The tyrosinekinases of the Src family are highly conserved signaling proteins involved in the regulation of cell growth whose catalytic activity can be modulated in response to specific cellular signals. The key role that the Src-family kinases play in the onset of many human diseases, particularly cancer, makes them important targets for therapeutic intervention. All nine members of the Src family are formed by a catalytic domain which is preceded by two peptide binding modules, the Src-homology domains SH2 and SF13. Phosphorylation of two tyrosines (Tyr527 and Tyr416) has opposing effects on catalytic activity: dephosphorylation of Tyr527 in the C-terminal tail results in the activation of the enzyme, while phosphorylation of Tyr416 which is located in a central activation loop"""""""" of the kinase domain opens the catalytic site and activates the enzyme. The available crystallographic structures do not, however, show readily how the catalytic activity is regulated by these two sites. To refine our understanding of the factors responsible for the regulation of Src tyrosine kinases, John Kuriyan and myself initiated a theoretical investigation using molecular dynamics simulations. Motivated by our preliminary results, we now seek to extend our collaborative effort. The goal of this research proposal is to characterize quantitatively the importance of conformational flexibility in the activation of Src tyrosine kinases. To overcome the sampling and timescale difficulties and obtain meaningful results, we will use special computational strategies based on molecular dynamics potential of mean force techniques with biased sampling. In particular, we will calculate the free energy barrier associated with the opening of the activation loop of the catalytic domain for various states of the tyrosine kinase. Lastly, the results of the computations based on atomic models will be contrasted and compared with a statistical analysis of the sequence patterns of members of the Src family.
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