Identifying the Regions Involved in the Interactions between CYP1A2 and CYP2B4

Abstract

The cytochromes P450s (P450 or CYP) that are involved in drug metabolism are membrane‐bound heme‐containing proteins that reside primarily in the endoplasmic reticulum (ER) of the liver, small intestine, and lung. P450s interact with limiting levels of cytochrome P450 reductase (CPR) to oxidize endogenous and exogenous substrates, converting them to water‐soluble products. Apart from competing for CPR, P450s also have been shown to form both homomeric and heteromeric complexes. Using Bioluminescence Resonance Energy Transfer (BRET), our lab has shown that CYP1A2 and CYP2B4 form physical complexes that, when formed, alter their metabolic characteristics. Interestingly, CYP1A2 and CYP2B4 form high affinity complexes. CYP1A2 also is capable of forming homomeric complexes; however, CYP2B4 does not appear to form such complexes. The goal of this study is to identify the regions that participate in the formation of CYP1A2•CYP1A2 and CYP1A2•CYP2B4 complexes, focusing on surface residues on both proteins. This will be accomplished by generating chimeric proteins and inserting a fragment of CYP2B4 into CYP1A2. The N terminus of the P450 sits in the ER membrane and we have divided the protein into 5 faces, namely the proximal face (region close to heme that binds to CPR), distal face (opposite to heme) and three lateral faces (DE, GE, and KL1). Using molecular cloning we have generated chimeric proteins. In these chimeric proteins, parts of the proximal, distal and lateral regions have been mutated. These chimeric proteins are predominantly CYP1A2 with fragments of CYP2B4 inserted in them and vice‐versa. These chimeras were expressed in HEK‐293T cell line followed by BRET to determine whether complexes are formed. Preliminary data shows that regions of the proximal and lateral faces that were tested did not appear to be involved in the complex formation. Other fragments in these regions remain to be examined. However, we inserted the region K59‐D75 of CYP2B4 into the corresponding region of CYP1A2 and tested for both CYP1A2•CYP1A2 and CYP1A2•CYP2B4 interactions. Surprisingly, substitution of the K59‐D75 fragment of CYP2B4 into CYP1A2 enhanced the BRET signal for formation of the CYP1A2‐CYP1A2 complex, suggesting that this lateral face participates in CYP1A2•CYP1A2 complex formation. Subsequent experiments will be performed to examine whether protein function is affected.Support or Funding InformationSupported by NIH GM123253 & ES013648This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.