Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their ‘visualization’ requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Our true-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.
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Detailed analysis of distorted retinal and its interaction with surrounding residues in the K intermediate of bacteriorhodopsin
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Atomic models built using X-ray crystallography data have been deposited in the Research Collaboratory for Structural Bioinformatics PDB with the codes 7Z09 (for the ground state of BR at 1.05 Å), 7Z0A (for the ground state of BR at 1.22 Å), 7Z0C (for the K state), 7Z0D (for the L state) and 7Z0E (for the M state). The UniProt gene database (uniport.org) was used for bioinformatic analysis of the BR clade of microbial rhodopsins.
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We acknowledge the Structural Biology Group of the ESRF for granting access to the synchrotron beamlines. We are deeply thankful to A. Round for his help with manuscript preparation. This work was supported by the common program of Agence Nationale de la Recherche (ANR), France and Deutsche Forschungsgemeinschaft, Germany (grant no. ANR-15-CE11-0029-02) and by funding from Frankfurt: Cluster of Excellence Frankfurt Macromolecular Complexes (to E.B.) by the Max Planck Society (to E.B.) and by the Commissariat à l’Energie Atomique et aux Energies Alternatives (Institut de Biologie Structurale)–Helmholtz-Gemeinschaft Deutscher Forschungszentren (Forschungszentrum Jülich) Special Terms and Conditions 5.1 specific agreement. V.G. greatly acknowledges his HGF Professorship. V.B. acknowledges DAAD Young Talents Programme Line A. This work used the icOS and HTX platforms of the Grenoble Instruct-ERIC center (ISBG; UMS3518 CNRS-CEA-UJF-EMBL) within the Grenoble Partnership for Structural Biology (PSB)98. Platform access was supported by FRISBI (grant no. ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (grant no. ANR-17-EURE-0003). Protein crystallization was supported by the Russian Foundation for Basic Research (RFBR) according to the research project no. 18-02-40020. Data collections for the K intermediate state structures were supported by RFBR (project no. 19-29-12022). Data collections for the L intermediate state structures were supported by the Russian Ministry of Science and Higher Education (grant no. 075-15-2021-1354). Data collection of ground state was supported by RFBR-CNRS (project no. 19-52-15017). Data treatment and structure solution were supported by the Russian Science Foundation (RSF) (project no. 21-64-00018). Bioinformatic analysis was supported by RSF-Helmholtz (grant no. 19-44-06302). The work is supported by Ministry of Science and Higher Education of the Russian Federation (project no. 075-00958-21-05/730000F.99.1.BV10AA00006 to A.R. for data analysis, and project 075-00337-20-03/FSMG-2020-0003 to V.B. for X-ray data treatment).
The authors declare no competing interests.
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A. Spectrum of the ground state of BR in a crystal at 100K. B. Difference absorption spectrum (final minus ground) of after cryotrapping of the K state in BR crystals at 100K. C. Difference absorption spectra (final minus ground) after the cryotrapping of the L and M states in BR crystals at 150 (black), 170 (red), 190 (green), 210 (blue), 230 (gold), and 250 K (orange).
A. Difference FoK-FoGR electron density maps built around the retinal cofactor and D212, W86, and W182 residues indicating structural rearrangements in the K state of BR. The maps are contoured at the level of 5σ. Views from two sides are shown. B,C. Difference FoL-FoGR electron density maps built around the central part of BR indicating structural rearrangements in the L state of BR. The maps are contoured at the level of 3σ. D. Difference FoL-FoGR electron density maps built around the R82 residue and proton release group of BR indicating structural rearrangements in the L state of BR. The maps are contoured at the level of 3σ.
A. Maps built around the central part of BR indicating structural rearrangements in the M state of BR. The maps are contoured at the level of 3σ. B. Maps built around the R82 residue and PRG of BR indicating structural rearrangements in the M state of BR. The maps are contoured at the level of 3σ. C. Maps built around retinal cofactor and W182 and W86 residues indicating structural rearrangements in retinal binding pocket in the M state of BR. The maps are contoured at the level of 3σ.
A. View on the crystal monolayer from the extracellular side of BR. The trimer of BR (cartoon representation) is contoured for clarity. B. Overall view of one BR trimer (white surface) surrounded by the ring of lipid molecules (green spheres). C,D. Side views of the BR trimer (white surface) surrounded by the ring of lipid molecules (green spheres).
A,B. Different side views of the PRG in the triple conformation obtained in the present work. The polder maps are contoured at the level of 3σ. C. Three conformations coexisting in the model from present work. D. Two conformations coexisting in the model from Hasegawa et al.54 (PDB ID: 5ZIL). E. Model of BR obtained using serial femtosecond crystallography (SFX) at XFEL at room temperature from Nogly et al.51 (PDB ID: 6G7H). F. Model of BR from Luecke et al.37 (PDB ID: 1C3W).
A. Side view of the ground state (violet). B. Side view of the K state (blue). C. Side view of the L state (salmon). D. Side view of the M state (yellow) of the photocycle. Retinal cofactor is colored teal. Cavities were calculated using HOLLOW and are shown with grey-blue surfaces. Helices F and G are hidden for clarity. Hydrophobic/hydrophilic membrane core boundaries were calculated using PPM server and are shown with black horizontal lines.
A. Alignment of the backbones of the central part of helix G in the ground (violet), L (salmon), and M (yellow) states. Black arrows indicated most critical rearrangements in the backbones of residues A215 and K216 during photocycle. Water molecules are shown with spheres with the color corresponding to each state. B. Backbone of helix G in the ground state of BR. C. Backbone of helix G in the L state of BR. D. Backbone of helix G in the M state of BR. H-bonds are shown with dashed black lines. Water molecules in panels B-C are shown with red spheres.
Extended Data Fig. 8 Comparison of the proton release group regions of BR in different structures of the M state.
A. The PRG of BR in the M state obtain in present work. B. The most similar organization of the PRG of BR found in the M state obtained with cryotrapping procedure44 (PDB ID: 2ZZL). C. The PRG organization in the highest-resolution structure of BR in the M state obtained using time-resolved serial femtosecond crystallography at X-ray Free Electron Laser51 (PDB ID: 6G7L). Cavities were calculated using HOLLOW and are shown with grey-blue surfaces.
A. The ground state of BR. B. The L state of BR. C. The M state of BR. H-bonds are shown with black dashed lines. A weak H-bond between T89 and D85 in the L state is shown with black solid line. The distance between RSB and D85 in the L and M states as well as the distance between T89 and D85 in the M state are shown with red arrows. Distance lengths are shown near the lines with bold italic numbers and are in Å. Retinal cofactor and K216 residue are colored teal.
A. The phylogenetic tree of archaeal outward proton-pumping rhodopsins. BR is shown with red dot. The group of 9 unusual rhodopsins having substitutions at the key functional positions (E194, E204, S193) compared to the other members of the group is contoured and labelled. Also, one of them (E194G, 5 of 9 proteins have this substitution) was shown not to have proton pumping activity. B. Amino acid alignment of the representatives of the group of archaeal proton pumps. Only key regions of the protein are shown. Last 4 proteins (marked with a black bracket) belong the group of 9 unusual proteins contoured in panel A. C. Fractions of proteins with specific amino acid at specific position according to the BR numeration. The group of 9 unusual rhodopsins is excluded for the calculation of the fractions. Total number of rhodopsins used is 261. D. Fractions of proteins with specific amino acid at specific position according to the BR numeration. All 270 proteins were used for calculations. Red arrows indicate essential residues forming the proton wires in course of the BR photocycle.
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Borshchevskiy, V., Kovalev, K., Round, E. et al. True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins. Nat Struct Mol Biol 29, 440–450 (2022). https://doi.org/10.1038/s41594-022-00762-2
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