The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180° with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP–peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR–Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.

The physical properties of solvents strongly affect the spectra of dissolved solutes, and this phenomenon can be exploited to gain insight into the solvent-solute interaction. The large solvatochromic shifts observed for many dye molecules in polar solvents are due to variations in the solvent reaction field, and these shifts are widely used to estimate the change in the dye’s dipole moment upon photoexcitation, which is typically on the order of ~1-10 Debye. In contrast, the change in dipole moment for vibrational transitions is approximately two orders of magnitude smaller. Nonetheless, vibrational chromophores display significant solvatochromism, and the relative contributions of specific chemical interactions and electrostatic interactions are debated, complicating the interpretation of vibrational frequency shifts in complex systems such as proteins. Here we present a series of substituted benzonitriles that display widely varying degrees of vibrational solvatochromism. In most cases, this variation can be quantitatively described by the experimentally determined Stark tuning rate, coupled with a simple Onsager-like model of solvation, reinforcing the view that vibrational frequency shifts are largely caused by electrostatic interactions. In addition, we discuss specific cases where continuum solvation models fail to predict solvatochromic shifts, revealing the necessity for more advanced theoretical models that capture local aspects of solute-solvent interactions.

Kinase inhibitors are important cancer drugs, but they tend to display limited target specificity, and their target profiles are often challenging to rationalize in terms of molecular mechanism. Here we report that the clinical kinase inhibitor bosutinib recognizes its kinase targets by engaging a pair of conserved structured water molecules in the active site, and that many other kinase inhibitors share a similar recognition mechanism. Using the nitrile group of bosutinib as an infrared probe, we show that the gatekeeper residue and one other position in the ATP-binding site control access of the drug to the structured water molecules, and that the amino acids found at these positions account for the kinome-wide target spectrum of the drug. Our work highlights the importance of structured water molecules for inhibitor recognition, reveals a new role for the kinase gatekeeper, and showcases an effective approach for elucidating the molecular origins of selectivity patterns.

Summary The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C-terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.9 Å resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site, used by most tyrosine kinases to recognize substrates, is destabilized in Csk by a deletion in the activation loop.