Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 A, which highlight its remarkable difference from all known rhodopsins. The interior of HeR's extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.

https://www.pnas.org/content/pnas/117/8/4131.full.pdf

High-resolution structural insights into the heliorhodopsin family

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

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Structure/Function Study of Photoreceptive Proteins by FTIR Spectroscopy

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Retinal Proteins: Photochemistry and Optogenetics

Dear Editor, Heliorhodopsins (HeRs) are a new class of Microbial rhodopsins (MRs), with a low homology (<15%) to all other MRs and a unique membrane topology in which their N-termini face the intracellular side and C-termini face the extracellular side. To explore the photoactivation mechanism of HeRs, we sought to solve the structure of HeR 48C12 (hereafter referred to as HeR). HeR protein was expressed in insect cells and purified by standard membrane purification methods (Supplementary information, Fig. S1a and Data S1). The purified HeR shows a monodispersed peak and exhibits a high thermostability (Tm= 74 °C, Supplementary information, Fig. S1b, c). Well-diffracting HeR protein crystals were grown in lipid cubic phase (Supplementary information, Fig. S1d, e), and the structure was solved at 2.7 Å resolution (Supplementary information, Data S1 and Table S1). The structure contains two HeR molecules in each asymmetric unit (designated as chains A and B) and the two molecules align very well with each other (RMSD= 0.361 Å, Supplementary information, Fig. S2a). We have successfully assigned almost the entire protein to the structure except 7 disordered residues in N/C-termini and 5 disordered residues (residues 94–97) in the intracellular loop 1. Despite the unique topology and low homology to other MRs, the structure of HeR shows a typical seven transmembrane helix bundle of MRs (Fig. 1a). The most striking feature of the HeR structure is the very long extracellular loop 1 (ECL1) that is mainly composed of two antiparallel β-strands, which has never been seen in other rhodopsins. An examination of surface electrostatics shows that the cytoplasmic side (N-terminus) is highly positively charged while the extracellular side is slightly negatively charged (Fig. 1c). The structure also revealed that HeR forms a dimer (Fig. 1b). The dimer interface is mainly formed by the extensive hydrophobic interaction between two perpendicular interfaces: 1) the interaction between the ECL1 of monomer A and ECL2/3 in monomer B at the extracellular side; 2) the interaction between transmembrane helices TM4 and TM5 of the two monomers (Supplementary information, Fig. S3a, b). The overall dimer interface is 2260 Å as calculated by ccp4 program PISA, which is the most extensive dimerization interface seen in MRs. We investigated the dimer formation in physiological environment by confocal microscopy. The eGFPand mRFP-fused HeRs were co-transfected into AD293 cells. The confocal images show that the eGFP/mRFP-labeled HeRs are tightly associated with each other on the membrane (Supplementary information, Fig. S4a). Next, we performed deletion/mutation experiments to study the role of dimerization in HeR’s function. We first analyzed the F144A/Y151S mutation, which interferes with the hydrophobic interaction between TM4 and TM5 of the two monomers. The F144A/Y151S mutation caused a dramatic decrease of the protein yield and a significant loss of purple color (Supplementary information, Fig. S4b). We then deleted the long ECL1 [Δ(38–63)] that has extensive interaction with ECL2/3 in the dimer formation. Deletion of ECL1 caused a complete loss of all-trans-retinal (ATR) binding as characterized by the complete loss of purple color, and a predominant aggregation with a small oligomer shoulder peak in the size column profiling. A combination of the ECL1 deletion with the F144A/Y151S mutation caused a complete aggregation and complete loss of purple color of the protein, suggesting that dimerization is needed for the function of HeR. Retinal is the ‘core’ of rhodopsin and is generally in all-trans configuration in the resting state. We then looked at the ATR positon in the hydrophobic core of the seven helix bundle. The ßionone ring and the long polyethylene chain of ATR are clearly defined by the electron density map (Supplementary information, Fig. S2b). Compared to the classic bacterial rhodopsin (BR), the hydrophobic core architecture for ATR binding is well conserved in HeR; in particular, the π-electron system formed by two aromatic residues (W182/W86 for BR, F206/Y108 for HeR) at both sides of ATR, is critical for stabilizing the protonated polyethylene chain of ATR (Fig. 1d). A previous study of HeR showed that HeR does not contain any pump or channel activity and suggested that protons are never being released from this protein. We therefore sought to understand these features at a structural level. First, we performed pump activity assay to confirm the lack of pump activity of HeR. Similar to a previous study, illumination of HeR-expressing E. coli cells did not change the pH of the external medium while green-absorbing proteorhodopsin-expressing cells, showed a quick drop of pH (Supplementary information, Fig. S5). The classic proton pumping chain of BR contains three parts: the proton uptake region, the Schiff base region and the proton release region. The proton uptake region is close to the cytoplasmic side, with two key residues, T46 and D96, that mediate the proton uptake. In the Schiff base region, the proton transferring is mediated by a series of polar and hydrogen network interactions (including water molecules), with key residues T89, D85, D212 and R82. The proton release region is close to the extracellular side, and contains two key residues, E204 and E194. Compared to BR, several key residues are missing in the HeR structure. For example, in the proton uptake region, the key proton donor D96 of BR is replaced by L118 in HeR. In the Schiff base region, the key residue D212 is changed to S237 in HeR; and most dramatically the key residues for proton release, E204 and E194, are missing in HeR (Fig. 1e). Together, the absence of these key residues likely explain the lack of pumping activity of HeR. Next, we explored whether HeR could function as a channel. A brief illumination of ChRmine induced a rapid depolarizing current (~1–2 nA). In contrast, illumination of HeR did not induce any measurable current across the membrane of AD293 cells (Supplementary information, Fig. S6). Unlike pumping, which is mainly mediated through the ‘tunnel’ formed by TM3–7 with ATR in the center, the channel in channelrhodopsins is mainly formed by TM1, 2, 3, and 7 and does not enclose ATR. Generally, charged residues lining the channel pore define the selectivity of the channel. For instance, in chanelrhodopsin C1C2, E122, E129 and E136 from TM2, E162 from TM3 and D292 from TM7 define the ion conductance and cation selectivity (Fig. 1f, top panel). In contrast, the ‘tunnel’ formed by TM1, 2, 3 and 7 of HeR is filled with hydrophobic residues, e.g., F88, V83, F72, V69 and V65 of TM2,

/pdf/crystal-structure-of-heliorhodopsin-48c12-gy6pf3gwvm.pdf

Crystal structure of heliorhodopsin 48C12.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-A-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the β-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Crystal structure of heliorhodopsin.

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H+ pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H+ release, the H+ is not metastably trapped in such a putative H+ acceptor, unlike the other H+ pumps. It remains elusive why SzR exhibits different kinetic behaviors in H+ release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 A resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the β-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H+ release; H+ is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.

https://www.pnas.org/content/pnas/118/14/e2016328118.full.pdf

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping.

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs reportedly work as light-driven inward H+ pumps, as xenorhodopsin. Here we report the crystal structure of SzR AM_5_00977 at 2.1 angstrom resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 in SzR are shorter than those in the other microbial rhodopsins. Thus, E81 is located near the cytosol, playing a critical role in the inward H+ release. We suggested the H+ is not metastably trapped in E81 and released through the water-mediated transport network from the retinal Schiff base to the cytosol. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.

https://biorxiv.org/content/10.1101/2020.07.28.224907v1.full.pdf

Akimitsu Higuchi

Papers

Crystal structure of heliorhodopsin.

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping.

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping