Global analysis of protein-protein interactions reveals multiple CYP2E1-reductase complexes.

Protein-ligand and protein-protein interactions are fundamental to biological processes such as signal transduction, chemical transformations, and electron transport. An understanding of the role of these processes in biological function requires the identification of the detailed interaction mechanisms. These details provide a framework in which to understand how the process of molecular recognition maintains proper homeostasis, or leads to deleterious conditions. In this study, we characterize the details of protein-protein interactions involving cytochrome P4501 (P450) and the cytochrome P450 reductase (CPR). Traditional experimental protocols and data-analytic methods applied to this two-component system failed to provide a clear answer regarding the binding stoichiometry. We describe an alternative data-analytic approach applicable to any multi-component system. The method is based on a general numerical analysis of simultaneous biochemical equilibria, without any restriction on the number of component molecular species or the number of complexes they form (1). Our results indicate the involvement of the quaternary complex (P450)2•(CPR)2, in addition to possible involvement of the ternary complex (P450)2•CPR, the ternary complex P450•(CPR)2, or both ternary complexes. Microsomal P450 enzymes are major catalysts in the oxidative transformation of a structurally diverse class of compounds including steroids, fatty acids, hormones, antibiotics, and a wide variety of artificially produced chemicals (xenobiotics), such as drugs, food additives, and environmental contaminants (2). P450s convert lipid-soluble molecules to more water-soluble forms and in effect, modulate transport and other chemical properties. To accommodate a wide array of compounds, typical P450 enzymes have evolved low specificity and activity toward substrates, making interpreting and predicting their catalytic properties difficult. Localized to the endoplasmic reticulum, the membrane-bound P450 is best considered to be an aggregate of multiple distinct P450s associating with redox partners, the obligatory cytochrome P450 reductase (CPR) and in some cases cytochrome b5 (cyt b5) (reviewed in (3)). The resulting protein-protein interactions ultimately define activity for P450s. Understanding the biological impact of P450 activity requires knowledge of the identity of the P450 functional complex and the mechanism modulating its formation. Because the estimated molar ratio for P450 and CPR in membranes is approximately 20:1 (4) to 40:1 (5), one of the first models of the functional P450 complex was a rigid cluster of multiple P450 molecules surrounding a single CPR molecule (4). However, rotational diffusion studies (6, 7), cross-linking efforts (8, 9), and catalytic studies with solubilized P450 and CPR (10–12) later favored a more dynamic mass action model whereby monomeric P450 and CPR were in equilibrium with a functional binary complex, as shown in Scheme 1. Predictions from this mechanism have influenced both the design and interpretation of studies elucidating the biological impact of P450 activity. Scheme 1 The formation and catalytic activity of the simplest (binary) functional complex between P450 and CPRa. P450 2E1 activity is highly dependent on the composition of the functional complex. P450 2E1 plays a central role in the metabolism of a large number of small molecular weight compounds (molecular weight < 100), such as aliphatic, aromatic, and halogenated hydrocarbons, many of which are solvents and industrial monomers, and some of which are suspected to cause cancer (13). Although the most notable P450 2E1 substrate is ethanol, p-nitrophenol (pNP) is regarded as a typical model substrate (14). The ability to transform these compounds to products depends on the coupling of electron-transfer processes between P450 2E1 and redox partners, CPR and cyt b5 (14). The mechanism by which the functional complex(es) form for these prospective partners remains to be resolved. Unlike other P450s, cyt b5 can even support certain reactions in the absence of CPR (15), further underscoring the complexity of the role of protein-protein interactions in P450 2E1 function. Poor coupling efficiency for P450 2E1 reactions leads to decreased transformation of organic substrates to products and the formation of reactive oxygen species, a precursor to oxidative stress. The biological significance of P450 2E1-induced oxidative stress has been implicated in alcohol-induced liver damage and roles in diabetes, obesity, fasting, cancer, and nonalcoholic steatohepatitis (reviewed in (16)). Due to the significance of protein-protein interactions in P450 2E1 activity, our goal in this study was to observe the effect of varied total concentrations of both proteins (P450 2E1 and CPR) on the overall catalytic activity of P450 2E1 toward the model substrate pNP. Because CPR is prone to degradation by contaminating proteases, we employed a proteolytically-resistant form of reductase, CPR-K56Q, which eliminated a known site of cleavage (17, 18). Initially, we employed the strategy of Miwa et al. (12) and performed a series of catalytic titrations for P450 2E1 and CPR-K56Q under conditions of excess titrant. Nevertheless, the choice of titrant yielded contradictory parameters for a binary complex mechanism, and a Job plot at 400 nM indicated the presence of a higher order complex. To reconcile these results, we eliminated titrant bias by expanding the experimental conditions and fit the data to models incorporating multiple complexes. For these experiments, both CPR and P450 were varied simultaneously and neither protein was in very large excess. Based on reports by others (19, 20), we proposed P450 2E1 and CPR-K56Q could form binary, ternary, and quaternary complexes in the absence or presence of P450 and CPR homodimers. Due to evidence for the binary functional complex, we required all models to include P450•CPR. For simplicity, we also assumed all complexes including both P450 and CPR to be catalytically active. The resulting 32 complexation models were used to perform global regression analysis of all pooled experimental data. Model discrimination analysis was performed on the basis of the second-order Akaike Information Criterion, which properly takes into account the fact that various fitting models contain a different number of adjustable model parameters. The approach enabled the ability to identify the most probable complexes present in the P450 2E1–CPR system.