Fig 2: Interaction between PRPK and TPRKB.a ConSurf style representation of the TPRKB surface involved in PRPK interaction. PRPK is shown as semi-transparent cartoon in white color. b ConSurf style representation of the PRPK surface involved in TPRKB interaction. TPRKB is shown as semi-transparent cartoon in white color. c Detailed interaction between PRPK and TPRKB. PRPK F52, F102, and F103 constitute a hydrophobic surface hosting the TPRKB helix a2. Hydrogen bonds are formed between PRPK Q53 and TPRKB D8, between PRPK R86 and TPRKB V174, and lastly between PRPK V101 and TPRKB S170 (dashed lines). Salt bridges link PRPK R55 with TPRKB D163, and link PRPK D105 with TPRKB H63 (dashed lines). d Pulldown of His-tagged TPRKB by GST-tagged PRPK mutants. GST-tagged PRPK was first immobilized on glutathione Sepharose beads and then incubated with a purified His-tagged TPRKB protein. After extensive washing, His-TPRKB bound to PRPK was detected using a His antibody. A representative result from at least three repetitions is shown. Uncropped images of gel and blot are shown in Supplementary Fig. S6.

Fig 3: 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 aF. In addition, PRPK does not have the elaborated helices after aF, seen in conventional kinases. b Close up view of the PRPK ATP binding pocket. The invariant K60 of ß3 holds the a-phosphate of AMPPNP. K60 itself is stabilized by a salt bridge with E84 of helix aC. 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.

Fig 4: A model of the human EKC/KEOPS complex.a The human EKC/KEOPS complex structural model, composed of TPRKB, PRPK, OSGEP, LAGE3, and GON7, is constructed by aligning our structure and the OSGEP–LAGE3–GON7 structure (PDB ID 6GWJ) to the mjBud32–Kae1 structure (PDB ID 3EN9). AMPPNP is shown as stick and Mg2+ as yellow spheres. b Detailed view of the interface between PRPK (violet) and OSGEP (yellow) in our model, aligned to the mjBud32–Kae1 crystal structure (gray). Three salt bridges found in mjBud32–Kae1 appear conserved at the PRPK–OSGEP interface. The involved residues in PRPK are K65, R80, and K205. The region deleted in the PRPK K238Nfs*2 mutant (residues 239-C) is colored light blue. c Pulldown of MBP-tagged OSGEP by GST-tagged PRPK mutants. GST-tagged PRPK was first immobilized on glutathione Sepharose beads and then incubated with a purified MBP-tagged OSGEP protein. After extensive washing, MBP-OSGEP bound to PRPK was detected using an MBP antibody. A representative result from at least 3 repetitions is shown. Uncropped images of gel and blot are shown in Supplementary Fig. S6.

Fig 5: Overall structure of the human PRPK–TPRKB–AMPPNP complex.PRPK adopts a kinase fold with a briefed C-lobe. TPRKB uses the ß1-ß2 loop, a2, a8, and a9 to interact with the PRPK N-lobe. PRPK is colored in violet and TPRKB in aquamarine. AMPPNP is shown as stick and Mg2+ as yellow spheres.