Probing an Ancient Protein's Dynamics with NMR

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The tyrosine kinases Abl and Src are very similar in function and fold. However, the cancer drug imatinib exhibits highly selective binding for Abl compared to Src. For a long time it was assumed that these differences arise through a conformational selection mechanism of a DFG-loop, which allows binding of imatinib only if it is in the correct conformation. Recently, an additional induced fit step in the process of imatinib binding has been shown to account for the differences in binding of the drug to Abl in comparison with Src. To investigate the binding mechanism of imatinib to Abl we used resurrected enzymes along the nodes in a phylogenetic tree between the common ancestor of Abl and Src and the contemporary Abl kinase. We show that the binding affinities of each of the four resurrected ancestor enzymes are gradually increasing from the common ancestor towards the contemporary Abl and that the mechanism of binding is comprised of a conformational selection step followed by an induced fit step. To probe the conformational changes the enzyme undergoes upon imatinib binding, we use CPMG relaxation dispersion experiments. We show that most of the exchanging residues are in an intermediate or fast time regime. Combining the CPMG data with molecular dynamics will allows us to get a more detailed look at which residues play an import role for the selectivity of imatinib towards Abl kinase.