Elstner M.
      • Hegemann P.
      • Brown L.S.
      • Kandori H.
      Microbial and animal rhodopsins: Structures, functions, and molecular mechanisms.
      ). Another structure of the same protein was also reported by Yun et al. (
      • Yun J.H.
      • Park J.H.
      • Jin Z.
      • Ohki M.
      • Wang Y.
      • Lupala C.S.
      • Liu H.
      • Park S.Y.
      • Lee W.
      Structure-based functional modification study of a cyanobacterial chloride pump for transporting multiple anions.
      ). They further focused on the difference in an extracellular loop between MastR and SyHR. The replacement of residues in the extracellular loop in the former with the corresponding sequence from the latter resulted in MastR being able to transport SO42. A structure of the SO42-transporting mutant revealed a more positively charged entrance to the putative extracellular ion pathway compared with the WT, suggesting that this entrance determines the anion selectivity of TSD-type HRs. Besaw et al. similarly point to differences in loops between MastR and BR that seem to explain their ion selectivity, suggesting an even broader role for the extracellular entrance.
      Figure thumbnail gr1
      Figure 1The structures of MastR and its H+-pumping mutant (MastR T74D) compared with the outward H+ pump, BR. Shown are the X-ray crystallographic structures of MastR (PDB code 6XL3), the H+-pumping MastR T74D mutant (PDB code 6WP8) (
      • Besaw J.E.
      • Ou W.L.
      • Morizumi T.
      • Eger B.T.
      • Sanchez Vasquez J.D.
      • Chu J.H.Y.
      • Harris A.
      • Brown L.S.
      • Miller R.J.D.
      • Ernst O.P.
      The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants.
      ), and BR (PDB code 1IW6) (
      • Matsui Y.
      • Sakai K.
      • Murakami M.
      • Shiro Y.
      • Adachi S.
      • Okumura H.
      • Kouyama T.
      Specific damage induced by x-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin.
      ) (top) with an enlarged view of the region around the retinal Schiff base and the first two motif residues (bottom). The motif residues are indicated by letters circled in red. Cl and water molecules are shown as green and cyan spheres, respectively. The dashed lines indicate hydrogen bonds connecting the retinal Schiff base and amino acids of each motif. CP, cytoplasmic side; EC, extracellular side. The structures of proteins were illustrated by CueMol software (RRID:SCR_019052).
      These two structural studies illuminate the long-sought functional determinants between H+ and Cl pumps and structural elements enabling divalent-anion pumping. Moreover, the new structures provide an exciting opportunity to revisit our understanding of rhodopsin evolution in general, in which the structural elements important for the function of the ancestral molecule were conserved in the descendants with diversified functions, as discussed by Besaw et al. There are, however, unsolved mysteries of ion transport by TSD-containing HRs remaining. For example, we do not know the structure of the SO42-bound state during SO42 transport. Also, although both studies suggested possible ion transport pathways in their respective proteins, these structures represent the dark state, so the precise structure of the transiently opened pathway has not been revealed. To solve these problems, further structural studies on the SO42-bound protein and photoactivated intermediates are required. Freeze-trapping methods will help to capture intermediate structures under light illumination (
      • Matsui Y.
      • Sakai K.
      • Murakami M.
      • Shiro Y.
      • Adachi S.
      • Okumura H.
      • Kouyama T.
      Specific damage induced by x-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin.
      ), but recently developed techniques, such as time-resolved serial millisecond crystallography (TR-SMX), available at synchrotron facilities (
      • Weinert T.
      • Skopintsev P.
      • James D.
      • Dworkowski F.
      • Panepucci E.
      • Kekilli D.
      • Furrer A.
      • Brünle S.
      • Mous S.
      • Ozerov D.
      • Nogly P.
      • Wang M.
      • Standfuss J.
      Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography.
      ), and the time-resolved serial femtosecond crystallography (TR-SFX) technique with X-ray free electron lasers (XFEL) (
      • Nango E.
      • Royant A.
      • Kubo M.
      • Nakane T.
      • Wickstrand C.
      • Kimura T.
      • Tanaka T.
      • Tono K.
      • Song C.
      • Tanaka R.
      • Arima T.
      • Yamashita A.
      • Kobayashi J.
      • Hosaka T.
      • Mizohata E.
      • et al.
      A three-dimensional movie of structural changes in bacteriorhodopsin.
      ), will provide not only structural insights into each photointermediate but also the dynamics of conformational change between them. It will be exciting to see how these and other explorations shed light on this fascinating protein family.

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