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

Abstract

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.

Keywords: Mastigocladopsis repens; X-ray crystallography; bacteriorhodopsin; bicelle crystallization; chloride pump; chloride transport; cyanobacterial rhodopsin; functional conversion; functional interconversion; membrane protein; microbial rhodopsin; protein structure; proton pump; retinal protein; structure–function.

Figure 1.

Microbial 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.

Figure 2.

Structure 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. 2F_c − F_o electron density maps are contoured at 0.8 σ. The direction of ion transport is indicated.

Figure 3.

Oligomeric 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.

Figure 4.

Topology 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 (65). 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.

Figure 5.

Putative 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 (65)) 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.

Figure 6.

Comparison 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.