title: Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.
abstract: Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.
authors.affiliation: Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama, 230-0045, Japan;
authors.forename: Mio
authors.initials: M
authors.lastname: Ohki
authors.affiliation: Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama, 230-0045, Japan; Drug Design Group, Kanagawa Academy of Science and Technology, Takatsu, Kawasaki 213-0012, Japan;
authors.forename: Kanako
authors.initials: K
authors.lastname: Sugiyama
authors.affiliation: Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama, 230-0045, Japan;
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Kawai
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, 274-8510, Japan;
authors.forename: Hitomi
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, 274-8510, Japan;
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Nihei
authors.affiliation: Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama, 230-0045, Japan;
authors.forename: Satoru
authors.initials: S
authors.lastname: Unzai
authors.affiliation: Central Research Laboratory, Hamamatsu Photonics K.K., Hamamatsu, Shizuoka 434-8601, Japan;
authors.forename: Masumi
authors.initials: M
authors.lastname: Takebe
authors.affiliation: Central Research Laboratory, Hamamatsu Photonics K.K., Hamamatsu, Shizuoka 434-8601, Japan;
authors.forename: Shigeru
authors.initials: S
authors.lastname: Matsunaga
authors.affiliation: Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan;
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Adachi
authors.affiliation: Division of Biophysics, Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan;
authors.forename: Naoya
authors.initials: N
authors.lastname: Shibayama
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Ryuta
authors.initials: R
authors.lastname: Koyama
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yuji
authors.identifier: 0000-0003-2260-8191
authors.initials: Y
authors.lastname: Ikegaya
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, 274-8510, Japan;
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama, 230-0045, Japan;
authors.forename: Jeremy R H
authors.initials: JR
authors.lastname: Tame
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, 274-8510, Japan; park@tsurumi.yokohama-cu.ac.jp mineo.iseki@phar.toho-u.ac.jp.
authors.forename: Mineo
authors.initials: M
authors.lastname: Iseki
authors.affiliation: Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama, 230-0045, Japan; park@tsurumi.yokohama-cu.ac.jp mineo.iseki@phar.toho-u.ac.jp.
authors.forename: Sam-Yong
authors.initials: SY
authors.lastname: Park
mesh_terms: D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics / Q000528:radiation effects; D057809:HEK293 Cells; D006801:Humans; D008027:Light; D054300:Oscillatoria / Q000201:enzymology* / Q000235:genetics; D000072417:Protein Domains; D015290:Second Messenger Systems / Q000235:genetics / Q000528:radiation effects; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D000242:Cyclic AMP; D000262:Adenylyl Cyclases
keywords: X-ray crystallography; allostery; blue light; optogenetics