Protein-Protein Interactions in the Cyanobacterial Kaiabc Circadian Clock

Williams

et al. 2008

EMBO J

Self Cite

143

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by the KaiA, KaiB and KaiC proteins in the presence of ATP. The principal clock component, KaiC, undergoes regular cycles between hyper‐ and hypo‐phosphorylated states with a period of ca. 24 h that is temperature compensated. KaiA enhances KaiC phosphorylation and this enhancement is antagonized by KaiB. Throughout the cycle Kai proteins interact in a dynamic manner to form complexes of different composition. We present a three‐dimensional model of the S. elongatus KaiB–KaiC complex based on X‐ray crystallography, negative‐stain and cryo‐electron microscopy, native gel electrophoresis and modelling techniques. We provide experimental evidence that KaiB dimers interact with KaiC from the same side as KaiA and for a conformational rearrangement of the C‐terminal regions of KaiC subunits. The enlarged central channel and thus KaiC subunit separation in the C‐terminal ring of the hexamer is consistent with KaiC subunit exchange during the dephosphorylation phase. The proposed binding mode of KaiB explains the observation of simultaneous binding of KaiA and KaiB to KaiC, and provides insight into the mechanism of KaiB's antagonism of KaiA.

Section: Introductionmentioning

confidence: 80%

Structural model of the circadian clock KaiB–KaiC complex and mechanism for modulation of KaiC phosphorylation

Williams

et al. 2008

EMBO J

Self Cite

143

“…This network of intramolecular interactions results in a “domino effect” of phosphorylations jumping from one subunit to the next. Therefore, it seems unnecessary to invoke a model with a KaiA dimer migrating from one KaiC subunit to the next or two or more KaiA dimers simultaneously binding to multiple KaiC tails. In fact, it was found early on in the study of the cyanobacterial clock that a single KaiA dimer is sufficient to upregulate phosphorylation of a KaiC hexamer to the maximal level .…”

Section: Discussionmentioning

confidence: 99%

Loop–Loop Interactions Regulate KaiA-Stimulated KaiC Phosphorylation in the Cyanobacterial KaiABC Circadian Clock

et al. 2013

Self Cite

The Synechococcus elongatus KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that runs in a temperature-compensated manner with a period of 24 hours. KaiA dimer stimulates phosphorylation of KaiC hexamer at two sites per subunit, T432 and S431, and KaiB dimers antagonize KaiA action and induce KaiC subunit exchange. Neither the mechanism of KaiA-stimulated KaiC phosphorylation nor that of KaiB-mediated KaiC dephosphorylation is understood in detail at the present time. We demonstrate here that the A422V KaiC mutant sheds light on the former mechanism. It was previously reported that A422V is less sensitive to dark pulse-induced phase resetting and has a reduced amplitude of the KaiC phosphorylation rhythm in vivo. A422 maps to a loop (422-loop) that continues toward the phosphorylation sites. By pulling on the C-terminal peptide of KaiC (A-loop), KaiA removes restraints from the adjacent 422-loop whose increased flexibility indirectly promotes kinase activity. We found in the crystal structure that A422V KaiC lacks phosphorylation at S431 and exhibits a subtle, local conformational change relative to wild-type KaiC. MD simulations indicate higher mobility of the 422-loop in the absence of the A-loop and mobility differences in other areas associated with phosphorylation activity between wild-type and mutant KaiCs. The A-loop···422-loop relay that informs KaiC phosphorylation sites of KaiA dimer binding propagates to loops from neighboring KaiC subunits, thus providing support for a concerted allosteric mechanism of phosphorylation.

“…In both the ‘tethered’ and the ‘engaged’ configurations this S-shaped loop has to unravel and this appears to increase the flexibility of the six KaiCII domains with respect to each other. As phosphorylation of the KaiC S431 and T432 residues occurs across subunit interfaces, increased flexibility would tend to enhance auto-kinase activity [4] , [5] , [27] , [35] . This is consistent with the observation that the S-shaped loop is important for locking KaiC in either the hypo- or the hyper-phosphorylated state [36] and the earlier observation that a single KaiA dimer is able to drive KaiC to the hyper-phosphorylated state [37] .…”

Section: Introductionmentioning

confidence: 99%

Combined SAXS/EM Based Models of the S. elongatus Post-Translational Circadian Oscillator and its Interactions with the Output His-Kinase SasA

et al. 2011

Self Cite

The circadian clock in the cyanobacterium Synechococcus elongatus is composed of a post-translational oscillator (PTO) that can be reconstituted in vitro from three different proteins in the presence of ATP and a transcription-translation feedback loop (TTFL). The homo-hexameric KaiC kinase, phosphatase and ATPase alternates between hypo- and hyper-phosphorylated states over the 24-h cycle, with KaiA enhancing phosphorylation, and KaiB antagonizing KaiA and promoting KaiC subunit exchange. SasA is a His kinase that relays output signals from the PTO formed by the three Kai proteins to the TTFL. Although the crystal structures for all three Kai proteins are known, atomic resolution structures of Kai and Kai/SasA protein complexes have remained elusive. Here, we present models of the KaiAC and KaiBC complexes derived from solution small angle X-ray scattering (SAXS), which are consistent with previous EM based models. We also present a combined SAXS/EM model of the KaiC/SasA complex, which has two N-terminal SasA sensory domains occupying positions on the C-terminal KaiC ring reminiscent of the orientations adopted by KaiB dimers. Using EM we demonstrate that KaiB and SasA compete for similar binding sites on KaiC. We also propose an EM based model of the ternary KaiABC complex that is consistent with the sequestering of KaiA by KaiB on KaiC during the PTO dephosphorylation phase. This work provides the first 3D-catalogue of protein-protein interactions in the KaiABC PTO and the output pathway mediated by SasA.