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The specificity of phosphorylation by protein kinases is essential to the integrity of biological signal transduction. While peptide sequence specificity for individual kinases has been examined previously, here we explore the evolutionary progression that has led to the modern substrate specificity of two non-receptor tyrosine kinases, Abl and Src. To efficiently determine the substrate specificity of modern and reconstructed ancestral kinases, we developed a method using mammalian cell lysate as the substrate pool, thereby representing the naturally occurring substrate proteins. We find that the oldest tyrosine kinase ancestor was a promiscuous enzyme that evolved through a more specific last common ancestor into a specific human Abl. In contrast, the parallel pathway to human Src involved a loss of substrate specificity, leading to general promiscuity. These results add a new facet to our understanding of the evolution of signaling pathways, with both subfunctionalization and neofunctionalization along the evolutionary trajectories.

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome., eLife digest Protein kinases are a family of enzymes found in all living organisms. These enzymes help to control many biological processes, including cell division. When particular protein kinases do not work correctly, cells may start to divide uncontrollably, which can lead to cancer. One example is the kinase Aurora A, which is over-active in many common human cancers. As a result, researchers are currently trying to design drugs that reduce the activity of Aurora A in the hope that these could form new anticancer treatments. In general, drugs are designed to be as specific in their action as possible to reduce the risk of harmful side effects to the patient. Designing a drug that affects a single protein kinase, however, is difficult because there are hundreds of different kinases in the body, all with similar structures. Because drugs often work by binding to specific structural features, a drug that targets one protein kinase can often alter the activity of a large number of others too. Gleevec is a successful anti-leukemia drug that specifically works on one target kinase, producing minimal side effects. It was recently discovered that the drug works through a phenomenon called ‘induced fit’. This means that after the drug binds it causes a change in the enzyme’s overall shape that alters the activity of the enzyme. The shape change is complex, and so even small structural differences can change the effect of a particular drug. Do other drugs that target other protein kinases also produce induced fit effects? To find out, Pitsawong, Buosi, Otten, Agafonov et al. studied how three anti-cancer drugs interact with Aurora A: two drugs specifically designed to switch off Aurora A, and Gleevec (which does not target Aurora A). The two drugs that specifically target Aurora A were thought to work by targeting one structural feature of the enzyme. However, the biochemical and biophysical experiments performed by Pitsawong et al. revealed that these drugs instead work through an induced fit effect. By contrast, Gleevec did not trigger an induced fit on Aurora A and so bound less tightly to it. In light of these results, Pitsawong et al. suggest that future efforts to design drugs that target protein kinases should focus on exploiting the induced fit process. This will require more research into the structure of particular kinases.

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.eLife digestThe Ser/Thr kinase Aurora A is an important target for the development of new anticancer therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in a background of over 550 protein kinases with very similar structures. To this end, understanding the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational change after drug binding (i.e., induced-fit step) dictates drug affinity.

Macromolecular function is rooted in energy landscapes, where sequence determines not a single structure but an ensemble of conformations. Hence, evolution modifies a protein’s function by altering its energy landscape. Here, we recreate the evolutionary pathway between two modern human oncogenes, Src and Abl, by reconstructing their common ancestors. Our evolutionary reconstruction combined with x-ray structures of the common ancestor and pre–steady-state kinetics reveals a detailed atomistic mechanism for selectivity of the successful cancer drug Gleevec. Gleevec affinity is gained during the evolutionary trajectory toward Abl and lost toward Src, primarily by shifting an induced-fit equilibrium that is also disrupted in the clinical T315I resistance mutation. This work reveals the mechanism of Gleevec specificity while offering insights into how energy landscapes evolve.

Protein kinases are obvious drug targets against cancer, owing to their central role in cellular regulation. Since the discovery of Gleevec, a potent and specific inhibitor of Abl kinase, as a highly successful cancer therapeutic, the ability of this drug to distinguish between Abl and other tyrosine kinases such as Src has been intensely investigated but without much success. Using NMR and fast kinetics, we establish a new model that solves this longstanding question of how the two tyrosine kinases adopt almost identical structures when bound to Gleevec but have vastly different affinities. We show that, in contrast to all other proposed models, the origin of Abl's high affinity lies predominantly in a conformational change after binding. An energy landscape providing tight affinity via an induced fit and binding plasticity via a conformational-selection mechanism is likely to be general for many inhibitors.

Human peptidyl-prolyl isomerase (PPIase) Pin1 plays key roles in developmental processes, cell proliferation, and neuronal function. Extensive phosphorylation of the microtubule binding protein tau has been implicated in neurodegeneration and Alzheimer's disease. For the past 15years, these two players have been the focus of an enormous research effort to unravel the biological relevance of their interplay in health and disease, resulting in a series of proposed molecular mechanism of how Pin1 catalysis of tau results in biological phenotypes. Our results presented here refute these mechanisms of Pin1 action. Using NMR, isothermal calorimetry (ITC), and small angle x-ray scattering (SAXS), we dissect binding and catalysis on multiple phosphorylated tau with particular emphasis toward the Alzheimer's associated AT180 tau epitope containing phosphorylated THR231 and SER235. We find that phosphorylated (p-) SER235-PRO, but not pTHR231-PRO, is exclusively catalyzed by full-length Pin1 and isolated PPIase domain. Importantly, site-specific measurements of Pin1-catalysis of CDK2/CycA-phosphorylated full-length tau reveal a number of sites that are catalyzed simultaneously with different efficiencies. Furthermore, we show that the turnover efficiency at pSER235 by Pin1 is independent of both the WW domain and phosphorylation on THR231. Our mechanistic results on site-specific binding and catalysis together with the lack of an increase of dephosphorylation rates by PP2A counter a series of previously published models for the role of Pin1 catalysis of tau in Alzheimer's disease. Together, our data reemphasize the complicated scenario between binding and catalysis of multiple phosphorylated tau by Pin1 and the need for directly linking biological phenotypes and residue-specific turnover in Pin1 substrates.

We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX21−25. While autophosphorylation is the key regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases. DOI: http://dx.doi.org/10.7554/eLife.02667.001, eLife digest The kinase, Aurora A, is a human protein that is needed for cells to divide normally. Kinases are enzymes that control other proteins by adding phosphate groups to these proteins; however, like other kinases, Aurora A must first be activated or ‘switched on’ before it can do this. Aurora A kinase can be switched on in two ways: by having a phosphate group added to its ‘activation loop’; or by binding to another protein called TPX2. Also like other kinases, Aurora A can self-activate, but the details of this process are not understood. Does a single Aurora A kinase add a phosphate group to its own activation loop, or does one Aurora A kinase activate a second? Furthermore, it is not clear how binding to TPX2 can activate an Aurora A kinase without adding a phosphate group to the activation loop. Zorba, Buosi et al. now show that Aurora A kinases that have been activated in different ways—via the addition of a phosphate group or binding to TPX2—are equally good at adding phosphate groups to other proteins. Zorba, Buosi et al. also worked out the three-dimensional shapes of the kinases activated in these two ways—since many proteins change shape when they are switched on—and found that they were also the same. Finally, it was shown that self-activation involves two Aurora A kinases binding to each other, and one kinase adding a phosphate group to the other, rather than a single kinase adding a phosphate group to itself. Since other protein kinases can be activated in similar ways to Aurora A, the findings of Zorba, Buosi et al. might also help us to understand how other protein kinases can be switched ‘on’ or ‘off’. And, as mutations in Aurora A have been linked to the development of cancer, uncovering how this kinase is controlled could help efforts to design new drugs to treat this disease. DOI: http://dx.doi.org/10.7554/eLife.02667.002

Recoverin, a 23-kDa Ca(2+)-binding protein of the neuronal calcium sensing (NCS) family, inhibits rhodopsin kinase, a Ser/Thr kinase responsible for termination of photoactivated rhodopsin in rod photoreceptor cells. Recoverin has two functional EF hands and a myristoylated N terminus. The myristoyl chain imparts cooperativity to the Ca(2+)-binding sites through an allosteric mechanism involving a conformational equilibrium between R and T states of the protein. Ca(2+) binds preferentially to the R state; the myristoyl chain binds preferentially to the T state. In the absence of myristoylation, the R state predominates, and consequently, binding of Ca(2+) to the non-myristoylated protein is not cooperative. We show here that a mutation, C39A, of a highly conserved Cys residue among NCS proteins, increases the apparent cooperativity for binding of Ca(2+) to non-myristoylated recoverin. The binding data can be explained by an effect on the T/R equilibrium to favor the T state without affecting the intrinsic binding constants for the two Ca(2+) sites.

International audience; Activation of the small GTP-binding protein Arf1, a major regulator of cellular traffic, follows an ordered sequence of structural events, which have been pictured by crystallographic snapshots. Combined with biochemical analysis, these data lead to a model of Arf1 activation, in which opening of its N-terminal helix first translocates Arf1-GDP to membranes, where it is then secured by a register shift of the interswitch β-strands, before GDP is eventually exchanged for GTP. However, how Arf1 rearranges its central β-sheet, an event that involves the loss and re-formation of H-bonds deep within the protein core, is not explained by available structural data. Here, we used Δ17Arf1, in which the N-terminal helix has been deleted, to address this issue by NMR structural and dynamics analysis. We first completed the assignment of Δ17Arf1 bound to GDP, GTP, and GTPγS and established that NMR data are fully consistent with the crystal structures of Arf1-GDP and Δ17Arf1-GTP. Our assignments allowed us to analyze the kinetics of both protein conformational transitions and nucleotide exchange by real-time NMR. Analysis of the dynamics over a very large range of timescale by (15)N relaxation, CPMG relaxation dispersion and H/D exchange reveals that while Δ17Arf1-GTP and full-length Arf1-GDP dynamics is restricted to localized fast motions, Δ17Arf1-GDP features unique intermediate and slow motions in the interswitch region. Altogether, the NMR data bring insight into how that membrane-bound Arf1-GDP, which is mimicked by the truncation of the N-terminal helix, acquires internal motions that enable the toggle of the interswitch.

Cancer is a serious illness and the second leading cause of death in the US. It is caused by misregulation of the signaling cascades and uncontrolled cell proliferation. Protein kinases are the key players in the regulatory machinery, and in the last 20 years they have become an increasingly important drug targets. The main requirement for such drugs is selectivity: ideally, only one kinase should be affected by an individual drug. The prominent success story is an FDA-approved chronic myeloid leukemia therapeutic Gleevec. Gleevec is a highly specific inhibitor of the Abl kinase and has very little potency towards a closely related Src subfamily of kinases. This specificity is particularly intriguing considering that the drug-binding pockets look nearly identical in these kinases. We combined NMR and pre-steady-state kinetic experiments to monitor Gleevec binding in real time and with residue-specific precision. Our data establish a novel model that fully and quantitatively explains the observed 3000-fold difference in Gleevec's affinities to Abl and Src, solving a long-standing paradox in the field. These results reveal the general principles of kinase-drug interactions and highlight the molecular mechanisms behind nanomolar affinities found in clinically relevant therapeutics.