KidA, a novel interactor of the cyanobacterial circadian oscillator that tunes its period.

Abstract

Composed of only three proteins, KaiA, KaiB, and KaiC, the cyanobacterial circadian clock is the simplest known biochemical oscillator that can be reconstituted in vitro. While the molecular mechanism of the three proteins has been studied in depth, how they are connected to the rest of the cellular physiology remains unclear. In order to identify previously unknown players in the broader network of the circadian system, we performed a coimmunoprecipitation (co-IP)/mass spec experiment on S. elongatus PCC 7942 cell lysate. Using KaiB in its nighttime form (fold-switched KaiB) as bait, we identified a novel clock interactor protein we named Kai-Interacting Diguanylate cyclase A (KidA), which is predicted to have four N-terminal PAS domains and C-terminal GGDEF/EAL domains. We found that KidA shortens the period of the in vivo rhythms in a dose-dependent manner and alters the entrainment to light-dark cycles. We found that an N-terminal triple-PAS domain fragment is sufficient for the period-shortening phenotype. Consistently, adding purified triple-PAS fragment to the in vitro Kai oscillator reconstituted the dose-dependent shortening of the period. Our in vitro co-IP showed that the N-terminal PAS domains of KidA can directly and specifically bind to the fold-switched form of KaiB. To understand the potential mechanism, we used a computational modeling approach where a fold-switched KaiB binder was added to the Kai oscillator model. The results showed that KidA can tune the period by shifting the equilibrium of KaiB fold-switching. The discovery of KidA suggests a potential role of PAS domain in prokaryotic timekeeping, which has been well documented only in eukaryotic clock systems. Future studies could unveil more potential of KidA, such as integration of time-of-day information and other signals via multiple PAS domains and regulation of cellular processes via cyclic-di-GMP.