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.
Highlights
With the increasing number of P450 structures that demonstrate conformational flexibility [1,2,3,4], conformational changes accompanying enzyme-substrate interactions are recognized as a common feature that contributes to the ability of drug-metabolizing cytochromes P450 to metabolize a vast range of substrates [5,6]
Our recent design of a P450-compatible luminescence resonance energy transfer (LRET) donor-acceptor pair consisting of the phosphorescent dye erythrosine iodoacetamide (ERIA) and nearinfrared fluorophore DY-731 maleimide (DYM) [13] provided a powerful tool for detecting these effector-induced conformational changes and probing their nature
An important aspect specific to the use of techniques based on RET to study conformational flexibility in cytochromes P450 is the absorbance of the heme chromophore
Summary
With the increasing number of P450 structures that demonstrate conformational flexibility [1,2,3,4], conformational changes accompanying enzyme-substrate interactions are recognized as a common feature that contributes to the ability of drug-metabolizing cytochromes P450 to metabolize a vast range of substrates [5,6]. In addition to the role in adaptation of the P450 active site to substrates of various shapes and sizes, ligand-induced conformational rearrangements in some cytochromes P450 are hypothesized to be a core component of an allosteric mechanism that regulates the function of the microsomal drug-metabolizing ensemble [7,8,9] This mechanism is thought to reveal itself in multiple instances of heterotropic cooperativity (activation of metabolism of one substrate by a second) observed with some drug-metabolizing P450s, of which human P450 3A4 (CYP3A4) is the most prominent. Important support of allosteric modulation of CYP3A4 is provided by recent observations of a peripheral ligand binding site in the enzyme [10,11,12] Interactions of this site located at the distal surface of the enzyme and surrounded by the F/F9 and G/ G9 loops [10,12] were hypothesized to be involved in CYP3A4 activation by steroids, a-naphthoflavone (ANF), and other heterotropic activators [11,12,13]. The above design allowed us to probe the conformational response of the doublelabeled protein variants to ligands exemplifying non-cooperative interactions, homotropic cooperativity, or heterotropic modulation of the enzyme
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