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| Subject Categories: Structural Biology |
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| The EMBO Journal
(2006) 25, 2017–2028, doi:10.1038/sj.emboj.7601086 Published online 20 April 2006 |
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| Analysis of KaiA–KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods |
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| Rekha Pattanayek1, Dewight R Williams2, Sabuj Pattanayek1, Yao Xu3, Tetsuya Mori3, Carl H Johnson3, Phoebe L Stewart2 and Martin Egli1 |
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1 Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN, USA 2 Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, TN, USA 3 Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA To whom correspondence should be addressed Martin Egli, Department of Biochemistry, School of Medicine, Vanderbilt University, 607 Light Hall, Nashville, TN 37232, USA. Tel.: +1 615 343 8070; Fax: +1 615 322 7122; E-mail: martin.egli@vanderbilt.edu Received 7 February 2006; Accepted 17 March 2006; Published online 20 April 2006. |
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| Abstract |
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| The cyanobacterial circadian clock can be reconstituted in vitro by mixing recombinant KaiA, KaiB and KaiC proteins with ATP, producing KaiC phosphorylation and dephosphorylation cycles that have a regular rhythm with a ca. 24-h period and are temperature-compensated. KaiA and KaiB are modulators of KaiC phosphorylation, whereby KaiB antagonizes KaiA's action. Here, we present a complete crystallographic model of the Synechococcus elongatus KaiC hexamer that includes previously unresolved portions of the C-terminal regions, and a negative-stain electron microscopy study of S. elongatus and Thermosynechococcus elongatus BP-1 KaiA–KaiC complexes. Site-directed mutagenesis in combination with EM reveals that KaiA binds exclusively to the CII half of the KaiC hexamer. The EM-based model of the KaiA–KaiC complex reveals protein–protein interactions at two sites: the known interaction of the flexible C-terminal KaiC peptide with KaiA, and a second postulated interaction between the apical region of KaiA and the ATP binding cleft on KaiC. This model brings KaiA mutation sites that alter clock period or abolish rhythmicity into contact with KaiC and suggests how KaiA might regulate KaiC phosphorylation. |
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| Keywords: cyanobacteria, electron microscopy, molecular clock, protein–protein interactions, phosphorylation |
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