A protein fold switch joins the circadian oscillator to clock output in cyanobacteria

Abstract

Organisms are adapted to the relentless cycles of day and night, because they evolved timekeeping systems called circadian clocks, which regulate biological activities with ~24-h rhythms. The clock of cyanobacteria is driven by a three-protein oscillator comprised of KaiA, KaiB, and KaiC, which together generate a circadian rhythm of KaiC phosphorylation. We show that KaiB flips between two distinct three-dimensional folds, and its rare transition to an active state provides a time delay that is required to match the timing of the oscillator to that of earth’s rotation. Once KaiB switches folds, it binds phosphorylated KaiC and captures KaiA, initiating a phase transition of the circadian cycle, and regulates components of the clock-output pathway, providing the link that joins the timekeeping and signaling functions of the oscillator.

Circadian Rhythms in Cyanobacteria

“…Moreover, expression of fs-KaiB in vivo abolishes rhythms when expressed either as the only copy of kaiB or in addition to the endogenous copy. The fact that expression of fsKaiB results in a dominant-negative phenotype supports the notion that fs-KaiB is the active form of KaiB (41). The transition from gs-KaiB to fs-KaiB is rare, and this low probability accounts for the slowness of the KaiB-KaiC complex association and for a significant element of the time delay necessary for 24-h timekeeping.…”

“…Major structural rearrangements in KaiB, specifically, rearrangement of the KaiB tertiary structure from a ground state (gs) to a fold-switched (fs) state, have recently come to light that play critical roles in driving the KaiC phosphorylation cycle in a clockwise direction, imparting time delay and linking the oscillator to output functions (41). X-ray crystallography has shown KaiB to exist as a homotetramer, organized as a dimer of dimers (42,43).…”

“…While KaiB exists as a tetramer in solution, it was believed that KaiB bound to KaiC as a dimer (34,(47)(48)(49). However, recent studies have shown that KaiB binds to KaiC as a monomer (41,47,50). Surprisingly, this monomeric form of KaiB no longer exhibits the typical gs fold found in the dimeric and tetrameric forms but rather has completely rearranged, or fold-switched (fs), its tertiary structure to have a thioredoxin-like fold, similar to the N terminus of SasA (41).…”

“…However, recent studies have shown that KaiB binds to KaiC as a monomer (41,47,50). Surprisingly, this monomeric form of KaiB no longer exhibits the typical gs fold found in the dimeric and tetrameric forms but rather has completely rearranged, or fold-switched (fs), its tertiary structure to have a thioredoxin-like fold, similar to the N terminus of SasA (41). Mutations that stabilize the fold-switched version of KaiB (fs-KaiB) cause very rapid binding to KaiC: complete binding is observed in minutes, in contrast to wild-type (WT) or gsKaiB, where hours transpire before an interaction is detected in vitro (41).…”

“…This trimolecular interaction sequesters KaiA away from the A-loop and promotes the dephosphorylation phase of the cycle. While there is some controversy regarding whether it is the CI or CII ring of KaiC that is bound by KaiB (51), new evidence clearly demonstrates physical binding of fs-KaiB to the CI ring (41); moreover, this binding is dependent on the presence of the B-loops, which are absent from CII (38,41). These data suggest that, regardless of interactions KaiB may have with the CII domain, it is the interactions with the CI ring that are required to inhibit KaiA and promote the dephosphorylation phase of the cycle.…”