The conformational changes accompanying L⇌R interconversion and how they compare with those of other kinases have not been elucidated.Ĭlues to defining R and L have been provided by small molecules with differential binding properties to 0P- and 2P-ERK2. Thus, allosteric regulation of ERK2 involves constraint of the unphosphorylated kinase in the L state and formation of the R state upon phosphorylation and associated activation. Global exchange could be modeled by an equilibrium between two conformational states, “L” (locked) and “R” (released), interconverting on a millisecond timescale. Nuclear magnetic resonance (NMR) ( 13C, 1H) multiple quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion measurements on ERK2 selectively labeled with Ile, Leu, and Val revealed global motions throughout the kinase core upon activation by phosphorylation ( 10, 11). Studies using hydrogen-exchange mass spectrometry (HX-MS) showed that phosphorylation of ERK2 alters rates of hydrogen exchange in localized regions, which can be ascribed to changes in conformational mobility ( 9).
Solution measurements of ERK2 have revealed changes in protein dynamics following kinase activation. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. inactive ERK2, where the extent of HX protection correlates with R state formation. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. Thus, the L→R shift in 2P-ERK2 is associated with movements needed to form a competent active site. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions.
Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. Conformational selection by small molecules expands inhibitory possibilities for protein kinases.
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