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bioRxiv · 10.64898/2026.09.01.748687

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

Orta, A. K.·Schäfer, J.-H.·Correy, G. J.·Norman, J. O.·Pampel, J.·Huddleston, K. K.·MacDermott-Opeskin, H.·Miller, E. B.·Reggiano, G.·Rodrigues, J. P.·Lander, G. C.·Walters, W. P.·Fraser, J. S.
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Abstract

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.

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Orta, A. K., Schäfer, J.-H., Correy, G. J., Norman, J. O., Pampel, J., Huddleston, K. K., MacDermott-Opeskin, H., Miller, E. B., Reggiano, G., Rodrigues, J. P., Lander, G. C., Walters, W. P., Fraser, J. S.. 2026-09-02. Structural Plasticity and Ligand Promiscuity of CYP3A4 Revealed by Cryo-EM. https://doi.org/10.64898/2026.09.01.748687

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