Regulation mechanism study of residues E318/319 for KaiC protein phosphorylation.

The biological clock is widely present in living organisms and regulates nearly all life processes. Cyanobacteria are known prokaryotes with the simplest biological clock for circadian rhythm. The KaiC hexamer serves as the core of the cyanobacterial clock and undergoes diurnal rhythm oscillations through phosphorylation and dephosphorylation to keep time. However, the precise roles of individual residues during KaiC phosphorylation remain unclear. Current experiments indicate that mutations at residues 318 and 319 completely inhibit the phosphorylation of the KaiC protein. However, the molecular mechanism by which these mutations disturb the protein's internal structure and subsequently shut down the phosphorylation reaction remains an unsolved mystery. Here, we conducted molecular dynamics simulations and found that the mutations reduced the stability of the protein (especially the CII domain), making the phosphorylation sites more accessible to solvent, weakening the rigidity of residues near the ATP-binding pocket, and disrupting the coordination network of Mg2+. Additionally, we discovered that efficient phosphorylation of KaiC requires the catalytic function of E318 and the mediation of Mg2+ coordination by E319, both of which are indispensable. This study clarified the roles of residues 318 and 319 in regulating KaiC phosphorylation, deepened understanding of the molecular mechanism of the circadian clock cycle in cyanobacteria, and is expected to provide new intervention strategies for diseases related to circadian rhythm disorders, such as sleep disorders and metabolic syndrome.