CIRCADIAN RHYTHMS - Cyanobacteria - page 311

portion. These domains probably correspond to the N-(amino acid residues 1-268) and C-terminal
halves (amino acid residues 269-518) which have a duplicate amino acid sequence (Hayashi et al .,
2003). Pattanayek et al . (2004) studied the crystal structure of KaiC that revealed a doughnut-shaped
structure with a central pore/channel. The hexameric structure showed an ATP-binding and a
scaffold portion for Kai-protein complex formation (Fig. 1C). The central channel runs through that
is wide open at N-terminal portion and narrows down at C-terminal region. The channel in CII half
represents the portion where it binds to DNA.
KaiC is a kinase that autophosphorylates its own Ser and Thr residues in vitro (Nishiwaki
et al ., 2000) and shows a clear circadian phosphorylation rhythm in vivo (Iwasaki et al ., 2002).
Mutation of either of these residues nullifi ed circadian rhythms in KaiBC gene expression. Two
autophosphorylation sites on KaiC have been identifi ed by Nishiwaki et al. (2004) by using mass
spectrometry. These are at Ser431 and Thr432. By site directed mutagenesis, these amino acids were
substituted by Ala to obtain single and double mutants. The phosphorylation of KaiC was reduced
in the former while in the latter it was completely abolished suggesting that these are the sites that
are phosphorylated in vivo as well. With loss of phosphorylation, the mutants lost circadian rhythm.
These proteins were expressed in E . coli BL21, purifi ed and in vitro complex formation studies were
conducted further to know the interactions. Though KaiC was able to form hexamers in vitro it was
unable to form complexes with KaiA, KaiB and SasA. These observations further signify that KaiC
phosphorylation regulates its own transcriptional repression activity by controlling its binding affi nity
to other clock proteins. On the other hand, Xu et al . (2004) identifi ed three potential phosphorylation
sites at Thr432, Ser431 and Thr426 residues in the KaiCII domains by crystallographic and mutagenic
analysis. Substitution of these residues by Ala singly or in combination altered phosphorylation
of KaiC with concomitant abolition of circadian rhythms. Analysis of the mutants suggested that
Ser431 and Thr426 may share a phosphate that can shuttle between these two residues there by
modulating the KaiC activity. So phosphorylation of Thr432 and Ser431/Thr426 appear to be the
key component for circadian clock to function in cyanobacteria. McClung (2007) suggested that
KaiC possesses at least three enzyme activities, i.e. an autokinase, autophosphatase and ATPase
and these three activities mutually infl uence each other. Evidences in favour of a weak and stable
ATPase activity of KaiC are presented by Terauchi et al . (2007). In presence of KaiA and KaiB the
ATPase activity oscillated with a circadian period in vitro . At different temperatures too the ATPase
activity of KaiC was not variable suggesting that temperature compensation of circadian period
seems to be mainly dependent on this reaction. They thus concluded that KaiC ATPase activity is
the most fundamental reaction underlying circadian periodicity in cyanobacteria.
D) Interaction between Kai proteins : Two types of interactions are known, i.e. homotypic and
heterotypic interactions (Iwasaki et al ., 1999). Based on studies on resonance energy transfer to
assay protein-protein interactions, Xu et al . (1999) demonstrated that KaiB proteins interact among
themselves (homotypic interaction). Based on the studies on a long period mutant that exhibits
heterotypic interaction between KaiA and KaiB, it was suggested that interaction between Kai proteins
is important for clock mechanism (heterotypic interaction). Further a negative feed back control of
KaiC expression by KaiC protein to induce the formation of circadian oscillation in cyanobacteria.
Two important points that emerge are that (i) protein-protein interactions are necessary for clock
expression and (ii) the oscillation is sustained by KaiA by enhancing KaiC expression.
The phosphorylation of KaiC constitutes a potential signal for two important processes. Firstly,
ectopic overexpression of KaiA leads to increased kaiBC transcription that in turn increases kaiC
gene expression patterns. Secondly, ectopic overproduction of KaiC represses kaiC gene expression