title: A model of a MAPK•substrate complex in an active conformation: a computational and experimental approach.
abstract: 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.
authors.affiliation: Division of Medicinal Chemistry, University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Sunbae
authors.initials: S
authors.lastname: Lee
authors.forename: Mangalika
authors.initials: M
authors.lastname: Warthaka
authors.forename: Chunli
authors.initials: C
authors.lastname: Yan
authors.forename: Tamer S
authors.initials: TS
authors.lastname: Kaoud
authors.forename: Andrea
authors.initials: A
authors.lastname: Piserchio
authors.forename: Ranajeet
authors.initials: R
authors.lastname: Ghose
authors.forename: Pengyu
authors.initials: P
authors.lastname: Ren
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D019295:Computational Biology / Q000379:methods*; D008024:Ligands; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D050784:Proto-Oncogene Protein c-ets-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D010455:Peptides; D050784:Proto-Oncogene Protein c-ets-1; D019950:Mitogen-Activated Protein Kinase 1