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A moving target Abl kinase is an important signaling protein that is dysregulated in leukemia and other cancers and is the target of inhibitors such as imatinib. Like other kinases, Abl kinase is dynamic, and regulating conformational dynamics is key to regulating activity. Xie et al. used nuclear magnetic resonance to show that the Abl kinase domain interconverts between one active and two inactive states. Imatinib stabilizes an inactive conformation, and several resistance mutations act by destabilizing this conformation. In a construct that includes the regulatory domain, depending on the relative arrangement of the kinase and regulatory domains, the kinase domain is stabilized in either the active state or one of the inhibited states. Understanding the conformational dynamics of kinases can be leveraged to design selective drugs. Science, this issue p. eabc2754 Nuclear magnetic resonance data allow dissection of regulatory and drug-resistance mechanisms in Abl kinase. INTRODUCTION Protein kinases mediate many cell signaling processes. Central to their physiological function is the regulation of their binding and enzymatic activities, which is typically achieved by conformational transitions between active and inactive states. Dysregulation of kinase activity by deletions or mutations often results in disease. Protein kinases are dynamic molecules that intrinsically sample a number of conformational states. However, it has been challenging to experimentally access their conformational ensemble and structurally characterize the discrete conformations associated with distinct activities. Such information could advance our understanding of activation and inhibition mechanisms in this protein family and aid in the development of selective inhibitors. RATIONALE We used nuclear magnetic resonance spectroscopy to monitor in atomic-level detail how Abl kinase transitions between distinct conformational states and to elucidate how the conformational ensemble is exploited by mutants, ligands, posttranslational modifications, and inhibitors to regulate the kinase activity and function. We combined structural and energetic approaches to quantitate the contribution of key structural elements such as the activation loop, the Asp-Phe-Gly (DFG) motif, the regulatory spine, and the gatekeeper residue to kinase regulation and provide the mechanistic basis for drug resistance. RESULTS We found that the Abl kinase domain interconverts between an active and two, transiently populated, conformational states that adopt discrete inactive structures. There are extensive differences in key structural elements between the conformational states that reveal multiple intrinsic regulatory mechanisms. The small energy difference between active and inactive states allows oncogenic mutations in the regulatory spine or the gatekeeper position to counteract inhibitory mechanisms and constitutively activate the kinase. By capturing and structurally characterizing the conformational state to which the cancer drug imatinib selectively binds, we explain a number of drug-resistance variants isolated in patients. These mutants confer resistance by depleting, through various mechanisms, the conformation to which imatinib binds. To determine the basis for allosteric regulation, we studied a construct that includes the kinase domain and the regulatory domains and that can adopt an assembled and an extended conformation. In the assembled conformation, in which the regulatory domains dock onto the back of the kinase domain, one of the inactive states is selectively stabilized, thereby suppressing catalytic activity. In the extended conformation, wherein the regulatory domains dock on top of the N-lobe, the inactive state is eliminated, thus explaining the increased leukemogenic activity associated with this conformational state. Only one of the detected inactive states appears to be physiologically relevant. The inactive state with no apparent biological function can nevertheless be leveraged for the design of selective inhibitors. Targeting nonphysiological conformational states may be an effective strategy in the design of drugs with increased selectivity and reduced selection pressure for the occurrence of drug-resistance mutations. Although the structure of inactive states can, in principle, vary considerably among kinases, structural comparison of the Abl inactive states with those previously determined for other kinases reveals that there may be a limited number of structurally divergent inactive states intrinsic to kinases. CONCLUSION Our data demonstrate that the detection and structural characterization of the distinct conformational states populated by a kinase, coupled to the energetic dissection of the contribution of key structural elements to the selective stability of these states, are essential to advance our understanding of the mechanisms underpinning kinase regulation and function. The approaches presented here can be extended to other kinases to characterize transiently populated conformational states, with the goal of revealing the full repertoire of regulatory and drug-resistance mechanisms in the kinome. Transitions of Abl kinase between conformational states. The Abl kinase domain adopts predominantly (~90%) an active state in solution, but it transiently switches between two low-populated (~5%) states that adopt distinct inactive conformations. Key structural elements that rearrange in the various states are highlighted. The conformational equilibrium is exploited by physiological and pathological stimuli to alter the function of Abl. Protein kinases intrinsically sample a number of conformational states with distinct catalytic and binding activities. We used nuclear magnetic resonance spectroscopy to describe in atomic-level detail how Abl kinase interconverts between an active and two discrete inactive structures. Extensive differences in key structural elements between the conformational states give rise to multiple intrinsic regulatory mechanisms. The findings explain how oncogenic mutants can counteract inhibitory mechanisms to constitutively activate the kinase. Energetic dissection revealed the contributions of the activation loop, the Asp-Phe-Gly (DFG) motif, the regulatory spine, and the gatekeeper residue to kinase regulation. Characterization of the transient conformation to which the drug imatinib binds enabled the elucidation of drug-resistance mechanisms. Structural insight into inactive states highlights how they can be leveraged for the design of selective inhibitors.