Computational and EPR studies of the conformational dynamics of the Aurora-A kinase protein activation loop

  • Maria Grazia Concilio

Student thesis: Phd


Aurora-A is a miotic serine/threonine protein kinase that controls many cellular pathways, its function is implicated in a wide range of diseases including cancer and its activity is tightly regulated by changes in conformation of a conserved region known as the activation loop. The structure of the Aurora-A has been extensively studied by X-ray crystallography and in many cases the position of the activation loop in solution is unknown. Therefore, studies of Aurora-A’s activation through characterisation of its conformations in solution are important to enhance understanding of molecular processes related to diseases and to support the discovery of small molecule kinase inhibitors. In this project, continuous-wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques were employed to characterize and resolve the conformational dynamics of the activation loop of Aurora-A kinase. The protein used for this study was spin labelled with the (1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl) methane-thiosulfonate spin label (MTSL) introduced via site-directed mutagenesis within the activation loop. Computational methods, including quantum mechanical (QM) calculations based on density functional theory (DFT) and classical molecular dynamics (MD) simulations were carried out to determine conformational states in which both the activation loop and spin label were engaged, as well as structural fluctuations, order parameters and rotational correlation times related to the motion of the full spin-labelled system. The theoretical data obtained were used for the interpretation of the room temperature CW EPR spectra of the spin labelled Aurora-A kinase. Comparisons made between simulated and experimental spectra revealed that the motion of the protein and spin label occurred on a comparable timescale and ranged between 0.1 ns and 10 ns, indicating that the dynamics of the Aurora-A was dominated by events occurring on long timescales. A comparison between MD data related to un-phosphorylated and phosphorylated activation loops revealed that the latter was endowed by less conformational movement. In both the cases, the region that showed the most conformational dynamics was that localized between residues 282 and 294, which includes residues 287 and 288 that are crucial for the catalytic activity of Aurora-A kinase. Pulsed electron–electron double resonance (PELDOR) measurements were carried out to determine the average distances and distance distributions between spin-label pairs. Broad distance distributions and multiple populations were identified, indicating high flexibility of the structure. Variations in the distance distributions were observed upon addition of different protein kinase inhibitors and binding partners, which were attributed to differences in conformational space accessible to the activation loop that were found to be in good agreement with previous studies using X-ray crystallography. This study provided evidence for structural adaptations that could be used for drug design and a methodological approach that has potential to characterize inhibitors in development.
Date of Award1 Aug 2017
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDavid Collison (Supervisor) & Alistair Fielding (Supervisor)

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