A Temperature-Accelerated Molecular Dynamics Study of the Insulin Receptor Kinase

Receptor tyrosine kinases are allosteric enzymes that catalyze the phosphorylation of specific tyrosines on protein substrates. The insulin receptor (IR) is a ligand-activated tyrosine kinase, whose biological function and ligand-stimulated catalytic activity depends upon trans-autophosphorylation of three activation loop (A-loop) tyrosines located in each of its cytoplasmic kinase domain (IRKD). The crystal structures of the excised inactive and active IRKD reveal that the A-loop is displaced by ~20 Å on activation. The highly conserved residues Asp¹¹⁵⁰, Phe¹¹⁵¹, and Gly¹¹⁵² at the N-terminus of the A-loop (the "DFG" motif) collectively "flip" to bury the Phe¹¹⁵¹ underneath αC-helix, and simultaneously present Asp¹¹⁵⁰ for ATP binding. However, the exact mechanism of the conformational change in the A-loop which leads to the "DFG-flip" remains elusive, chiefly due to the unavailability of structural data on intermediate conformations of the A-loop. In this work, we have studied the inactive to active transition of the A-loop at the atomistic level using temperature accelerated molecular dynamics (TAMD). Starting with the inactive A-loop conformation, TAMD (50-ns) generated a target A-loop conformation within ~8 Å (RMSD) of the known active A-loop conformation. A further 20-ns MD-equilibration of this structure in the presence of ATP and phosphotyrosines stabilizes the A-loop conformation to an RMSD of ~4 Å with respect to its active state. Significantly, we also capture the DFG-flip during TAMD, and observe that this flip is facilitated by a transient three-turn helical conformation of the A-loop, the folding of which draws the placement of the side-chains of Phe¹¹⁵¹ and Asp¹¹⁵². Such transient helical conformations of the A-loop can potentially be exploited for the design of novel inhibitors that target a specific DFG conformation, and have been observed in crystal structures of related kinase families.