A Novel Mechanism by Which Small Molecule Inhibitors Induce the DFG Flip in Aurora A

Martin, Mathew P.; Zhu, J.; Lawrence, Harshani R.; Pireddu, Roberta; Luo, Yunting; Alam, R.; Özcan, Sevil; Sebti, Saı̈d M.; Lawrence, Nicholas J.; Schönbrunn, E.

doi:10.1021/cb200508b

Cited by 58 publications

(90 citation statements)

References 49 publications

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“…X-ray structures show AurA adopting a variety of DFG-Out states, with the activation loop displaced across the active site cleft from its position in the DFG-In state by as little as 1 or by up to 4 nanometers 22,23,35,36 . For each biochemical state of AurA we measured the distance between the dyes from the degree of donor quenching in the presence of acceptor (see Online Methods).…”

Section: Resultsmentioning

confidence: 99%

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A water-mediated allosteric network governs activation of Aurora kinase A

et al. 2017

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The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift towards the active DFG-In state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium. Tpx2 further activates AurA by stabilizing a water-mediated allosteric network that links the C-helix to the active site through an unusual polar residue in the regulatory spine. The polar spine residue and water network of AurA are essential for phosphorylation-driven activation, but an alternative form of the water network found in related kinases can support Tpx2-driven activation, suggesting that variations in the water-mediated hydrogen bond network mediate regulatory diversification in protein kinases.

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Section: Resultsmentioning

confidence: 99%

“…X-ray structures show AurA adopts the DFG-Out state when bound to a variety of kinase inhibitors 22,23 , and the DFG-In state when bound to Tpx2 24 . However, structures also show that AurA can adopt the DFG-In state when bound to nucleotide alone 24–26 , and it remains unclear whether the DFG-In/Out transition occurs during activation.…”

Section: Introductionmentioning

confidence: 99%

A water-mediated allosteric network governs activation of Aurora kinase A

et al. 2017

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“…The αC-helix rotation also results in movement of F275 of the conserved DFG motif from the DFG-out into the DFG-in position (Martin et al, 2012) (Figure 2C, bottom inset). This positions the catalytic D274 in the correct orientation to carry out phosphoryl transfer.…”

Section: Resultsmentioning

confidence: 99%

Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2

Zorba

Buosi

Kutter

et al. 2014

eLife

117

150

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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

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“…Thus it was surprising to observe that CD532 binds Aurora A as DFG-in, yet still induces a conformational disruption not achieved by nonselective tool inhibitors which induce a DFG-out conformation in Aurora A in vitro (Martin et al, 2012). Comparing CD532-bound Aurora A to the Apo structure shows the activation loop in the inactive orientation, accompanied by a shift in the entire N-terminal domain.…”

Section: Discussionmentioning

confidence: 99%

“…Purification and expression of Aurora A was performed as described previously (Martin et al, 2012), with the following modifications. Aurora A (residues 123–390, T287D) was cloned into a pET28a plasmid providing fusion with a PreScission Protease-cleavable hexahistidine tag.…”

Section: Methodsmentioning

confidence: 99%

Drugging MYCN through an Allosteric Transition in Aurora Kinase A

et al. 2014

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Summary MYC proteins are major drivers of cancer, yet are considered undruggable, as their DNA binding domains are composed of two extended alpha helices with no apparent surfaces for small molecule binding. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora A. We describe a class of inhibitors that disrupts the native conformation of Aurora A, and drives degradation of MYCN protein across MYCN-driven cancers. Comparison of co-crystal structures with structure-activity relationships across multiple inhibitors and chemotypes, coupled with mechanistic studies and biochemical assays, delineates an Aurora A conformation-specific effect on proteolytic degradation of MYCN, rather than simple nanomolar-level inhibition of Aurora A kinase activity.

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A Novel Mechanism by Which Small Molecule Inhibitors Induce the DFG Flip in Aurora A

Cited by 58 publications

References 49 publications

A water-mediated allosteric network governs activation of Aurora kinase A

A water-mediated allosteric network governs activation of Aurora kinase A

Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2

Drugging MYCN through an Allosteric Transition in Aurora Kinase A

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