Interactions among Cytochromes P450 in Microsomal Membranes: Oligomerization of Cytochromes P450 3A4, 3A5 and 2E1 and its Functional Consequences [Membrane Biology]
December 22nd, 2014 by Davydov, D. R., Davydova, N. Y., Sineva, E. V., Halpert, J. R.
The body of evidence of physiologically-relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5 and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer (LRET) we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1 and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the inter-subunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov et al., Biochem. J., 2013: 453, 219-230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the LRET measurements of inter-probe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures . According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species.