Why an A-loop phospho-mimetic fails to activate PAK1: understanding an inaccessible kinase state by molecular dynamics simulations

Structure
Yuen-Wai NgE Manser

Abstract

Crystal structures of inactive PAK1(K299R) and the activation (A)-loop phospho-mimetic PAK1(T423E) have suggested that the kinase domain is in an active state regardless of activation loop status. Contrary to a large body of literature, we find that neither is PAK1(T423E) active in cells, nor does it exhibit significant activity in vitro. To explain these discrepancies all-atom molecular dynamics (MD) simulations of PAK1(phospho-T423) in complex with ATP and substrate were performed. These simulations point to a key interaction between PAK1 Lys308, at the end of the alphaC helix, and the pThr423 phosphate group, not seen in X-ray structures. The orthologous PAK4 Arg359 fulfills the same role in immobilizing the alphaC helix. These in silico predictions were validated by experimental mutagenesis of PAK1 and PAK4. The simulations explain why the PAK1 A-loop phospho-mimetic is inactive, but also point to a key functional interaction likely found in other protein kinases.

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Citations

Jun 29, 2011·The Journal of Cell Biology·Violaine D Delorme-WalkerGary M Bokoch
May 4, 2011·The Journal of Physical Chemistry. B·Priyadarshi SatpatiThomas Simonson
Nov 27, 2015·Nature Communications·Yohendran BaskaranRobert C Robinson
Jun 2, 2012·EMBO Reports·Yohendran BaskaranEd Manser
Jan 3, 2013·Journal of Computational Chemistry·Thomas Simonson, Priyadarshi Satpati
Mar 13, 2012·Expert Opinion on Therapeutic Patents·James J CrawfordJoachim Rudolph
Nov 20, 2012·Cellular Logistics·Zhuo-Shen Zhao, Ed Manser
Nov 6, 2012·Cellular Logistics·Jeffrey Field, Ed Manser
Jan 22, 2019·Journal of Biomolecular Structure & Dynamics·Jayashree BiswalJeyakanthan Jeyaraman

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