Extended Data Fig. 6: Different conformational states of PI3KC2α.
From: Structural basis of phosphatidylinositol 3-kinase C2α function
(a) PI3KC2αΔN was crosslinked with DSSO chemical crosslinker and separated by 4–15% gradient SDS-PAGE. The monomeric band of crosslinked PI3KC2αΔN indicated as dashed red square was cut out for further MS analysis. (b) Identified crosslinked peptides using DSSO as chemical crosslinker. (c) Model of PI3KC2αΔN based on the cryo-EM map without the PX domain. The PX domain was docked onto the distal C2 domain using restraints imposed by the obtained crosslinks between the PX and distal C2 domain, using the HADDOCK server. The obtained model was subsequently verified manually using all identified crosslinking sites between the kinase core, PX, and distal C2 domains and the C- to N- terminal connections between these domains. Green line: distance < 30 Å; red line; distance > 30 Å. Distant crosslinks (>30 Å) suggest different conformational states of PI3KC2α. (d) Interaction between the kinase core and lipid binding C2 domains of PI3KC2α. GST pull down assays using GST-PX, GST-C2 or GST-PX-C2 as baits to capture PI3KC2αΔN+ΔPX−C2 Wild-type (WT) or RBD mutant (K426A; W458A; D461A; D462A) versions. Samples were analyzed by SDS-PAGE and Coomassie Blue staining and quantified using ImageJ software. Binding of PI3KC2αΔN+ΔPX−C2 WT was set to 100%. Data represent SEM from triplicate (n = 3) experiments.
