Structural Plasticity and Ligand Promiscuity of CYP3A4 Revealed by Cryo-EM.

Cytochrome P450 3A4 (CYP3A4) metabolizes roughly half of all marketed drugs, and its inhibition can cause clinically significant drug-drug interactions. The enzyme accommodates chemically diverse ligands, making binding modes and metabolic outcomes difficult to predict. Previous X-ray crystallography efforts have leveraged a truncated construct without the N-terminal segment that tethers CYP3A4 to the membrane. Here we show that the same construct assembles into a symmetric trimer that can be resolved by cryo-EM and determine structures of both unliganded and ligand-bound CYP3A4. Multiple ligands are resolved with density consistent with several mutually exclusive conformations. Protein remodeling to reshape the binding pocket is concentrated in the F/G loop, which is poorly resolved and unmodeled in many X-ray structures. These features likely underlie the poor predictive performance of co-folding methods on this target. The routine use of cryo-EM to resolve CYP3A4 ligand-bound complexes will provide the ground truth data needed to make predictive models of drug metabolism useful in practice.