Extended Data Fig. 6: FIKK13 D379N dead mutant crystal structure informs on ATP binding.

a, FIKK13 WT and FIKK13 D379N phosphorylating activity on cyclic peptide FIKK13_4. Results are represented as mean ± SEM fold change compared to the no substrate luminescent signal. Statistical significance was determined using a two-tailed t-test (FIKK13 D379N versus FIKK13, p < 0.0001). n = 3 biological replicates. b, FIKK13 kinase domain – in grey – bound to ATPƔS and complexed with Nb9F10 (olive, CDR3 in magenta) and Nb2G9 (orange, CDR3 in olive wrapping around the kinase C-lobe) c, FIKK13 D379N crystal structure with ATPƔS. The FIKKs N-lobe is compact with more features than ePKs including two α-helices packed above the conserved C-helix. The A-helix, rarely observed in kinase structures apart from the defining cAMP-dependent kinase PKA⁴⁷, marks the beginning of the N-lobe with the conserved Trp-162 (Extended Data Fig. 10) buried in a pocket between the narrow ends of the aligned A and B-helices positioned above the C-helix. The arrangement is capped by an FIKK-specific α-helix between the β4 and β5 strands. The mainly α-helical C-lobe contains, compared to ePKs, three additional α-helices inserted after the activation loop (A-loop). These helices directly interact with the A-loop, potentially limiting its conformational flexibility upon phosphorylation, as seen in various ePKs¹²⁰. The FIKK13 kinase domain catalytic machinery is conserved from ePKs with notable changes; the HRD motif where Asp acts as a general base during phospho-transfer, is conserved as ³⁷⁷HLD³⁷⁹. The DFG motif, which can switch between active “DFG-in” and inactive “DFG-out” conformations¹²¹, is present in FIKK13 as ³⁹⁸DLS⁴⁰⁰, although conserved as DFG in FIKK1 and FIKK9.1 (Extended Data Fig. 10) and adopts the “DFG-in” conformation in the FIKK13 kinase domain structure. d, Close-up representation of FIKK13 kinase domain ATP-binding pocket containing ATPƔS, focusing on the F-I-K-K motif. The size and hydrophobicity of Phe-228 restrict the ATP-binding pocket volume while the Lys-230 coordinates the nucleotide α- and β-phosphates and forms a salt bridge with Glu-261 of the C-helix, characteristic of active ePKs⁴⁷. Taken together, the first experimentally determined FIKK kinase structure reveals strong resemblance to ePKs with conservation of the essential elements for catalysis. However, FIKK-specific features, such as additional α-helices in both the N- and C-lobe suggest differences in regulation.