Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC

Abstract

The cyanobacterial circadian oscillator can be reconstituted in vitro. In the presence of KaiA and KaiB, the phosphorylation state of KaiC oscillates with a periodicity of ∼24 h. KaiC is a hexameric P-loop ATPase with autophosphorylation and autodephosphorylation activities. Recently, we found that dephosphorylation of KaiC occurs via reversal of the phosphorylation reaction: a phosphate group attached to Ser431/Thr432 is transferred to KaiC-bound ADP to generate ATP, which is subsequently hydrolyzed. This unusual reaction mechanism suggests that the KaiC phosphorylation rhythm is sustained by periodic shifts in the equilibrium of the reversible autophosphorylation reaction, the molecular basis of which has never been elucidated. Because KaiC-bound ATP and ADP serve as substrates for the forward and reverse reactions, respectively, we investigated the regulation of the nucleotide-bound state of KaiC. In the absence of KaiA, the condition in which the reverse reaction proceeds, KaiC favored the ADP-bound state. KaiA increased the ratio of ATP to total KaiC-bound nucleotides by facilitating the release of bound ADP and the incorporation of exogenous ATP, allowing the forward reaction to proceed. When both KaiA and KaiB were present, the ratio of ATP to total bound nucleotides exhibited a circadian rhythm, whose phase was advanced by several hours relative to that of the phosphorylation rhythm. Based on these findings, we propose that the direction of the reversible autophosphorylation reaction is regulated by KaiA and KaiB at the level of substrate availability and that this regulation sustains the oscillation of the phosphorylation state of KaiC.in vitro reconstitution | self-sustained oscillation | nucleotide exchange

Timing the day: what makes bacterial clocks tick?

“…At the beginning of the nanocomplex cycle, KaiA rapidly interacts intermittently with the CII tentacles of KaiC 59–62 . This transient interaction stimulates the phosphorylation of KaiC (or inhibits dephosphorylation, thereby shifting the reaction towards phosphorylation) 57,58,63 , and when S431 of KaiC achieves the (T432/pS431) state in the phosphorylation sequence, KaiB binds to KaiC. A recent study reported that KaiB exists in two diametrically different conformations: a ground state (gsKaiB) that does not interact readily with KaiC, and a substantially fold-shifted state (fsKaiB) that binds to the CI domain 18 (FIG.…”

Sufficiency of unidirectional allostery in KaiC in generating the cyanobacterial circadian rhythm

“…It has been experimentally suggested that the change of the ADP/ATP exchange in C2 induces an equilibrium shift toward (de)phosphorylated states of C2 [16]. The present model designs the sequential phosphorylation of C2 as schematically shown in Fig.…”

“…Moreover, we assume that KaiA can bind to C2 only when C2 is in the exposed state. To realize the experimental result that KaiA facilitates the ADP/ATP exchange in C2 [16], we also assume that the ADP/ATP exchange in C2 is allowed only in the exposed state. With these assumptions, the KaiA binding promotes the ADP/ATP exchange through the stabilization of the exposed state, i.e.…”

“…The detailed procedure of the optimization, the experimental data used in the optimization, and the optimized parameter values are summarized in Method. Figure 3G shows the ratio of the bound ATP in KaiC to the bound nucleotide, in which the corresponding experimental data [16] at time t = 8 h are considered in the optimization. Although the result of the model in the presence of KaiA deviates from the experimental data to some extent, the model reproduces the experimental result that the ratio in the presence of KaiA is larger than in the absence of KaiAB due to the promotion of the ADP/ATP exchange in C2 by KaiA [16].…”

Intricate Protein-Protein Interactions in the Cyanobacterial Circadian Clock

“…The former are composed of kinase, phospho-transferase (ATP synthase (47,48)), and ATPases in CII and CI. KaiB may modulate phosphorylation, dephosphorylation, or ATP hydrolysis, or perhaps influence the ratio between bound ADP and ATP (49). Because of the rotational symmetry of the KaiC particle (Fig.…”