Fig. 2: JM forms an intermolecular interaction in an asymmetric KD dimer.

a JM-KD construct with progressively increasing pY residues on KD were run on size exclusion chromatography (SEC; Superdex 75 26/100) at 80–100 µM (inset: dimer/monomer ratio of KD with different phosphorylation levels). b Dimerization of KD^pY1 is reduced in the presence of JM. The dimerization of JM-KD^pY1 constructs with JM deletions (JM-KD^pY1; JM^Δ35-KD^pY1; and KD^pY1) was determined using 10 µM injected on a size exclusion column (Superdex 75 10/30). c The ‘apparent’ dimerization K_d of JM-KD^pY1 (red) and KD^pY1 (blue) determined using MST. JM-KD^pY1 and KD^pY1were labeled with Atto488 dye then titrated with unlabeled JM-KD^pY1 and KD^pY1. The error bars represent the standard deviation of 2 technical replications. d Under basal conditions the presence of JM in the enzyme-acting molecule (JM-KD^pY1) is required for the recruitment of substrate-acting molecule (JM-KD^K518I). This sustains the asymmetric dimer configuration required for the enhancement of transphosphorylation as the phosphorylation levels of substrates (left side: enzyme: JM-KD^pY1, right side: enzyme: KD^pY1) were examined using a phosphotyrosine antibody (pY99). An anti-6xHis tag antibody was used to probe total proteins as the loading control. e The JM from JM-KD^K518I cannot recruit KD^pY1 to form an active dimer. Phosphorylation of substrate is at the same level as the independent monomers. Together Fig. 2d, e demonstrate a role of kinase activation under basal conditions where the JM interacts in trans, recruiting and phosphorylating a substrate molecule.