275009 Understanding the Activation Mechanism of the Insulin Receptor Kinase Domain Using Enhanced Conformational Sampling and Free-Energy Calculations

Thursday, November 1, 2012: 9:51 AM
411 (Convention Center )
Harish Vashisth, Chemistry and Biophysics, University of Michigan, Ann Arbor, MI and Cameron F. Abrams, Department of Chemical and Biological Engineering, Drexel University, Philadelphia, PA

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.

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