Dynamic fluctuations lubricate the circadian clockMing-Tao Pai and Charalampos Kalodimos1

Department of Chemistry and Chemical Biology and BioMaPS Institute for Quantitative Biology, Rutgers University,Piscataway, NJ 08854

Several biological processes relatedto metabolism and cell division arecontrolled by biochemical oscil-lators that underpin circadian

clock systems. The function of such circa-dian oscillators is modulated by rhythmic,phosphorylation-dependent interactionsbetween proteins of the clock system. Theself-sustained circadian clocks oscillatethrough different phases characterized bydistinct phosphorylation states (1–3). Un-derstanding the mechanisms underlyingthis process has been very challenging. Anew study in PNAS (4) provides intriguingdata implicating changes in intrinsic pro-tein dynamics in the modulation of thecircadian timing and rhythms.A major breakthrough in the field has

been the finding that the simple mixture ofthree proteins from cyanobacteria, KaiA,KaiB, and KaiC, together with ATP, issufficient to generate a self-sustained ≈24-hrhythm of KaiC phosphorylation (5).KaiC autophosphorylation and autode-phosphorylation follow the ordered pat-tern ST→SpT→pSpT→pST→ST, withS/pS and T/pT corresponding to the un-phosphorylated/phosphorylated forms ofresidues Ser431 and Thr432, respectively(Fig. 1). This oscillator is optimally suitedfor mechanistic studies and has provideda model system for understanding thefundamental underlying mechanisms ofthis biological process. In their work re-ported in PNAS, Chang et al. (4) useprimarily NMR spectroscopy to charac-terize the conformational and dynamicproperties of KaiC in its differentphosphorylation states.KaiC is a large kinase comprising two

domains, CI and CII, that forms a homo-hexameric oligomer with a molecular massof ≈350 kDa (1). Because of the large sizeof this supramolecular system, Changet al. resorted to using methyl-TROSYand methyl labeling schemes, an ap-proached pioneered by the group of LewisKay (6–8) that has recently enabled thestructural and dynamic NMR character-ization of large systems (6, 9). Althoughthis approach has proven to be very robustfor recording spectra of large proteins withhigh sensitivity and resolution, a majorhurdle in obtaining site-specific informa-tion remains the difficulty in obtainingresonance assignment. The only approachcurrently available is to “disassemble” thesupramolecular system: for higher-orderoligomeric systems, such as the protea-some (6), by preparing the subunit in its

monomeric form; and for large single-chain proteins, such as the SecA ATPase(9), by preparing isolated domains orfragments. Although to date the chal-lenging nature of the KaiC system hasimpeded site-specific assignment, the au-thors were able to distinguish several ofthe Ile methyl signals between the CI andCII domains. The Ile methyl groups arelocated at strategic locations and thusprovided excellent probes for monitoringthe effect of the different phosphorylationstates on the conformational and dynamicproperties of KaiC.The signals in an NMR spectrum carry

information about both the averagestructure (signal position) as well as thedynamic properties (line broadening) ofthe protein (10). Signal line broadening isparticularly sensitive to conformationalexchange processes, which take place onthe micro- to millisecond (μs-ms) timescale, and may report on important dy-namic and conformational phenomena inthe protein. NMR analysis of the KaiCNMR signals indicated that the oscillationof the kinase among the different phos-

phorylation states is accompanied by sig-nificant changes in the protein intrinsicmotions. Using phosphomimetics as ameans to mimic the phosphorylation statesand produce homogenous samples, Changet al. observed that there is a pattern inthe change of slow motions (μs-ms) in theCII ring that follows the pattern STflexible→SpTflexible→pSpTrigid→pSTvery-rigid→STflexible (Fig. 1). The CII ring containsboth phosphorylation sites (Ser431 andThr432) as well as the A-loops (11). TheA-loop alternates between an exposed anda buried conformation. In the exposedconformation, KaiA binds to the A-loopand induces autophosphorylation of KaiC.The authors suggest that the flexibilityobserved in the ST phase, which is re-tained in the SpT phase, stimulates KaiAbinding and thus phosphorylation by

CI

CII

P

ST

SpT

pSpT

pST

P P

P

phosphorylation phase

dephosphorylation phase

flexible

rigid

very rigid

X

Fig. 1. KaiC phosphorylation and dephosphorylation cycle for the cyanobacteria circadian clock.Phosphorylation of Ser431 is denoted by the orange circle, whereas phosphorylation of Thr432 is de-noted by the green circle. Flexibility of the CII rings was inferred from NMR signal broadening analysisand reflects primarily changes in μs-ms protein motions.

Author contributions: M.-T.P. and C.K. wrote the paper.

The authors declare no conflict of interest.

See companion article on page 14431.1To whom correspondence should be addressed: E-mail:babis@rutgers.edu.

www.pnas.org/cgi/doi/10.1073/pnas.1111105108 PNAS | August 30, 2011 | vol. 108 | no. 35 | 14377–14378

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promoting the exposed conformation ofthe A-loop. Interestingly, they observedthat when Ser is first phosphorylated(ST→pST, mimicked by phosphomi-metics) there is a notable rigidification ofthe CII ring that may suppress phosphor-ylation of Thr432. Chang et al. put forwardthe intriguing hypothesis that the changein KaiC dynamics may in fact be the forcethat drives the ST→SpT reaction and in-hibits the ST→pST one (Fig. 1).Another interesting mechanistic aspect

of this study is the observation that expo-sure of the A-loop seems to stimulateautophosphorylation at sites that are lo-cated more than 15 Å away. The notablechanges in protein motions accompanyingthis process suggest that dynamics-drivenallostery may be at play (12–14). Themodulation of the CII ring flexibility isalso suggested to set the correct timing forthe phosphorylation, followed by dephos-

phorylation, by regulating the interactionbetween KaiC and KaiB. In addition, itwas observed that when the CII ring isrigid, the ATPase activity of the CI ringis suppressed.Over the recent years the intimate link

between internal motions and function inprotein–protein and protein–ligand inter-actions has been demonstrated (15–17).NMR has been instrumental in establish-ing the important role of protein dynamicsin mediating allosteric and binding inter-actions. NMR, in addition to providingstructural information of a biological sys-tem in solution, is unique in providing site-specific information about the amplitudeand time scale of motions and accessinglowly populated conformational states(18). Recent breakthrough studies havenow shown that such information canbe accessed in supramolecular systemsas well (6–9).

The work by Chang et al. is significantbecause it highlights the importance ofintrinsic motions, completely overlookeduntil now, in regulating the rhythm of thisinteresting biochemical oscillator. Thework raises several intriguing hypothesesthat will certainly need to be followedup with a more detailed NMR character-ization. Site-specific assignment, as wellas the use of more sophisticated ap-proaches to determine the conformationaldynamic properties at different timescales, will be required to shed light intothe mechanistic basis of the circadianclock system.

ACKNOWLEDGMENTS. Research in protein allo-stery and NMR of supramolecular systems in theC.K. laboratory is supported by National Institutesof Health Grants GM073854 and AI094623 andNational Science Foundation Grants MCB0543698and MCB0842491.

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14378 | www.pnas.org/cgi/doi/10.1073/pnas.1111105108 Pai and Kalodimos

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