Aided by molecular modeling, we establish three major conformations, the relative abundances of which were dependent on the Aur A activation status: one highly populated compact conformer similar to that observed in most crystal structures, a second highly populated conformer possessing a more open structure infrequently found in crystal structures, and an additional low-abundance conformer not currently represented in the protein databank.
#Aurora with screens unveiled Activator#
Using gas-phase ion-mobility mass spectrometry (IM-MS), we characterize changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition. Evaluating the effects of binding of small molecule inhibitors on kinase conformational dynamics can assist in understanding both inhibition and resistance mechanisms. Protein kinase inhibitors are highly effective in treating diseases driven by aberrant kinase signaling and as chemical tools to help dissect the cellular roles of kinase signaling complexes.
f) Example trace of ad ouble-labeled K224C/S283C Aurora As ingle molecule showing the background-subtracted fluorescence intensity over time. The differencein activity between pseudo-wildtype and labeled protein cannot be accountedf or by incomplete protein labeling (see the SupportingI nformationf or labeling efficiency). Activity shown as the produced over the course of a1hreaction. Labeling sites (K224 in orange and S283 in pink/cyan) shown as filled spheres.P DBs 1OL5 and 2WTV.e)Kinase activity assay for unlabeled pseudo-wildtype Aurora A( &)a nd TMR-labeled K224C/S283C (~). Activation loop shown in cyan/bright pink. Active T-loop:b lue, inactive T-loop:p ink. d) Conformational change and labeling sites in Aurora A. c) Proposed equilibrium model showing TMR-labeled sites (pink stars) and the expected fluorescence signal. b) Current equilibrium model of type II inhibitor binding:A ctive apo kinase is in equilibriumwith inhibited kinase in an inactive T-loop conformation. A) Current model of kinase activation:P hosphorylation on the activation loop (black line) locks the kinase in an active T-loop conformation.
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