CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

JOURNAL OF MOLECULAR BIOLOGY

卷:425

Article

Villarreal, Seth A.^1,2  Pattanayek, Rekha³  Williams, Dewight R.⁴  Mori, Tetsuya⁵  Qin, Ximing⁵  Johnson, Carl H.⁵  Egli, Martin³  Stewart, Phoebe L.^1,2 

[1] Case Western Reserve Univ, Dept Pharmacol, Cleveland, OH 44106 USA

[2] Case Western Reserve Univ, Cleveland Ctr Membrane & Struct Biol, Cleveland, OH 44106 USA

[3] Vanderbilt Univ, Sch Med, Dept Biochem, Nashville, TN 37232 USA

[4] Vanderbilt Univ, Sch Med, Dept Mol Physiol & Biophys, Nashville, TN 37232 USA

[5] Vanderbilt Univ, Dept Biol Sci, Nashville, TN 37235 USA

关键词: circadian oscillator;    cryoEM;    MDFF;    protein-protein interface;    Synechococcus elongatus;   

DOI  :  10.1016/j.jmb.2013.06.018

来源: Elsevier

【 摘 要 】

The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Delta 489, which is persistently phosphorylated. The KaiB-KaiC-Delta 489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein-protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC. (C) 2013 Elsevier Ltd. All rights reserved.

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