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.
Highlights
The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC
KaiCDT mimicked an optimally phosphorylated form of KaiC in terms of KaiB binding in comparison with KaiCWT and the other KaiC mutants, KaiCDE, KaiCAA, and KaiCSE (Fig. 1)
Our native mass spectrometry (MS) data indicated that upon titration with KaiB, the KaiCDT hexamer formed a uniform complex with a molecular mass of 428,600 ± 410 Da, which corresponded to a 6:6 stoichiometry (Fig. 2 and Supplemental Fig. S2), consistent with previous native MS data obtained using hyperphosphorylated KaiCWT18
Summary
The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. To further understand the circadian clock mechanisms of KaiB–KaC assembly, we collected detailed structural information on the protein complexes using inverse contrast-matching small-angle neutron scattering (iCM-SANS) in conjunction with native MS analyses. KaiCAA, KaiCSE, KaiCDT, and KaiCDE were used as phospho-mimicking mutants to examine which phosphorylation form of KaiC exhibits the highest binding affinity to KaiB.
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