(A) Overlay of WT kinase domain (gray, PDB: 1CKJ, chain B) with tau (maroon, PDB: 6PXN, chain A). The 3 anion binding sites (WO42-, from 1CKJ) are labeled. (B) View of Site 1 in WT (top, gray) and tau (bottom, maroon). Polar interactions, dashed black lines. (C) Overlaid view of Site two in WT and tau as above. Polar interactions, dashed black lines. Asterisk, hinge point for conformational change at G175. Note: Site two anion is only bound in WT, as it is blocked by the activation loop in tau. (D) Representation depicting the left-hand configuration of G175 and subsequent rotation (solid arrow) of upstream residues T174 and L173. (E) Alignment of the activation loop of CK1δ with representatives of other serine/threonine kinase families. Residues that coordinate anion binding on CK1 are indicated above in gray. (F) Superposition of the Site two anion binding site in CK1 with the binding site for the phosphorylated activation loop of other serine/threonine kinases. Depicted are: PKB (PDB: 1O6K, pale cyan), PDK1 (1H1W, aquamarine), CDK2 (1QMZ, green cyan), ERK2 (2ERK, teal) and CK1δ (5 × 17, dark gray). Residues that coordinate the anion are depicted in sticks, as are phosphoserine or phosphothreonine residues from other kinases; the SO42- coordinated at Site two by CK1δ (PDB: 5 × 17) is shown in transparent spheres. (G) Enzymatic efficiency on the Degron (n = 3 with s.d.). Significance assessed relative to WT with an unpaired Student’s two-sided t-test: **, p<0.01; ***, p<0.001; ****, p<0.0001. (H) Ratio of enzymatic efficiency on FASP relative to Degron (n = 3 with s.d.). Equivalent activity on FASP and Degron, dashed line. Significance assessed as above. See also Figure 2—figure supplement 1.