Figure 2. CK1 follows an ordered distributive mechanism on the human PER2 FASP region.

a, Schematic of ordered distributive kinetic model used for panels b and c (modeled using Wolfram Mathematica, see Supplement). b, Reaction coordinate for ordered distributive kinetic mechanism with differential rates for priming and sequential kinase activity. c, Reaction coordinate for ordered distributive kinetic mechanism with similar rates for priming and sequential kinase activity. d, Zoom of ¹⁵N/¹H HSQC hPER2 FASP phosphoserine region looking at peaks corresponding to different states arising from at 3 hr incubation with CK1 WT (red). States A-D (inset) correspond to unique chemical shift states observed throughout the kinase assay. e, Schematic of unique states A-D observed in d: A, unphosphorylated FASP; B, primed FASP; C, all serines in FASP phosphorylated; D, all serines plus T675 phosphorylated. Numbering indicates order of phosphorylation. f, Traces of accumulating peak volume from the NMR kinase assay. Letters C-D with superscript numbers correspond to specific phosphoserines observed in the NMR assay. g, Same traces as in f, with peak volumes corresponding to states C and D for the last two serines (4–5) summed to show that all phosphoserines in the FASP report on state C with similar kinetics. h, Phosphoserine region showing unique chemical shift environment for singly phosphorylated (priming only) FASP S665A (blue) overlayed with WT FASP with CK1 WT (red) or K224D (gray) after 3 hr incubation. B-D lettering corresponds to unique states observed in NMR kinase assay as in d. i, Traces of accumulating peak volume from NMR kinase assay with CK1 K224D, resolving the transient accumulation of primed FASP and a clear lagging phase for subsequent phosphorylation states.