title: Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering.
abstract: The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Sugiyama
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Yagi
authors.affiliation: Okazaki Institute for Integrative Bioscience and 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Ishii
authors.affiliation: Institut Laue-Langevin, 71, Avenue des Martyrs, Grenoble 38042, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Porcar
authors.affiliation: Institut Laue-Langevin, 71, Avenue des Martyrs, Grenoble 38042, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Martel
authors.affiliation: Graduate School of Life Sciences, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan.
authors.forename: Katsuaki
authors.initials: K
authors.lastname: Oyama
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Noda
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yunoki
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Murakami
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan.
authors.forename: Rintaro
authors.initials: R
authors.lastname: Inoue
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Sato
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan.
authors.forename: Yojiro
authors.initials: Y
authors.lastname: Oba
authors.affiliation: Graduate School of Life Sciences, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Terauchi
authors.affiliation: Okazaki Institute for Integrative Bioscience and 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.|Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.|Okazaki Institute for Integrative Bioscience and 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.|Institute for Molecular Sciences, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057906:Circadian Clocks; D056925:Circadian Rhythm Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D053838:Scattering, Small Angle; D046940:Synechococcus / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114269:KaiA protein, cyanobacteria; C114270:KaiB protein, cyanobacteria; C114271:KaiC protein, cyanobacteria; D046912:Multiprotein Complexes; D010770:Phosphotransferases; C083143:SasA protein, Synechococcus