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, NMR studies and enzyme kinetics were used to understand ERK2:Substrate interactions as follows : 1) Computer modeling approaches were used to predict how the N-terminus of Ets-1 associates with 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). 2) Detailed investigation of the structural perturbations of inactive ERK2 that accompany interactions involving both canonical and noncanonical recruitment events were performed using solution NMR studies. The chemical shift perturbations in inactive ERK2, indicates the structural changes in the presence of canonical and noncanonical motifs, are not restricted to the recruitment sites but also involve the linker that connects the N- and C-lobes. The canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions 3) Two docking interactions mediated peptides were used to understand the coupling of D-recruitment site interactions (DRS) and F-recruitment interactions (FRS). These studies facilitate the potential inhibitor design targeting individual docking interactions of ERK2.

Delineating MAPK-substrates interactions using molecular modeling, NMR, and enzyme kinetics

244th National Meeting (2012)

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