Dynamic fluctuations lubricate the circadian clock.

S everal biological processes related to metabolism and cell division are controlled by biochemical oscillators that underpin circadian clock systems. The function of such circadian oscillators is modulated by rhythmic, phosphorylation-dependent interactions between proteins of the clock system. The self-sustained circadian clocks oscillate through different phases characterized by distinct phosphorylation states (1–3). Understanding the mechanisms underlying this process has been very challenging. A new study in PNAS (4) provides intriguing data implicating changes in intrinsic protein dynamics in the modulation of the circadian timing and rhythms. A major breakthrough in the field has been the finding that the simple mixture of three proteins from cyanobacteria, KaiA, KaiB, and KaiC, together with ATP, is sufficient to generate a self-sustained ≈24-h rhythm of KaiC phosphorylation (5). KaiC autophosphorylation and autodephosphorylation follow the ordered pattern ST→SpT→pSpT→pST→ST, with S/pS and T/pT corresponding to the unphosphorylated/phosphorylated forms of residues Ser431 and Thr432, respectively (Fig. 1). This oscillator is optimally suited for mechanistic studies and has provided a model system for understanding the fundamental underlying mechanisms of this biological process. In their work reported in PNAS, Chang et al. (4) use primarily NMR spectroscopy to characterize the conformational and dynamic properties of KaiC in its different phosphorylation states. KaiC is a large kinase comprising two domains, CI and CII, that forms a homohexameric oligomer with a molecular mass of ≈350 kDa (1). Because of the large size of this supramolecular system, Chang et al. resorted to using methyl-TROSY and methyl labeling schemes, an approached pioneered by the group of Lewis Kay (6–8) that has recently enabled the structural and dynamic NMR characterization of large systems (6, 9). Although this approach has proven to be very robust for recording spectra of large proteins with high sensitivity and resolution, a major hurdle in obtaining site-specific information remains the difficulty in obtaining resonance assignment. The only approach currently available is to “disassemble” the supramolecular system: for higher-order oligomeric systems, such as the proteasome (6), by preparing the subunit in its monomeric form; and for large singlechain proteins, such as the SecA ATPase (9), by preparing isolated domains or fragments. Although to date the challenging nature of the KaiC system has impeded site-specific assignment, the authors were able to distinguish several of the Ile methyl signals between the CI and CII domains. The Ile methyl groups are located at strategic locations and thus provided excellent probes for monitoring the effect of the different phosphorylation states on the conformational and dynamic properties of KaiC. The signals in an NMR spectrum carry information about both the average structure (signal position) as well as the dynamic properties (line broadening) of the protein (10). Signal line broadening is particularly sensitive to conformational exchange processes, which take place on the microto millisecond (μs-ms) time scale, and may report on important dynamic and conformational phenomena in the protein. NMR analysis of the KaiC NMR signals indicated that the oscillation of the kinase among the different phosphorylation states is accompanied by significant changes in the protein intrinsic motions. Using phosphomimetics as a means to mimic the phosphorylation states and produce homogenous samples, Chang et al. observed that there is a pattern in the change of slow motions (μs-ms) in the CII ring that follows the pattern STflexible→ SpTflexible→pSpTrigid→pSTvery-rigid→ STflexible (Fig. 1). The CII ring contains both phosphorylation sites (Ser431 and Thr432) as well as the A-loops (11). The A-loop alternates between an exposed and a buried conformation. In the exposed conformation, KaiA binds to the A-loop and induces autophosphorylation of KaiC. The authors suggest that the flexibility observed in the ST phase, which is retained in the SpT phase, stimulates KaiA binding and thus phosphorylation by CI CII P