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Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

ABSTRACT: KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadian rhythm, we investigated structural changes of an inner-radius side of the CII ring using time-resolved Trp fluorescence spectroscopy. A KaiC mutant harboring a Trp fluorescence probe at a position of 419 exhibited a robust circadian rhythm with little temperature sensitivity in the presence of KaiA and KaiB. Our fluorescence observations show a remarkable environmental change at the inner-radius side of the CII ring during circadian oscillation. Crystallographic analysis revealed that a side chain of Trp at the position of 419 was oriented toward a region undergoing a helix-coil transition, which is considered to be a key event to allosterically regulate the CI ring that plays a crucial role in determining the cycle period. The present study provides a dynamical insight into how KaiC generates circadian oscillation.

SUBMITTER: Mukaiyama A

PROVIDER: S-EPMC9342895 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

Mukaiyama Atsushi A Furuike Yoshihiko Y Yamashita Eiki E Akiyama Shuji S

The Biochemical journal 20220701 14

KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadi ...[more]

PMID: 35771042

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Project description:The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between α/β heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.

Dataset Information

Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

Publications

Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

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