Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB

Atsushi Mukaiyama,Shuji Akiyama,Yoshihiko Furuike,Shin-Ichi Koda,Takao Kondo,Jun Abe,Eiki Yamashita

doi:10.1038/s41598-018-27131-8

Abstract

KaiC, the core oscillator of the cyanobacterial circadian clock, is composed of an N-terminal C1 domain and a C-terminal C2 domain, and assembles into a double-ring hexamer upon ATP binding. Cyclic phosphorylation and dephosphorylation at Ser431 and Thr432 in the C2 domain proceed with a period of approximately 24 h in the presence of other clock proteins, KaiA and KaiB, but recent studies have revealed a crucial role for the C1 ring in determining the cycle period. In this study, we mapped dynamic structural changes of the C1 ring in solution using a combination of site-directed tryptophan mutagenesis and fluorescence spectroscopy. We found that the C1 ring undergoes a structural transition, coupled with ATPase activity and the phosphorylation state, while maintaining its hexameric ring structure. This transition triggered by ATP hydrolysis in the C1 ring in specific phosphorylation states is a necessary event for recruitment of KaiB, limiting the overall rate of slow complex formation. Our results provide structural and kinetic insights into the C1-ring rearrangements governing the slow dynamics of the cyanobacterial circadian clock.

Highlights

KaiC, the core oscillator of the cyanobacterial circadian clock, is composed of an N-terminal C1 domain and a C-terminal C2 domain, and assembles into a double-ring hexamer upon ATP binding
We found that W157 fluorescence is sensitive to the conformational change of the C1 ring, and to KaiB binding that is known to occur in the C1 ring[15,16]
To the previous works[22,23,24], these results demonstrate that the binding affinity of KaiB to the C1 ring is dependent on the phosphorylation state

Summary

Introduction

KaiC, the core oscillator of the cyanobacterial circadian clock, is composed of an N-terminal C1 domain and a C-terminal C2 domain, and assembles into a double-ring hexamer upon ATP binding. Cyanobacteria are the simplest organisms known to exhibit a circadian rhythm[1] Their clock oscillator is composed of three proteins, KaiA, KaiB, and KaiC2. In the presence of KaiA and KaiB, KaiC rhythmically alters its own ATPase activity[3], auto-phosphorylation/auto-dephosphorylation activities[4], and assembly state with other Kai proteins[5] with a period of approximately 24 h (Kai oscillator). The period of these rhythmic phenomena is minimally dependent on the temperature; this property, termed temperature compensation, is common to circadian systems from multiple species. Using a series of KaiC mutants harboring a fluorescence probe for the C1-ring structure, we obtained evidence that the structural transition of the C1 ring is coupled with ATPase activity and the phosphorylation state and is the origin of the basic timing cue for assembly with KaiB

Methods

Results

Conclusion