<scp>ERK</scp> autoinhibition mechanism informs a drug combination strategy

Abstract 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 L 16‐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.