Regions of CYP1A2 and CYP2B4 that are Involved in the Homomeric and Heteromeric Complex Formation: The Search Continues

Abstract

Cytochromes P450 (CYP or P450) represent a superfamily of enzymes that interact with limited levels of NADPH‐cytochrome P450 reductase (POR) and are responsible for the oxidation of endogenous and exogenous compounds, converting them into water‐soluble products. This superfamily of heme‐containing enzymes are membrane‐bound and reside primarily in the endoplasmic reticulum (ER) of the liver, small intestine, and lung. As previously established, apart from interacting with limited levels of POR, the cytochrome P450s form homomeric and heteromeric complexes that alter the metabolic activities of the P450s involved. This study focuses on the interaction of CYP1A2 and CYP2B4. Previous work has shown that CYP1A2 is capable of forming homomeric and heteromeric complexes whereas CYP2B4 can form only heteromeric complexes. Simple binary systems containing CYP2B4 and POR exhibit typical Michaelis‐Menten saturation curves as a function of [POR], which is consistent with simple interactions between monomeric proteins. However, CYP1A2 activities for CYP1A2/POR binary systems exhibit a sigmoidal response as a function POR, which is consistent with cooperativity resulting from CYP1A2•CYP1A2 complexes. The heteromeric interaction of the two P450s causes the activity of CYP1A2 to increase and that of CYP2B4 to decrease. The objective of this study is to identify the surface regions of the proteins that interact in the CYP1A2•CYP1A2 and CYP1A2•CYP2B4 complexes. Using molecular cloning we substituted protein regions of CYP1A2 with regions of CYP2B4 and vice‐versa and expressed the chimera in the HEK‐293T cells as fusion proteins with C‐terminal protein tags that were capable of bioluminescence resonance energy transfer (BRET) when the chimeras interacted. We focused on the proximal face of the P450 as a potential region for P450•P450 complex formation. The proximal face is the region close to the heme that binds to the POR when electrons are transferred to the P450s. We have divided the proximal region into three regions designated as P1, P2, and P3. The amino acids L92‐K107 constituting P1, P132‐S156 representing P2 and T438‐K451 making up the P3 region of CYP1A2 were substituted with the respective regions of CYP2B4: T92‐S107 for P1, E132‐E163 for P2 and D438‐R448 for P3. The substitution of the P2 region of CYP1A2 with the corresponding region of CYP2B4 did not affect either CYP1A2•CYP1A2 or CYP1A2•CYP2B4 complex formation as indicated by the BRET measurements. Interestingly, the substitution of the P1 region of CYP1A2 with the corresponding region of CYP2B4 into the CYP1A2 protein disrupted the CYP1A2•CYP1A2 complex. This indicates that P1 is an important region involved in CYP1A2 homomeric complex formation. The role of P1 in the CYP1A2•CYP2B4 complex formation is currently being investigated along with the role of P3 in both the CYP1A2•CYP1A2 and the CYP1A2•CYP2B4 complexes.Support or Funding InformationSupported by NIH GM123253 & ES013648