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 single phospho-Thr-288, while paradoxically, the presence of a 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 has a significant effect on the direction of the A-loop motions. 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.

Phosphorylation-Induced Conformational Dynamics and Inhibition of Janus Kinase 1 by Suppressors of Cytokine Signaling 1

“…Surprisingly, from Figure B, we find that due to Tyr 1034 residue phosphorylation, dPCA suggests a single global conformation for the A-loop, which means pTyr 1034 stabilizes the A-loop structure, as is evident from our rmsd analysis (Figure ). Similarly, it was observed in previous studies that the single phosphorylation in WNK1­(Ser 382 ), , JNK­(Thr 221 ), , and Aurora-A­(Thr 288 ) stabilize the flexible A-loop. Similarly, Figure C suggests four low-energy basins corresponding to four different conformations, in which conf1 is active and well-matched with its native X-ray crystal.…”

“…This RMSF plot shows the flexible subdomain of the protein. It is evident from Figures S4 and S5 that the loop regions are relatively more flexible, and the evolutionarily conserved part shows high rigidity, as expected and consistent with previous theoretical 78,79 and experimental 23 studies of tyrosine kinases. It is evident from Figure S4A−C that after the complex formation with SOCS1, the overall flexibility of JAK1 increases except for the SOCS1-binding residues and the GQM motif.…”