Activation Loop Dynamics Are Coupled to Core Motions in Extracellular Signal-Regulated Kinase-2.

The activation loop segment in protein kinases is a common site for regulatory phosphorylation. In extracellular signal-regulated kinase 2 (ERK2), dual phosphorylation and conformational rearrangement of the activation loop accompany enzyme activation. X-ray structures show the active conformation to be stabilized by multiple ion pair interactions between phosphorylated threonine and tyrosine residues in the loop and six arginine residues in the kinase core. Despite the extensive salt bridge network, nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiments show that the phosphorylated activation loop is conformationally mobile on a microsecond to millisecond time scale. The dynamics of the loop match those of previously reported global exchange within the kinase core region and surrounding the catalytic site that have been found to facilitate productive nucleotide binding. Mutations in the core region that alter these global motions also alter the dynamics of the activation loop. Conversely, mutations in the activation loop perturb the global exchange within the kinase core. Together, these findings provide evidence for coupling between motions in the activation loop and those surrounding the catalytic site in the active state of the kinase. Thus, the activation loop segment in dual-phosphorylated ERK2 is not held statically in the active X-ray conformation but instead undergoes exchange between conformers separated by a small energetic barrier, serving as part of a dynamic allosteric network controlling nucleotide binding and catalytic function.

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2.

Conformational selection by small molecules expands inhibitory possibilities for protein kinases. 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. 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. 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. 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. Thus, the L→R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. 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.