title: X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION.
abstract: Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.
authors.affiliation: From the Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsukamoto
authors.affiliation: the Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan, the Molecular Chirality Research Center, Chiba University, Chiba 263-8522, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Mizutani
authors.affiliation: the Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Hasegawa
authors.affiliation: the Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: From the Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Honda
authors.affiliation: the Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Hashimoto
authors.affiliation: the Faculty of Pharmaceutical Sciences, Toho University, Funabashi 274-8510, Japan, and.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Shimono
authors.affiliation: the RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Yamashita
authors.affiliation: the RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: the Faculty of Pharmaceutical Sciences, Toho University, Funabashi 274-8510, Japan, and.
authors.forename: Seiji
authors.initials: S
authors.lastname: Miyauchi
authors.affiliation: the Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Takagi
authors.affiliation: the Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Shigehiko
authors.initials: S
authors.lastname: Hayashi
authors.affiliation: the Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan, the Molecular Chirality Research Center, Chiba University, Chiba 263-8522, Japan, t.murata@faculty.chiba-u.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.affiliation: From the Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan, sudo@pharm.okayama-u.ac.jp.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sudo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites / Q000235:genetics; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006358:Hot Temperature; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D062308:Optogenetics / Q000379:methods; D000072417:Protein Domains; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025604:Rhodopsins, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism
chemical_list: D017494:Proton Pumps; D025604:Rhodopsins, Microbial
keywords: MD simulation; X-ray crystallography; membrane protein; optogenetics; photoreceptor; proton pump; thermal stability