Mixtures of opposing phosphorylations within hexamers precisely time feedback in the cyanobacterial circadian clock
Mixtures of opposing phosphorylations within hexamers precisely time feedback in the cyanobacterial circadian clock
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DOI：
10.1073/pnas.1408692111
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发表时间：
2014-09-16
影响因子：
11.1
通讯作者：
Rust, Michael J.
中科院分区：
文献类型：
--
作者：
Lin, Jenny;Chew, Justin;Rust, Michael J.
昼夜节律振荡是由纯化的蓝藻时钟蛋白KaiA、KaiB和KaiC通过依赖于KaiC多位点磷酸化的节律性相互作用产生的。然而，允许这些磷酸化反应在一系列蛋白质化学计量上强有力地控制振荡时间的机制尚不清楚。我们发现，当KaiC六聚体由不同磷酸化亚基的混合物组成时，两个磷酸化位点对每个六聚体结合负调节因子KaiB的能力具有相反的影响。我们同样表明，正调节因子KaiA作用于KaiC的能力取决于六聚体的磷酸化状态，并且KaiA和KaiB识别KaiC环的可变变构状态。利用从实验数据中获取的动力学参数的数学模型，我们发现两个KaiC磷酸化位点的拮抗作用产生了负反馈强度的超灵敏开关，这对于在一定浓度范围内稳定的昼夜节律振荡是必要的。基于相反修改的类似策略可用于支持其他定时系统和更一般的细胞信号的鲁棒性。
Circadian oscillations are generated by the purified cyanobacterial clock proteins, KaiA, KaiB, and KaiC, through rhythmic interactions that depend on multisite phosphorylation of KaiC. However, the mechanisms that allow these phosphorylation reactions to robustly control the timing of oscillations over a range of protein stoichiometries are not clear. We show that when KaiC hexamers consist of a mixture of differentially phosphorylated subunits, the two phosphorylation sites have opposing effects on the ability of each hexamer to bind to the negative regulator KaiB. We likewise show that the ability of the positive regulator KaiA to act on KaiC depends on the phosphorylation state of the hexamer and that KaiA and KaiB recognize alternative allosteric states of the KaiC ring. Using mathematical models with kinetic parameters taken from experimental data, we find that antagonism of the two KaiC phosphorylation sites generates an ultrasensitive switch in negative feedback strength necessary for stable circadian oscillations over a range of component concentrations. Similar strategies based on opposing modifications may be used to support robustness in other timing systems and in cellular signaling more generally.