A Model of a MAPK•Substrate Complex in an Active Conformation: A Computational and Experimental Approach
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The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)(2-3)-X(2-6)-Φ(A)-X-Φ(B), where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus (7)RK-X(2)-Φ(A)-X-Φ(B) (13). This results in a 2-fold increase in k(cat)/K(m) for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves k(cat)/K(m) by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in k(cat)/K(m), with little apparent change in k(cat). A peptide that binds to the DRS of ERK2 affects K(m), but not k(cat). Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.
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NMR CSP experiments can validate models of MAPK:MAPK regulatory/substrate protein interactions generated using alternative methods, as was done for the pTpYERK2:ETS-1 complex.
“These NMR experiments can be used to validate models of MAPK:MAPK regulatory/substrate protein interactions generated using alternative methods, such as molecular modeling, as was done for the pTpYER-K2:ETS-1 complex.70”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [70]
The SAM domain of Ets-1 binds the FRS or FxF docking site on ERK2, which is located 15 Å away from the ERK2 KIM binding groove.
“For example, the SAM domain of Ets-1 binds the FRS or FxF docking site on ERK2, which is located 15 A ˚away from the ERK2 KIM binding groove [Fig.4(C), lower right].70,74”
From Molecular basis of MAP kinase regulation (2013) · cites this paper as [70]
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Regulation and inhibition of MAP kinases
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- Authors
- Lee, S., Warthaka, M., Yan, C., Kaoud, T. S., Piserchio, A., Ghose, R., Ren, P., & Dalby, K. N.
- Affiliations
- The University of Texas at Austin (US)
- City College of New York (US)
- The Graduate Center, CUNY (US)
- Corresponding author
- Ren, Pengyu
- Dalby, Kevin N.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2011-04-11
- Volume
- 6
- Issue
- 4
- Pages
- e18594
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3073974
- OpenAlex
- W2107292855
- Cited by
- 26
- References
- 37
- Citations by year
- 2026: 2 · 2025: 1 · 2023: 2 · 2022: 1 · 2020: 1 · 2019: 3 · 2018: 1 · 2017: 1 · 2015: 1 · 2014: 2 · 2013: 2 · 2012: 7
- FWCI
- 1.69
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- CUNY Academic Works (City University of New York) (submittedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Texas ScholarWorks (Texas Digital Library) (submittedVersion, cc-by)
- Funders
- Welch Foundation (F‐1390)
- National Institute of General Medical Sciences (R01GM059802, R01GM084278, GM079686, GM084278, R01GM079686)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Melanoma and MAPK Pathways
- Keywords
- ERK2, ETS1, substrate recognition, phosphorylation kinetics, docking site, N-terminal disordered region, Elk-1, MKK1, mutagenesis, protein-protein interaction
- MeSH terms
- Amino Acid Sequence, Animals, Ligands, Models, Molecular, Molecular Sequence Data, Mutation, Peptides, Phosphorylation, Protein Binding, Protein Conformation, Substrate Specificity, Computational Biology, Mitogen-Activated Protein Kinase 1, Catalytic Domain, Proto-Oncogene Protein c-ets-1, Mice, Biocatalysis
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