Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex.
KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 A and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95-108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria.
Iwase
Ryo
R
Center for Gene Research, Nagoya University, Furo, Chikusa, Nagoya, Japan.
Imada
Katsumi
K
Hayashi
Fumio
F
Uzumaki
Tatsuya
T
Morishita
Megumi
M
Onai
Kiyoshi
K
Furukawa
Yukio
Y
Namba
Keiichi
K
Ishiura
Masahiro
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002850:Chromatography, Gel; D002940:Circadian Rhythm; D056925:Circadian Rhythm Signaling Peptides and Proteins; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D018014:Gene Transfer Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; D003432:Cross-Linking Reagents; C114270:KaiB protein, cyanobacteria