Biochemical analysis of a catalytic core construct of PIKfyve, and comparison with PPIn kinases. (A) Gel filtration elution profile of the PIKfyve^CC, with size standards indicated. The SDS-PAGE of this fraction is shown to the right. (B) Structures of the type I PI4P5Kα and type II PI5P4Kβ dimers (PDB: 4TZ7, 3X03) (Hu et al., 2015; Sumita et al., 2016) compared with the monomeric PIKfyve^CC. Steric clashes, as indicated, prevent dimerization seen in type I and type II PIP kinases. (C) Relative percentage of deuterium incorporation after 3 s of D₂O exposure at 19°C. Each point represents a single peptide graphed in the x axis according to its central residue, with the domain architecture of the PIKfyve^CC above. (D) Schematic of liposomes generated for PIKfyve^CC activity assays. ATPase conditions contained ATP but no liposomes in solution; PS liposomes contained 45% PC, 25% PE, and 30% PS; PI liposomes contained 45% PC, 20% PI, 25% PE, and 10% PS; and PI3P liposomes contained 45% PC, 20% PI3P, 25% PE, and 10% PS. (E) Measurement of ATP turnover performed with different liposomes in solution with the PIKfyve^CC (see D for liposome conditions). Experiments were performed with 1,000–4.11 nM PIKfyve^CC with a final concentration of 0.2 mg/ml of liposomes and 100 µM ATP. (F) Specific activity values for the ATPase assays performed with different liposomes in solution with the PIKfyve^CC (technical replicates; data are presented as mean values, and error bars are SD, n = 3). Specific activity was calculated using the concentrations where kinase activity was in range (111 and 37 nM for ATPase, PI, and PS, and 37 and 12 nM for PI3P). Two-tailed t test P values represented by the symbols are as follows: * < 0.0001.