(650e) Understanding the Activation Mechanism of the Insulin Receptor Kinase Domain Using Enhanced Conformational Sampling and Free-Energy Calculations

Receptor tyrosine kinases (RTKs) are tightly regulated
ligand-activated transmembrane glycoproteins that catalyze the
phosphorylation of specific tyrosines on protein substrates.
Activation of the insulin receptor (IR), an RTK, is dependent upon
trans-autophosphorylation of three activation loop (the A-loop)
tyrosines located in each cytoplasmic kinase domain of IR
(IRKD). Crystal structures of the inactive and active states of
bi-lobal IRKD have revealed critical differences in the conformations
of the A-loop and the alpha-C-helix in the N-lobe, both of which are
significantly displaced 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
alpha-C-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'' as well as
assembly/disassembly of the regulatory (R) and catalytic (C) spines
remains elusive, chiefly due to the unavailability of structural data
on intermediate conformations of the A-loop. In this work, we have
characterized the inactive-to-active conformational change in the
A-loop via a judicious combination of untargeted
temperature-accelerated molecular dynamics (TAMD) and free-energy
calculations using the string method. TAMD simulations consistently
show folding of the A-loop into a helical conformation followed by
unfolding to an active conformation, causing the highly conserved
DFG-motif to switch from the inactive “D-out/F-in” to the active
“D-in/F-out” conformation. The minimum free-energy path (MFEP)
computed from the string method confirms existence of these helical
intermediates along the inactive-to-active path, and the thermodynamic
free-energy differences are consistent with previous work on various
kinases. The conformational change in the A-loop also suggests that
the R-spine can be dynamically assembled/disassembled both via
DFG-flip or the movement of the alpha-C-helix.