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.optogenetics | X-ray crystallography | blue light | allostery

Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.

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Euglena tumble and change their swimming direction in response to an abrupt increase or decrease in light intensity, called step-up or step-down photophobic responses, respectively. Iseki et al. successfully isolated the photosensing receptor, named PAC, responsible for step-up photophobic responses from a photosensing organelle, the paraflagellar body (PFB), in Euglena gracilis and identified it to be a flavoprotein that has adenylyl cyclase activity regulated by blue light. We recently revealed how PFB is constructed from PAC using cutting edge cryo-EM techniques, such as CEMOVIS. From the view of structural biology, we draw ever closer to understanding the mechanism behind how Euglena senses light, leading to a change of swimming direction. 1SAP-03 チューブリン様蛋白質 TubZ によるプラスミド分配の分子 機構 Plasmid segregation driven by the tubulin-like GTPase TubZ Ikuko Hayashi (Yokohama City University)Segregation of low-copy-number plasmids relies on partitioning systems that contain plasmid-encoded cytoskeletal proteins. Tubulin/FtsZ-like GTPase TubZ was identified as a partitioning factor of the pXO1-like plasmids in virulent Bacillus. TubZ exhibits high GTPase activity and assembles into polymers both in vivo and in vitro, and its activation is suggested to be regulated by the DNA-binding protein TubR and the centromeric DNA site. However the molecular mechanism of plasmid segregation by TubZ assembly is not well understood. Based on our recent progress in structural and biochemical studies, I would like to discuss the molecular recognition mechanism of TubR as an adaptor between the TubZ filament and DNA. 1SAP-04 黄色ブドウ球菌のコロニースプレッディングにおける毒素の 役割 Role of toxin in Staphylococcus aureus colony spreadingChikara Kaito, Kazuhisa Sekimizu (Grad. Sch. Phar., Univ. Tokyo) S. aureus, a human pathogen, spreads on soft agar plates. We call the phenomenon "colony spreading". High virulence S. aureus strains produce higher amount of toxins and exhibit higher colony-spreading abilities than low virulence strains. Deletion of a toxin encoding-gene diminishes the colony spreading. The toxin is present in both culture supernatant and cellular extract. In this study, we isolated mutants altering the toxin distribution and found that a mutant with less amount of cellular toxin decreased the colony spreading, whereas another mutant with less amount of extracellular toxin did not decrease the colony spreading ability. These findings suggest that cellular toxin has a significant role in S. aureus colony spreading.1SAP-05 らせん細菌 Leptospira の遊泳力学とエネルギー論 Swimming dynamics and energetics of the spirochete LeptospiraShuichi Nakamura (Grad. Sch. Eng., Tohoku Univ.) Leptospira are spirochetes and pathogenic species cause a zoonotic disease.Leptospira have a right-handed and short-pitch helical cell body, which is called protoplasmic cylinder. When a cell swims in liquid, the anterior and the posterior cell ends are transformed into a left-handed and long-pitch helix (spiral), and a half circle (hook), respectively. The spiral end rotates counterclockwise and ...