2023 Volume 20 Issue Supplemental Article ID: e201014
The title of this session was “Structural mechanism of microbial rhodopsins”, but the actually presented topics were very diverse (cryo-electron-microscopy, time-resolved X-ray crystallography, computer simulation & theory; microbial rhodopsins vs. visual rhodopsins). Here, the five presentations in this session are separately overviewed.
Since the light-driven proton pump bacteriorhodopsin was isolated from the purple membrane of Halobacteirum salinarum by Oesterhelt and Stoeckenius in 1971 [1], an increasing number of microbial rhodopsins have been discovered in archaea, eubacteria, eukarya, and viruses. Some of them have proved useful for optical control of cell activity (optogenetics). In this session, Dr. Shalev-Benami (Weizman Institute of Science) discussed the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem are C-terminally fused to a bestrophin channel [2]. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UV-absorbing forms in the different variants. Bestrhodopsin behaves as a light-modulated anion channel and the retinal chromophore photoisomerizes from all-trans to 11-cis form. More surprisingly, the structural analysis by cryo-electron-microscopy revealed that bestrhodopdin forms a pentameric megacomplex (~700 kDa) with ten rhodopsin units surrounding a central channel. Their findings increase the degree of freedom in designing new optogentic tools.
Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light. Dr. Nureki (Univ. Tokyo) reported time-resolved serial femtosecond crystallography of channelrhodopsin [3]. Nureki and his colleagues showed that the isomerized retinal adopts a twisted conformation and laterally shifts toward helix TM3, consequently inducing an outward shift of TM3 and a local deformation in TM6 and TM7. These early conformational changes in the pore-forming helices are suggested to be the triggers that lead to opening of the ion-conducting pore. He also presented the crystal structures of the red-light-activated channelrhodopsins Chrimson [4]. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for the red-light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, they engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics. Their study will help to design more versatile optogentics tools.
Dr. Schertler (Paul Scherrer Institute, Swiss) discussed “Femtosecond-to-millisecond structural biology at the Swiss X-ray Free electron laser – Rhodopsins pave the way” [5] and presented time-resolved X-rat crystallography of visual rhodopsin at room temperature [6]. As compared to structural analyses of reaction intermediates trapped at cryogenic temperature, time-resolved serial femtosecond crystallography has the advantage that the problem of a dose limit (i.e., radiation damage) can be avoided and, hence, light-induced structural changes can be analyzed at a higher spatial and very high temporal resolution. By using SwissFEL, his group investigated how an isomerised twisted all-trans retinal stores the photon energy required to initiate protein conformational changes associated with the formation of the G protein-binding signalling state. Their result showed that the distorted retinal at 1 ps time-delay of photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Their work sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated G-Protein Coupled Receptor (GPCR) activation. Look also to Dr. Panneels presentation on the same topic in Session 8.
Graph theory has become an important tool for analyses of biological networks such as protein-protein interaction networks. Dr. Bondar (Univ. Bucharest) presented C-Graphs, an efficient tool with graphical user interface that was designed to identify the functionally conserved hydrogen-bond network between different protein moieties [7]. In the case of GPCRs, dynamic hydrogen-bond networks are thought to provide proteins with structural plasticity required to translate signals into a cellular response. Visual rhodopsins are GPCRs that respond to light. At the end of their photoreaction cycle, monostable rhodopsins (e.g., bovine rhodopsin) release retinal, whereas bistable rhodopsins (e.g., squid rhodopsin) absorb a second photon and re-isomerize the retinal chromophore. Using C-Graphs, they dissected hydrogen-bond networks in a set of 24 high-resolution structures of visual rhodopsins and adenosin A2A receptors. Graph analyses combined with clustering of crystallographic waters revealed that some of the internal waters of squid rhodopsin are conserved in structures of jumping spider rhodopsin-1 (bistable rhodopsin) and adenosine A2A receptor, whereas the conserved hydrogen-bond trajectory of the bovine rhodopsin structures is rather sparse. Their result seems to suggest that monostable rhodopsins acquired a distinct activation mechanism during the evolution from ancestral bistable rhodopsins. Since the reaction sequences of GPCRs are thought to involve protonation changes, pH-dependent computations would be required to elucidate the activation mechanisms.
Internal water molecules play a crucial role in the functional processes of proton pumping proteins. Recent progress in infrared spectroscopy provided new information on water molecules inside bacteriorhodopsin. Dr. Yagi (RIKEN) presented an anharmonic vibrational method to exploit the locality of molecular vibrations. Yagi and Sugita divided the potential energy surface of a system into intra- and inter-group contributions and solved the vibrational Schrödinger equation based on a potential energy surface, in which the inter-group coupling is truncated at the harmonic level while accounting for the intra-group anharmonicity [8]. The method is applied to a pentagonal hydrogen bond network composed of internal water molecules and charged residues in the active center of bacteriorhodopsin. The infrared spectrum is computed using a set of coordinates localized to each water molecule and amino acid residue by second-order vibrational quasi-degenerate perturbation theory. They showed that the incorporation of anharmonicity as well as structural samplings are of essential importance to reproduce the experimental infrared spectrum. The computational spectrum paves the way for decoding the infrared signal of strong hydrogen-bonded networks and helps to elucidate their functional roles in biomolecules.
