Abstract
Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys64 and Cys377) and the alpha-domain, where Cys121 and Cys468 are located.