Molecular Mechanisms of T221 Phosphorylation in Modulating SIK3 Kinase Function and ATP Binding

Abstract

Phosphorylation of Thr221 (T221) in salt-inducible kinase 3 (SIK3) is a key determinant of its catalytic activity, with broad implications ranging from sleep homeostasis to tumorigenesis. Despite its physiological significance, however, the underlying molecular mechanism by which this phosphorylation event regulates enzymatic activity remains poorly understood. Here, we combine all-atom molecular dynamics (MD) simulations, QM/MM steered molecular dynamics (SMD) simulations, MM/GBSA binding free-energy analyses, protein structure–network mapping, and principal component analysis (PCA) to systematically elucidate the allosteric effects of T221 phosphorylation. We show that a highly occupied pT221–Arg112 salt bridge stabilizes the αC-helix in its “in” conformation and strengthens the conserved αC-Glu113–β3-Lys95 interaction, thereby biasing the conformational ensemble toward active-like states. This inward orientation of the αC-helix, directed toward both the ATP-binding pocket and the catalytic center, further positions Lys109 to maintain a persistent and energetically favorable salt bridge with ATP, consistent with enhanced ATP affinity. Consistent with these atomistic observations, PCA and MM/GBSA analyses reveal a phosphorylation-induced population shift toward a lower free-energy ensemble and substantially stronger ATP binding, jointly indicating a coordinated allosteric enhancement of catalytic activity. Further QM/MM MD simulations indicate that T221 phosphorylation pre-organizes the SIK3 active site to position HDAC4-Ser245(Oγ) closer to the ATP γ-phosphate in a reaction-competent arrangement, thereby facilitating Ser245–O–P phosphoester bond formation and promoting Ser245 phosphorylation. Taken together, these findings define—at atomic resolution—the detailed structural and dynamic principles by which T221 phosphorylation regulates SIK3 function, thus providing mechanistic insight into sleep-need homeostasis and offering a foundation for structure-guided development of SIK3-targeted cancer therapeutics.