Structure of the C-terminal domain of the clock protein KaiA in complex with a KaiC-derived peptide: Implications for KaiC regulation

Abstract

Circadian clocks are widespread endogenous mechanisms that control the temporal pattern of diverse biological processes, including gene transcription. KaiA is the positive element of the cyanobacterial clock because KaiA overexpression elevates transcription levels of clock components. Recently, we showed that the structure of KaiA is that of a domain-swapped homodimer. The N-terminal domain is a pseudo-receiver; thus, it is likely to be involved in signal transduction in the clock-resetting pathway. The C-terminal domain of KaiA is structurally novel and enhances the KaiC autokinase activity directly. Here, we report the NMR structure of the C-terminal domain of KaiA (ThKaiA180C) in complex with a KaiC-derived peptide from the cyanobacterium Thermosynechococcus elongatus BP-1. The protein-peptide interface is revealed to be different from a model that was proposed earlier, is stabilized by a combination of hydrophobic and electrostatic interactions, and includes many residues known to produce a circadian-period phenotype upon substitution. Although the structure of the monomeric subunit of ThKaiA180C is largely unchanged upon peptide binding, the intersubunit dimerization angle changes. It is proposed that modulation of the C-terminal KaiA domain dimerization angle regulates KaiA-KaiC interactions.

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

“…The highly conserved homologs from the thermophile T. elongatus, on the other hand, were superior with respect to expression, stability, and solubility. The T. elongatus and S. elongatus Kai proteins are highly similar with respect to sequence, structure, and biochemistry (21,27,(36)(37)(38)(39). Thus, the results herein were obtained on T. elongatus proteins, which will henceforth be referred to as simply KaiA, KaiB, and KaiC.…”

“…KaiA has three segments, an N-terminal domain (residues 1-129), a linker segment (residues 130-179), and a C-terminal dimerization domain (residues 180-283) (47). The C-terminal domain binds to the A loops and C-terminal tails of KaiC during the phosphorylation phase (20,21), but is apparently prevented from doing so by KaiB during the dephosphorylation phase (15,23). The site of sequestration on KaiA was unknown.…”

“…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.…”

Meshing the gears of the cyanobacterial circadian clock

“…Some phosphorylation-dependent changes in the hexamer itself can be inferred from the crystal structure (11). A glimpse into the conformational changes that Kai heterotypic interactions impart is evident in the rotation of the C-terminal dimeric domain of KaiA when it binds a specific peptide of KaiC (4).…”

“…With fitting élan, a small community of scientists has ripped open the packaging of the cyanobacterial circadian clock, compiled the parts list, examined the gears, and begun to piece together the mechanism. Over the past 2 years, the 3D molecular structures have been solved for the core components of the cyanobacterial circadian clock: KaiA, KaiB, and KaiC (1)(2)(3)(4)(5)(6). In a surprisingly literal analogy to mechanical timepieces, the protein that seems to be at the heart of the clock mechanism, KaiC, forms a hexameric ring that even looks like a cog: the escape wheel, perhaps (5,7,8).…”