Analysis of KaiA–KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods

Abstract

The cyanobacterial circadian clock can be reconstituted in vitro by mixing recombinant KaiA, KaiB and KaiC proteins with ATP, producing KaiC phosphorylation and dephosphorylation cycles that have a regular rhythm with a ca. 24-h period and are temperature-compensated. KaiA and KaiB are modulators of KaiC phosphorylation, whereby KaiB antagonizes KaiA's action. Here, we present a complete crystallographic model of the Synechococcus elongatus KaiC hexamer that includes previously unresolved portions of the C-terminal regions, and a negativestain electron microscopy study of S. elongatus and Thermosynechococcus elongatus BP-1 KaiA-KaiC complexes. Site-directed mutagenesis in combination with EM reveals that KaiA binds exclusively to the CII half of the KaiC hexamer. The EM-based model of the KaiA-KaiC complex reveals protein-protein interactions at two sites: the known interaction of the flexible C-terminal KaiC peptide with KaiA, and a second postulated interaction between the apical region of KaiA and the ATP binding cleft on KaiC. This model brings KaiA mutation sites that alter clock period or abolish rhythmicity into contact with KaiC and suggests how KaiA might regulate KaiC phosphorylation.

A Cyanobacterial Circadian Clock Based on the Kai Oscillator

“…We discuss this issue in a later section of this chapter. Similar results of Kai protein interaction and KaiC monomer exchange were also reported using different methods (Pattanayek et al 2006). The shuffling of KaiC monomers between different phosphorylation states was also predicted to be important for the stability of the oscillator (Emberly and Wingreen 2006).…”

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

“…The A loops of free KaiC are in a dynamic equilibrium that favors the buried positions, thereby maintaining KaiC in a hypophosphorylated state (20). KaiA selectively captures the A loops of KaiC in their exposed positions, thereby shifting the population of KaiC proteins to autophosphorylate (ST → SpT → pSpT) (20)(21)(22). However, the allosteric mechanism by which A-loop exposure activates autophosphorylation at sites >15 Å away was unclear.…”

Insight into cyanobacterial circadian timing from structural details of the KaiB–KaiC interaction

“…KaiC forms a hexamer with a double ring structure, the CI and CII domain each forming one ring, stacked together (4,25,26). Free KaiB has been crystallized as a tetramer in four studies and has once been described as a dimer (27)(28)(29)(30)(31)(32).…”