A Model of a MAPK center dot Substrate Complex in an Active Conformation: A Computational and Experimental Approach

作者：Lee Sunbae^*; Warthaka Mangalika; Yan Chunli; Kaoud Tamer S; Piserchio Andrea; Ghose Ranajeet; Ren Pengyu; Dalby Kevin N

来源：PLos One, 2011, 6(4): e18594.

DOI：10.1371/journal.pone.0018594

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)-X2-6-Phi(A)-X-Phi(B), where Phi 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 (RK)-R-7-X-2-Phi(A)-X-Phi(13)(B). 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 center dot Ets center dot MgATP complex and intermediates of the enzymatic reaction.

• 出版日期2011-4-11