title: Why an A-loop phospho-mimetic fails to activate PAK1: understanding an inaccessible kinase state by molecular dynamics simulations.
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.
authors.affiliation: sGSK Group at IMCB, Astar Neuroscience Research Partnership, Proteos Building, 61 Biopolis Drive, Singapore 138673.
authors.forename: Yuen-Wai
authors.initials: YW
authors.lastname: Ng
authors.forename: Devanathan
authors.initials: D
authors.lastname: Raghunathan
authors.forename: Perry M
authors.initials: PM
authors.lastname: Chan
authors.forename: Yohendran
authors.initials: Y
authors.lastname: Baskaran
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Smith
authors.forename: Chung-Hung
authors.initials: CH
authors.lastname: Lee
authors.forename: Chandra
authors.initials: C
authors.lastname: Verma
authors.forename: Ed
authors.initials: E
authors.lastname: Manser
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D020125:Mutation, Missense / Q000235:genetics; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D054462:p21-Activated Kinases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010710:Phosphates; C116367:PAK4 protein, human; C515906:Pak1 protein, rat; D054462:p21-Activated Kinases