Dynamic single-molecule binding of carbamazepine explains diffuse cryo-EM density in the SUR1 drug-binding pocket

Abstract

Carbamazepine (CBZ), an anticonvulsant drug and pharmacochaperone of pancreatic ATP-sensitive potassium (KATP) channels, binds within the SUR1 drug-binding pocket. However, cryo-electron microscopy (cryo-EM) studies revealed an unusually diffuse ligand-associated density that could not be unambiguously interpreted. Despite being substantially smaller than glibenclamide (GBM), CBZ produced a density of comparable size, leading to the proposal that either two CBZ molecules occupy the binding pocket simultaneously or that a single molecule adopts multiple binding configuresions. Here, we combine molecular dynamics simulations, density reconstruction from simulation ensembles, quantum chemical calculations, and binding free-energy analyses to investigate the molecular origin of this diffuse cryo-EM density. Density functional theory calculations indicate that CBZ dimers are only weakly stabilized and do not exhibit a strong intrinsic preference for dimerization. Molecular dynamics simulations further show that two simultaneously bound CBZ molecules fail to form a stable dimeric complex and generate density distributions inconsistent with the experimental cryo-EM map. In contrast, a single CBZ molecule remains localized within the SUR1 cavity while sampling a broad ensemble of positions and orientations. Remarkably, density reconstructed directly from the simulation trajectories closely reproduces the extent and shape of the experimentally observed cryo-EM density. Our results demonstrate that the diffuse cryo-EM density associated with CBZ arises from dynamic binding of a single molecule rather than ligand dimerization. More broadly, this study highlights how ligand dynamics can shape cryo-EM densities and illustrates the value of integrating molecular simulations with experimental structural data to interpret heterogeneous ligand-binding states.