Fig. 7: A crystallographic snapshot of c-terminal Tyr 530 intermolecular autophosphorylation.
a Cartoon representation of the two molecules (chain A and B) of the asymmetric unit of the c-Src KD- Ponatinib crystal structure where the back side of the c-lobe of one protomer faced the substrate binding area located at the base of the active site cleft of the other. Selected residues and secondary structure elements are color-coded as in Fig. 5. b Close-up view of the interface between enzyme and substrate molecules showing secondary structural elements and interacting residues from (a). c Superimposition of c-Src KD-Ponatinib crystal structure, with previously described structures of protein kinases in complex with different peptides substrates: PKA (PDB 1ATP, light blue), PKB (PDB 1O6K blue, 1O6L cyan), PhK (PDB 2PHK, soft gray) and IRK (PDB 1RK3, magenta) in surface semitransparent representation. d WB analyses of an in vitro time-course autophosphorylation assay with a c-Src WT 3D- increasing concentrations (from 0.25 to 2 μM) of c-Src WT (3D-construct) in the presence of saturating concentrations of ATP (1 mM) and MgCl2 (2 mM) for 0–60 min. Total c-Src protein was evaluated by Coomassie staining. Data are representative of 3 independent experiments. e Three time-course plots show molecular interactions across a molecular dynamics (MD) simulation. The top two plots show the minimum distance between R463 to E520 (top) and between R463 to D521 (middle). Distance is represented by color, as indicated by the legend in the top-right. The line graph (bottom) shows the minimum distance between Tyr 530 and ATP. Distance is represented across the Y-axis. For all three graphs, time is represented by the shared X-axis at the bottom. Snapshots from four time points in the MD simulation are indicated at the top-left. For each snapshot, the substrate molecule is shown in salmon, while the enzyme molecule is shown in white. The top circle encapsulates Y530 and ATP, while the bottom circle encapsulates R463, E520, and D521, lower panel. Source data are provided in the Source Data file.
