Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2
| Autor: | Natalie G. Ahn, Weiru Wang, Yao Xiao, Dylan B. Iverson, Jennifer C Liddle, Darin Smith, Laurel M. Pegram, John Moffat, Johannes Rudolph, Hailong Zhang, Guy Vigers, Maria Hoh |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
MAPK/ERK pathway Mitogen-activated protein kinase kinase MAP kinase phosphatase activity Crystallography X-Ray Models Biological 01 natural sciences Mass Spectrometry Protein Structure Secondary Dephosphorylation 03 medical and health sciences Allosteric Regulation Catalytic Domain Humans Nucleotide Phosphorylation Extracellular Signal-Regulated MAP Kinases Protein Kinase Inhibitors 030304 developmental biology chemistry.chemical_classification 0303 health sciences Binding Sites Multidisciplinary biology Nucleotides 010405 organic chemistry Chemistry Kinase Deuterium Exchange Measurement Active site Biological Sciences Small molecule 0104 chemical sciences 030104 developmental biology Mitogen-activated protein kinase Biocatalysis MAP kinase phosphatase biology.protein Biophysics |
| DOI: | 10.1101/639567 |
| Popis: | Modulating the dynamics of protein kinases expands the inhibitory mechanisms for small molecules. NMR measurements of the 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, while inhibitor GDC-0994 and ATP analogue AMP-PNP retain L⇌R exchange. 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 analogue. 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 modes for Vertex-11e, GDC-0994 and AMP-PNP to active vs inactive ERK2, where the extent of HX protection matches their degree of R-state formation. In addition, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop, suggesting that L⇌R exchange involves coupling between the activation loop and the active site. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity towards 2P-ERK2. We conclude that global motions in ERK2 promote productive nucleotide binding, and couple with the activation loop to allow control of dephosphorylation by conformation-selective inhibitors.SIGNIFICANCE STATEMENTProtein kinases in the RAF/MKK/ERK signaling pathway are dysregulated in cancer and are important targets for inhibitor development. Catalytic activation of the MAP kinase, ERK2, induces global motions involving exchange between two conformational states. Using nuclear magnetic resonance (NMR) and hydrogen-exchange mass spectrometry, we show that inhibitors exploit these motions to trap ERK2 in distinct states. Our findings reveal motions of the activation loop coupled to the active site. Inhibitor binding can control these activation loop dynamics to alter its rate of dephosphorylation by MAP kinase phosphatase. |
| Databáze: | OpenAIRE |
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