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

Pegram, Laurel M. and Liddle, Jennifer C. and Xiao, Yao and Hoh, Maria and Rudolph, Johannes and Iverson, Dylan B. and Vigers, Guy P. and Smith, Darin and Zhang, Hailong and Wang, Weiru and Moffat, John G. and Ahn, Natalie G. (2019) Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2. Proceedings of the National Academy of Sciences of the United States of America, 116 (31). pp. 15463-15468. ISSN 0027-8424. PMCID PMC6681744.

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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.

Item Type:Article
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URLURL TypeDescription Information Paper CentralArticle
Pegram, Laurel M.0000-0003-3131-9199
Rudolph, Johannes0000-0003-0230-3323
Vigers, Guy P.0000-0003-4451-8453
Ahn, Natalie G.0000-0002-2690-2630
Additional Information:© 2019 National Academy of Sciences. Published under the PNAS license. Contributed by Natalie G. Ahn, June 14, 2019 (sent for review April 23, 2019; reviewed by Amy Andreotti and Elizabeth J. Goldsmith). We are indebted to Drs. Thomas Lee and Danijel Djukovic for assistance with LC-MS instrumentation and analysis; to Dr. Marcelo Sousa and Sandra Metzner for the gift of His6-SUMO-ratERK2; and to the 2015 CCP4/APS Summer School program for use of the Argonne National Laboratory beamline and help with X-ray data collection. This work was supported by NIH grant awards R01GM114594, S10RR026641 (N.G.A.), and T32GM008759 (J.C.L. and D.B.I.). J.C.L. gratefully acknowledges support by the Eugene Huffman Memorial Scholarship and the Sheryl R. Young Memorial Scholarship, University of Colorado. Author contributions: L.M.P., J.C.L., Y.X., M.H., J.R., G.P.V., D.S., H.Z., W.W., J.G.M., and N.G.A. designed research; L.M.P., J.C.L., Y.X., M.H., J.R., G.P.V., D.S., H.Z., W.W., and J.G.M. performed research; L.M.P., J.C.L., Y.X., M.H., J.R., D.B.I., G.P.V., D.S., H.Z., W.W., J.G.M., and N.G.A. analyzed data; and L.M.P. and N.G.A. wrote the paper. Reviewers: A.A., Iowa State University; and E.J.G., University of Texas. Conflict of interest statement: G.P.V., D.S., and H.Z. are employees of Array BioPharma Inc.; W.W. and J.G.M. are employees of Genentech Inc.; and H.Z. is the CEO of Blueray Biopharma. Data deposition: PDB ID 6OPG (Deposition ID: D_1000241111) Phosphorylated ERK2 with AMP-PNP; PDB ID 6OPH (Deposition ID: D_1000241141) Phosphorylated ERK2 with GDC-0994; PDB ID 6OPI (Deposition ID: D_ 1000241142) Phosphorylated ERK2 with SCH-CPD336; and PDB ID 6OPK (Deposition ID: D_ 1000241143) Phosphorylated ERK2 with VERTEX-11E. This article contains supporting information online at
Funding AgencyGrant Number
NIH Predoctoral FellowshipT32GM008759
University of ColoradoUNSPECIFIED
Subject Keywords:ERK2; dynamics; NMR; hydrogen-exchange mass spectrometry; conformational selection
Issue or Number:31
PubMed Central ID:PMC6681744
Record Number:CaltechAUTHORS:20190516-072949718
Persistent URL:
Official Citation:Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2. Laurel M. Pegram, Jennifer C. Liddle, Yao Xiao, Maria Hoh, Johannes Rudolph, Dylan B. Iverson, Guy P. Vigers, Darin Smith, Hailong Zhang, Weiru Wang, John G. Moffat, Natalie G. Ahn. Proceedings of the National Academy of Sciences Jul 2019, 116 (31) 15463-15468; DOI: 10.1073/pnas.1906824116
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:95525
Deposited By: Tony Diaz
Deposited On:16 May 2019 15:15
Last Modified:02 Jun 2020 18:34

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