Fig. 2: The PRPK ATP binding pocket is in an active conformation.

a Conserved kinase elements are color coded in the PRPK structure. PRPK lacks the conventional kinase activation loop between the DFG loop and helix αF. In addition, PRPK does not have the elaborated helices after αF, seen in conventional kinases. b Close up view of the PRPK ATP binding pocket. The invariant K60 of β3 holds the α-phosphate of AMPPNP. K60 itself is stabilized by a salt bridge with E84 of helix αC. Two Mg²⁺ ions are coordinated by D183 of the DFG loop and N167 of the catalytic loop, respectively. D162 is the catalytic residue, and M113 is the gatekeeper residue. This configuration satisfies the requirement for an active kinase. Charged interactions are labeled with dashed lines and the distances are indicated. c Close up view of the PRPK ATP binding pocket in surface representation. E114 and I116 from the hinge region form hydrogen bonds with one edge of the adenine ring. The adenine base is surrounded by hydrophobic resides V47, V58, M113, L169, and I182 (V47 is on the roof of the pocket and could not be seen from this angle). Notably, the first glycine of the GxGxxG G-loop motif is replaced with K40 in human PRPK. The surface area of K40 is colored blue, and it may constitute a hindrance to ATP binding. Hydrogen bonds are labeled with dashed lines and the distances are indicated. d In vitro kinase assay showing the autophosphorylation activity of the wild type and mutant PRPK–TPRKB complexes. Uncropped images of gel and autoradiograph are shown in Supplementary Fig. S6.