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Proc Natl Acad Sci U S A. 2016 Jun 14;113(24):6659-64. doi: 10.1073/pnas.1517520113. Epub 2016 May 31.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

Author information

1
Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama, 230-0045, Japan;
2
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;
3
Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, 274-8510, Japan;
4
Central Research Laboratory, Hamamatsu Photonics K.K., Hamamatsu, Shizuoka 434-8601, Japan;
5
Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan;
6
Division of Biophysics, Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan;
7
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
8
Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, 274-8510, Japan; park@tsurumi.yokohama-cu.ac.jp mineo.iseki@phar.toho-u.ac.jp.
9
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.

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.

KEYWORDS:

X-ray crystallography; allostery; blue light; optogenetics

PMID:
27247413
PMCID:
PMC4914150
DOI:
10.1073/pnas.1517520113
[Indexed for MEDLINE]
Free PMC Article

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