ERK autoinhibition mechanism informs a drug combination strategy

ERK is a key regulator in the MAPK pathway, controlling essential cell processes through dual-phosphorylation-based activation. We investigate the conformational equilibrium between inactive and active ERK states, which is controlled allosterically by its structurally unique C-terminal L16 segment. Using molecular dynamics simulations of full-length and truncated ERK variants across phosphorylation states, we demonstrate that activation is achieved through a lateral αC-helix rotation initiated by dual-phosphorylation. The L16 segment intrinsically stabilizes the inactive kinase conformation by restricting αC-helix orientation, counteracting its natural tendency to adopt an active alignment. Removal of the L16 loop disrupts the unphosphorylated state, whereas all phosphorylated systems remain stable. The α_L16-helix restricts the movements of αC-helix, ensuring controlled conformational transitions. Disruption of L16 segment's constraints through modification or cellular processes lowers the activation barrier, rendering kinase activation under partial phosphorylation. Importantly, we discovered an L16 segment-associated cryptic pocket, whose (allosteric) targeting in combination with orthosteric inhibitors that selectively bind the active state, appears a promising therapeutic strategy. Currently, no direct ERK allosteric and orthosteric drug combination is available. This discovery has been potentiated by a mechanistic understanding of ERK autoinhibition which informs L16's potential as a cryptic binding site for ERK dysregulation.