Jonathan Valencia Swain

The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria

Background: The function of KaiB remains to be solved. Results: Dimeric KaiB1–94 generated circadian oscillation in vitro, but it did not in cells. Conclusion: KaiB tetramer-dimer transformation is responsible for the regulation of the SasA-mediated clock output pathway. Significance: We demonstrated the role of KaiB in the regulation of the SasA-KaiC interaction, involved in the transmission of time-information from KaiABC-machinery to transcription apparatus. The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB1–94), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB1–94 interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB1–94 also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB1–94 showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB1–94.

Site-directed spin labeling-electron spin resonance mapping of the residues of cyanobacterial clock protein KaiA that are affected by KaiA–KaiC interaction

The cyanobacterial clock proteins KaiA, KaiB and KaiC interact with each other to generate circadian oscillations. We have identified the residues of the KaiA homodimer affected through association with hexameric KaiC (KaiC6mer) using a spin‐label‐tagged KaiA C‐terminal domain protein (KaiAc) and performing electron spin resonance (ESR) analysis. Cys substitution and/or the attachment of a spin label to residues located at the bottom area of the KaiAc concave surface, a KaiC‐binding groove, hindered the association of KaiAc with KaiC6mer, suggesting that the groove likely mediates the interaction with KaiC6mer. The residues affected by KaiC6mer association were concentrated in the three areas: the concave surface, a lobe‐like structure (a mobile lobe near the concave surface) and a region adjacent to both the concave surface and the mobile lobe. The distance between the two E254, D255, L258 and R252 residues located on the mobile lobe decreased after KaiC association, suggesting that the two mobile lobes approach each other during the interaction. Analyzing the molecular dynamics of KaiAc showed that these structural changes suggested by ESR analysis were possible. Furthermore, the analyses identified three asymmetries in KaiAc dynamic structures, which gave us a possible explanation of an asymmetric association of KaiAc with KaiC6mer.