Furuike Yoshihiko, Onoue Yasuhiro, Saito Shinji, Mori Toshifumi, Akiyama Shuji
Research Center of Integrative Molecular Systems (CIMoS), Institute for Molecular Science, National Institutes of Natural Sciences, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan.
Molecular Science Program, Graduate Institute for Advanced Studies, SOKENDAI, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan.
PNAS Nexus. 2025 Apr 28;4(5):pgaf136. doi: 10.1093/pnasnexus/pgaf136. eCollection 2025 May.
KaiC, a cyanobacterial circadian clock protein with autokinase activity, catalyzes the dual phosphorylation of its own S431 and T432 residues in a circadian manner in the presence of KaiA and KaiB. Priming phosphorylation at T432 is a key step that promotes secondary phosphorylation at S431. Although KaiA binding is considered essential for KaiC phosphorylation, the mechanisms underlying the activation and inactivation of priming phosphorylation remain elusive. We found that although the priming phosphorylation is autoinhibited within KaiC, it actually proceeds at a rate constant of 0.019 h even in the absence of KaiA. The autoinhibition of KaiC and the mechanism underlying the release from autoinhibition by KaiA were examined by KaiC structural analysis and by classical molecular dynamics and quantum mechanics/molecular mechanics simulations. We found that the side chain of T432 adopts two rotamers in dephosphorylated KaiC, one of which places T432 in a position suitable for a nucleophilic attack on the terminal phosphate of adenosine triphosphate. However, the nucleophilicity of T432 was insufficient to overcome an energy barrier of ∼21 kcal mol because the catalytic function of a nearby base, E318, was self-suppressed by hydrogen bonding to positively charged R385. Biochemical assays of KaiC mutants showed that the autoinhibition of KaiC autokinase activity is attenuated by conferring T432 high nucleophilicity through the KaiA-assisted release of R385 from E318 to E352. During the circadian cycle, R385 switches interacting partners to inactivate/activate the autokinase function and to ensure the unidirectionality of the KaiC phosphorylation cycle.
KaiC是一种具有自激酶活性的蓝藻生物钟蛋白，在KaiA和KaiB存在的情况下，以昼夜节律的方式催化自身S431和T432残基的双重磷酸化。T432位点的起始磷酸化是促进S431位点二次磷酸化的关键步骤。尽管KaiA的结合被认为是KaiC磷酸化所必需的，但起始磷酸化激活和失活的潜在机制仍然不清楚。我们发现，尽管起始磷酸化在KaiC内部受到自身抑制，但即使在没有KaiA的情况下，它实际上仍以0.019 h的速率常数进行。通过KaiC结构分析以及经典分子动力学和量子力学/分子力学模拟，研究了KaiC的自身抑制作用以及KaiA从自身抑制中释放的潜在机制。我们发现，在去磷酸化的KaiC中，T432的侧链采取两种旋转异构体，其中一种将T432置于适合对三磷酸腺苷末端磷酸进行亲核攻击的位置。然而，T432的亲核性不足以克服约21 kcal mol的能垒，因为附近的碱基E318的催化功能通过与带正电荷的R385形成氢键而自我抑制。对KaiC突变体的生化分析表明，通过KaiA辅助R385从E318释放到E352，赋予T432高亲核性，可减弱KaiC自激酶活性的自身抑制作用。在昼夜节律循环中，R385切换相互作用伙伴以失活/激活自激酶功能，并确保KaiC磷酸化循环的单向性。
