Antagonistic insulin mimetics lock the insulin receptor in an alternative apo-state

Despite significant advancements in high-resolution structural analysis of activated human insulin receptor (IR), the molecular mechanisms underlying its conformational plasticity that govern the transition from the apo state to the activated state are still not well understood. This leaves critical aspects of IR regulation unclear. Here, we reveal the mechanism by which the insulin mimetics Ada, Trim, and S661 fully inhibit the insulin receptor. The receptor is stabilized in a yet structurally un–described ∩–shaped conformation which is induced by antagonist binding between the L1 and FnIII-1’ domains. In contrast to insulin-bound IR structures, the α-CT helix is not observable in the ∩ conformation, and the membrane-proximal regions of the FnIII-3 domains are >10 nm apart, which prohibits transmembrane signal transduction and kinase domain activation. Analysis of apo-IR electron cryo-microscopy data indicates that the ∩-shaped state is one of several metastable apo-IR conformations. These findings underscore the intrinsic conformational dynamics of apo-IR and its role in integrating insulin binding and receptor activation.