Role Of The Proximal Region Of Cyp1a2 In The Homomeric P450 Complex Formationthe

Previous studies have established the formation of the CYP1A2-CYP1A2 homomeric complex and its effect on metabolic function. Preliminary evidence has shown that CYP1A2 activity exhibits a sigmoidal response in the CYP1A2/POR binary system as a function of POR. As the CYP1A2‑CYP1A2 homomeric complex is disrupted, CYP1A2 activity is increased, which is consistent with cooperativity. The goal of this study was to identify the regions of CYP1A2 involved in the homomeric complex formation. Structural evidence suggested that the proximal face of CYP1A2 may play a role in the homomeric complex formation. Consequently, a part of the proximal face (residues L91-K106: P1 region) was examined. Residues in this region of CYP1A2 were replaced with the homologous region of CYP2B4 (T81‑S96) using molecular cloning and was expressed in HEK-293T cells. Bioluminescence resonance energy transfer (BRET) was used to measure complex formation and disruption. The chimeric CYP1A2 (with the mutated P1 region) showed a decreased BRET signal when co‑transfected with wild-type CYP1A2, thereby indicating a decrease in complex formation. This was further verified by competition studies, which showed that the CYP1A2-P1 chimera was less effective at disrupting the CYP1A2 homomeric complexes as compared to wild‑type CYP1A2. This effectively demonstrated the impaired binding of the CYP1A2-P1 chimera to wild-type CYP1A2. Lower activity was observed as well with the CYP1A2‑P1 chimera as compared to wild‑type CYP1A2. As POR associates with CYP1A2 on the proximal face, the decreased activity of the chimera indicated that the ability of the chimera to interact with POR was affected. The chimera exhibited a decreased BRET signal when co‑transfected with wild‑type CYP1A2. Competition studies demonstrated that the chimera was less effective than wild‑type CYP1A2 at disrupting the CYP1A2‑POR heteromeric complex. Therefore, the binding of the chimera to POR was impaired, which was consistent with the decreased activity of the chimera. Therefore, these results not only establish the P1 region as a contact region in the CYP1A2‑CYP1A2 homomeric complex formation but also that modification of P1 region also affects formation of the CYP1A2‑POR heteromeric complex.