Thesis title: Phosphorylation, Mg-ADP, and Inhibitors Differentially Shape the Conformational Dynamics of the A-loop of Aurora-A
Abstract
The conformational state of the activation loop (A-loop) is pivotal for the activity of most protein kinases. Hence, the characterization of the conformational dynamics of the A-loop is important to increase our understanding of the molecular processes related to diseases and to support the discovery of small molecule kinase inhibitors. Here, we carry out a combination of molecular dynamics (MD) and essential dynamics (ED) analyses to fully map the effects of phosphorylation, ADP, and conformation disrupting (CD) inhibitors (i.e., CD532 and MLN8054) on the dynamics of the A-loop of Aurora-A.
MD revealed that the stability of the A-loop in an open conformation is enhanced by a single phospho-Thr-288, while paradoxically, the presence of second phosphorylation at Thr-287 decreases such stability and renders the A-loop more fluctuant in time and space. Moreover, we found that this post-translational modification significantly affects the direction of the A-loop motions.
Additionally, the free energy landscape from MD simulation is a very useful approach to gain an overall view of the conformational landscape accessible to a protein and identify representative protein substates. The main free energy well in the global free energy minimum region was completely changed after the binding of inhibitors. These free energies well indicate the stable conformational states of this molecule. The binding of CD532 compounds to Aurora-A leads to different global minima of Aurora-A during the 100 ns MD simulations as compared to Aurora-A in complex with MLN8054.
ED analysis suggests that the presence of the phosphate moiety induces the dynamics of Aurora-A to sample two distinct energy minima, instead of a single large minimum, as in unphosphorylated Aurora-A states. This observation indicates that the conformational distributions of Aurora-A with both single and double phospho-threonine modifications are remarkably different from the unphosphorylated state. In the closed states, binding of CD532 and MLN8054 inhibitors has the effect of increasing the distance of the N- and C-lobes of the kinase domain of Aurora-A, and the angle analysis between those two lobes during MD simulations showed that the N- and C-lobes are kept more open in presence of CD532, compared to MLN8054. As the A-loop is a common feature of Aurora protein kinases, our studies provide a general description of the conformational dynamics of this structure upon phosphorylation and different ligands binding.
Keywords: Aurora-A; molecular dynamics simulation; activation loop; conformational dynamics; free energy landscape