Cooperative binding of aflatoxin B1 by cytochrome P450 3A4: a computational study

Aflatoxin B1 (AFB1)-the most potent natural carcinogen known to men-is metabolized by cytochrome P450 3A4 (CYP3A4), either to the genotoxic AFB1 exo-8,9-epoxide or to the detoxified 3α-hydroxy AFB1. The activation of the procarcinogen proceeds in a highly cooperative fashion, which differs from common allosteric regulation in the sense that it can be attributed to simultaneous occupancy of a single large and malleable active site by multiple ligand molecules. Unfortunately, unlike in the case of ketoconazole, there is currently no experimental structure available for the doubly ligated CYP3A4-AFB1 complex. Therefore, we employed a sequential molecular docking protocol to create various possible doubly ligated complexes and subsequently performed molecular dynamics simulations and free-energy calculations to check for their consistency with the available experimental data on regio- and stereoselectivity of both AFB1 oxidations as well as with available kinetic data. Only the system in which the first AFB1 molecule was bound in a face-on C8-C9 epoxidation mode and the second AFB1 molecule was bound in a side-on 3α-hydroxylation mode-a result of an unconstrained molecular docking protocol-has successfully fulfilled all the imposed criteria and is therefore proposed as the most likely structure of the doubly ligated complex of CYP3A4 with AFB1. The empirical Linear Interaction Energy method revealed that shape complementarity through nonpolar dispersion interactions between the two bound AFB1 molecules is the main source of the experimentally observed positive homotropic cooperativity. The reported study represents a nice example of how state-of-the-art molecular modeling techniques can be used to study complicated macromolecular complexes, whose structures have not yet been experimentally determined, and to validate these against the available experimental data. The proposed structure will facilitate future studies on the rational design of successful AFB1 modulators or on human subpopulations characterized by specific CYP3A4 polymorphisms that are especially sensitive to AFB1.