2023 年 20 巻 Supplemental 号 論文ID: e201009
Rhodopsins are membrane-embedded photoreceptor proteins that contain a covalently-linked retinal chromophore. Rhodopsins are classified into two types. Type-1 rhodopsins are microbial rhodopsins (formerly archaeal rhodopsins) derived from archaea, eubacteria, eukaryotes, or viruses. Type-2 rhodopsins are animal rhodopsins derived from higher organisms that are evolutionarily distinct from microbial rhodopsins. Apart from a few exceptions, the chromophores of microbial rhodopsins are all-trans retinal. All-trans retinal is photoisomerized to 13-cis retinal when light is absorbed by microbial rhodopsin. Following that, the protein moiety undergoes structural changes, and the retinal is thermally isomerized before returning to its initial state. This photochemical reaction, known as the photocycle, is one of the characteristics of microbial rhodopsins and is responsible for their function. The functions of microbial rhodopsins are classified into four categories. Light-driven ion pumps, light-gated ion channels, photosensors, and enzymatic rhodopsins are examples. In light-driven pumps such as bacteriorhodopsin (BR), the molecular mechanism of microbial rhodopsin has been extensively studied. Many microbial rhodopsins’ functional mechanisms have recently been elucidated using advances in physicochemical methods such as spectroscopy, structural biology, computational chemistry, and electrophysiology [1]. We discussed the most recent findings on the molecular mechanisms that generate the functional diversity of microbial rhodopsins in session 6. Even at atomic level, remarkable progress has been made in our understanding of the mechanistic steps.
Heliorhodopsins (HeRs) are unique rhodopsins with an orientation that is opposite to that of Type-1 or Type-2 rhodopsins [2]. They are embedded in the membrane with their N-termini facing the cytoplasmic side of the cell. Until recently, the function of HeR was unknown. Dr. Kwang-Hwan Jung (Sogang University) reported that HeRs regulate corresponding enzymes in a light-dependent manner. He looked for enzymes that HeR might regulate and were located in the same operon as the helio-opsin gene. He discovered two HeRs and the enzymes that regulate them. Their complexes included Actinobacteria bacterium IMCC26103 heliorhodopsin (AbHeR) and glutamine synthetase (AbGS), Trichococcus flocculiformis heliorhodopsin (TfHeR), and class 2 cyclobutane pyrimidine dimer (CPDII) Photolyase (TfPHR). He showed that HeRs bind to their regulated enzymes in vitro and in vivo using isothermal titration calorimetry. Mutational studies revealed that the cytoplasmic surface amino acid residues of HeRs bind to the enzyme. He also presented evidences that enzyme activities are regulated in vivo in a light-dependent manner. These findings were published in two papers in 2022 [3,4], revealing that previously unknown HeRs function as photosensors. His findings pave the way for future research into the photo-regulation of cellular functions by HeRs.
Solid-state NMR is a sensitive technique for investigating local structures and a powerful technique for determining the functional effects of minor structural changes [5]. Solid-state NMR data of sodium ion pumping rhodopsin Krokinobacter rhodopsin 2 (KR2) and HeR embedded in the membrane were presented by Dr. Izuru Kawamura (Yokohama National University). To begin, he emphasized that the 15N chemical shifts of retinal protonated Schiff base (RPSB), which relate to the electronic environment of nitrogen, are highly correlated with the maximum absorption of some microbial rhodopsins (e.g., BR, sensory rhodopsin II, and middle rhodopsin) based on the strength of hydrogen bonding with counterion [6]. Using the 15N RPSB signal of KR2 and H30A, he and his colleagues discovered that changes in RPSB with Asp116 in helix C (counterion) were induced by Na+ binding at the extracellular protein interface [7]. He is also studying the retinal and RPSB NMR data to better understand the unique property of HeR. Based on the local structure of ion selectivity of ion transporting rhodopsins, solid-state NMR data can provide important insight into the molecular mechanism.
It is worth noting that the absorption maxima of microbial rhodopsins differ from one another, despite the fact they all have same chromophore, all-trans retinal. The spectral range used ranges from 370 nm to 750 nm. The environment around the chromophore causes this spectral diversity. Neorhodopsin (NeoR) is an enzyme rhodopsin that forms a heterodimer with another enzyme rhodopsin to absorb near-infrared light [8]. Dr. Matthias Broser (Humboldt-Universität zu Berlin) described the mechanism by which NeoR absorbs near-infrared light and its enzymatic activity in the aquatic fungus Rhizoclosmatium globosum. In NeoR, there is a counterion triad for RPSB that consists of E136, D140, and E262. He concluded that the two counterions E136 and E262 are primarily responsible for NeoR’s near-infrared absorption based on a comprehensive mutational study of the retinal binding site supported by theoretical calculations [9]. Furthermore, he discovered that the isolated cyclase core of NeoR is inactive, whereas Rhodopsin-guanylyl cyclases 1 (RGC1) in heterodimeric complex with NeoR is active (Km=5.43 mM; Vmax=0.97 μmol min–1 mg–1). He emphasized that the discovery of heterodimeric rhodopsin cyclase, including NeoR, greatly expands our understanding of the spectral and functional diversity of this ancient and ubiquitous photoreceptor based on these findings.
Microbial rhodopsins with high light sensitivity and ion conductance are suitable as optogenetic tools for manipulating neural activity [10]. Cation-conducting and anion-conducting channels were discovered in channel rhodopsins (ChRs) [11]. Dr. Satoshi Tsunoda (Nagoya Institute of Technology) presented the electrophysiological properties of KnChR from the algae Klebsormidium nitens and GtCCR from the cryptophyte algae Guillardia theta, showing that they are light-gated cationic channels (CCRs). He discussed the function of the large C-terminal cytoplasmic domain found in all ChRs. Electrophysiological analysis of KnChR with various lengths of C-terminus revealed that the shorter the C-terminal domain, the longer the channel open lifetime [12]. GtCCRs are particularly intriguing channel rhodopsins because they are more homologous to haloarchaeal rhodopsins, such as the proton-pumping BR, than to chlorophyte CCRs, such as ChR2 from Chlamydomonas reinhardtii (ChR2) [13]. One of the most noticeable characteristics of GtCCRs (GtCCR4) is that their light sensitivity is greater than that of ChR2, while their channel open lifetime is in the same range (20–30 ms). He discussed the application of GCCR4 for optical manipulations of cultured neurons due to its high sensitivity. His findings could aid in the improvement of light-gated channel function and the development of more advanced optogenetics tools.
Natural inward-directed light-driven proton pumps were discovered in 2016, as opposed to outward pumps like BR [14]. One powerful approach for understanding the functional mechanism is to artificially convert the function. Dr. María del Carmen Marín (The University of Tokyo) who was chosen as a speaker from the poster presenters, reported the successful conversion of a light-driven outward proton pumping rhodopsin into an inward proton pump. The pumping direction of a triple mutant (D73F, T77S, and T78C) outward proton pump PspR of Pseudomonas putida was converted to inward. In the dark, the absorption maximum and retinal composition of the PspR triple mutant are nearly identical to those of the wild-type. The triple mutant’s photoreaction is much slower than that of the wild-type. Moreover, she showed that the proton directly binds to the retinal chromophore from the extracellular solvent in the process of light-driven pumping of the triple mutant that was converted to an inward proton pump. These findings suggest that amino acid residues at these positions are important in determining vectorial proton transport direction. Understanding the mechanism of ion transport direction determination will lead to the development of light-driven ion-pumping rhodopsins for specific applications, potentially expanding the possibilities of applied research on microbial rhodopsins.
Optogenetics has made remarkable progress in the study of microbial rhodopsins as powerful tools. The mechanisms of various functions of microbial rhodopsins are being elucidated at the atomic level, as reported in this session. It may be possible in the near future to convert absorption wavelengths and/or functions of any microbial rhodopsins as desired. Furthermore, microbial rhodopsins are expected to evolve not only as optogenetic tools for controlling neural activity, which is a medical application, but also for industrial applications.
