Pivotal role of P450–P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone

Abstract

SYNOPSIS We investigated the relationship between oligomerization of cytochrome P450 3A4 (CYP3A4) and its response to α-naphthoflavone (ANF), a prototypical heterotropic activator. Addition of ANF resulted in over a two-fold increase in the rate of CYP3A4-dependent debenzylation of 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC) in human liver microsomes (HLM) but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-cytochrome P450 reductase (CPR). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behavior observed with HLM. The activation in this system was dependent on the surface density of the enzyme. While no activation was detectable at a lipid:P450 (L/P) ratio ≥ 750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization we probed P450-P450 interactions with a new technique based on luminescence resonance energy transfer (LRET). The amplitude of LRET in mixed oligomers of the heme protein labeled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes. Addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore, the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions.

Interactions among Cytochromes P450 in Microsomal Membranes

“…Preparation of Proteoliposomes-Proteoliposomes were obtained by the described octyl glucoside dialysis technique with some modifications (23). Specifically, we used a 2:1:0.6 mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid.…”

“…Site-directed Mutagenesis, Protein Purification, and Modification with Thiol-reactive Probes-The cysteine-depleted mutants CYP3A4(C58,C64) (36), CYP3A4(C468) (23) and CYP3A4(C166) were generated using the QuikChange site-directed mutagenesis kit and a template consisting of the cDNA of the N-terminally truncated (⌬3-12) S18F CYP3A4 variant with a tetrahistidine tag attached at the C terminus. This construct is referred to in this work as the "wild type CYP3A4" to distinguish it from the cysteine-depleted mutants.…”

“…In particular, we demonstrated that the stimulation by ␣-naphthoflavone (ANF), a prototypical activator of CYP3A4, is observed only with enzyme oligomers and becomes suppressed upon their dissociation (23). In the present work, we apply our new methods for the analysis of interactions between dissimilar P450 enzymes.…”

“…However, the integrative connections in microsomal monooxygenase go far beyond that and involve functional effects of physical interactions of multiple P450 molecules that result in oligomerization (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Besides evidence of homooligomerization of various P450 species (10,12,15,(17)(18)(19)(20)(21)(22)(23), there are also multiple indications of interactions between dis-similar P450s in membranes of proteoliposomes (13), microsomes (14), and living cells (16). The body of evidence of profound functional effects exerted by P450-P450 interactions continues to grow (24,25).…”

“…It capitalizes on the results of our recent work (23), where we developed a simple technique that produces catalytically competent membranous systems with variable content of cytochromes P450 via incorporation of the purified enzymes into insect cell microsomes containing recombinant NADPHcytochrome P450 reductase. This innovative approach is further complemented by assessing the degree of P450 oligomerization through monitoring P450-P450 interactions with luminescence resonance energy transfer (LRET) (23).…”