Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single‐Molecule Spectroscopy

Gilburt, James; Sarkar, Hajrah; Sheldrake, Peter; Blagg, Julian; Ying, Liming; Dodson, Charlotte A.

doi:10.1002/anie.201704654

Cited by 40 publications

(74 citation statements)

References 32 publications

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“…10D). We find that mant-ATP can bind to both the DFG-in or -out conformations, consistent with 342 our nucleotide-bound crystal structures ( Figure 1A-D) and recent single-molecule fluorescence 343 spectroscopy data that indicates that nucleotide binding does not significantly affects this 344 equilibrium (Gilburt et al, 2017). To confirm the model, the kinetic data were globally fit to a two-345 step binding mechanism ( Figure 10H, G).…”

supporting

confidence: 76%

Dynamics of human protein kinases linked to drug selectivity

Pitsawong

Buosi

Otten

et al. 2018

Preprint

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15Protein kinases are major drug targets, but the development of highly-selective inhibitors has 16 been challenging due to the similarity of their active sites. The observation of distinct structural 17 states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational 18 selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that 19Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after 20 drug binding. Here, we investigate whether protein dynamics after binding is a more general 21 paradigm for drug selectivity by characterizing the binding of several approved drugs to the 22 Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal 23 drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time 24 for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays 25 the crucial role, can be applied to inhibitor design of targets outside the kinome.26 27 eLife digest 28The Ser/Thr kinase Aurora A is an important target for the development of new anticancer 29 therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in 30 a background of over 550 protein kinases with very similar structures. To this end, understanding 31 the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the 32 kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) 33 and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely 34 exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. 2Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational 36 change after drug binding (i.e., induced-fit step) dictates drug affinity. 37 38 93 after drug binding. Notably, such conformational changes have evolved for its natural substrates, 94 and the drugs take advantage of this built-in protein dynamics. 95 96 Results 97 Dephosphorylated Aurora A samples both an inactive and active structure 98 A plethora of X-ray structures and functional assays led to the general notion that 99 dephosphorylated Aurora A and, more universally, Ser/Thr kinases are in an inactive 100 conformation and that phosphorylation or activator binding induces the active structure. A 101 comparison of many X-ray structures of inactive and active forms of Ser/Thr kinases resulted in 102 an elegant proposal of the structural hallmarks for the active state by Taylor and collaborators: 103 4 the completion of both the regulatory and catalytic spines spanning the N-and C-terminal 104 domains, including the orientation of the DFG-motif (Kornev & Taylor, 2010, 2015.

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supporting

confidence: 76%

Dynamics of human protein kinases linked to drug selectivity

Pitsawong

Buosi

Otten

et al. 2018

Preprint

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“…Our results show that phosphorylation can drive catalytic activation of a protein kinase without restraining the activation loop in the DFG-In state, providing a contrasting and highly dynamic view of an activated kinase in which major conformational changes of catalytic elements may occur continuously during the catalytic cycle. A recent single-molecule fluorescence study also reported that phosphorylated AurA dynamically transitions between multiple structural states 51 .…”

Section: Discussionmentioning

confidence: 98%

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation

Ruff

Muretta

Thompson

et al. 2017

Preprint

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23Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved 24 residue in the regulatory activation loop, a post-translational modification thought to stabilize the 25 active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods 26. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/205260 doi: bioRxiv preprint first posted online 2 that track different catalytic elements of the kinase domain to show that the ~100-fold activation 27 of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift to the 28 DFG-In state, and that the activation loop of the activated kinase remains highly dynamic.

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“…Our results show that phosphorylation can drive catalytic activation of a protein kinase without restraining the protein in the DFG-In state, providing a contrasting and highly dynamic view of an activated kinase in which major conformational changes of catalytic elements may occur continuously during the catalytic cycle. Of note, a recent single-molecule fluorescence study also reported that phosphorylated AurA dynamically transitions between multiple structural states ( Gilburt et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation

Ruff

Muretta

Thompson

et al. 2018

eLife

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Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases.

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Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single‐Molecule Spectroscopy

Cited by 40 publications

References 32 publications

Dynamics of human protein kinases linked to drug selectivity

Dynamics of human protein kinases linked to drug selectivity

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation

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