Flexibility and charge asymmetry in the activation loop of Src tyrosine kinases

Regulated activity of Src kinases is critical for cell growth. Src kinases can be activated by trans-phosphorylation of a tyrosine located in the central activation loop of the catalytic domain. However, because the required exposure of this tyrosine is not observed in the down-regulated X-ray structures of Src kinases, transient partial opening of the activation loop appears to be necessary for such processes. Umbrella sampling molecular dynamics simulations are used to characterize the free energy landscape of opening of the hydrophilic part of the activation loop in the Src kinase Hck. The loop prefers a partially open conformation where Tyr416 has increased accessibility, but remains partly shielded. An asymmetric distribution of the charged residues in the sequence near Tyr416, which contributes to shielding, is found to be conserved in Src family members. A conformational equilibrium involving exchange of electrostatic interactions between the conserved residues Glu310 and Arg385 or Arg409 affects activation loop opening. A mechanism for access of unphosphorylated Tyr416 into an external catalytic site is suggested based on these observations.