Identification of the contact region responsible for the formation of the homomeric CYP1A2•CYP1A2 complex.
CYP1A2
NADPH-cytochrome P450 reductase
bioluminescence resonance energy transfer
cytochrome p450
protein–protein interactions
Journal
The Biochemical journal
ISSN: 1470-8728
Titre abrégé: Biochem J
Pays: England
ID NLM: 2984726R
Informations de publication
Date de publication:
11 06 2021
11 06 2021
Historique:
received:
19
04
2021
revised:
21
05
2021
accepted:
25
05
2021
pubmed:
26
5
2021
medline:
23
11
2021
entrez:
25
5
2021
Statut:
ppublish
Résumé
Previous studies showed that cytochrome P450 1A2 (CYP1A2) forms a homomeric complex that influences its metabolic characteristics. Specifically, CYP1A2 activity exhibits a sigmoidal response as a function of NADPH-cytochrome P450 reductase (POR) concentration and is consistent with an inhibitory CYP1A2•CYP1A2 complex that is disrupted by increasing [POR] (Reed et al. (2012) Biochem. J. 446, 489-497). The goal of this study was to identify the CYP1A2 contact regions involved in homomeric complex formation. Examination of X-ray structure of CYP1A2 implicated the proximal face in homomeric complex formation. Consequently, the involvement of residues L91-K106 (P1 region) located on the proximal face of CYP1A2 was investigated. This region was replaced with the homologous region of CYP2B4 (T81-S96) and the protein was expressed in HEK293T/17 cells. Complex formation and its disruption was observed using bioluminescence resonance energy transfer (BRET). The P1-CYP1A2 (CYP1A2 with the modified P1 region) exhibited a decreased BRET signal as compared with wild-type CYP1A2 (WT-CYP1A2). On further examination, P1-CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared with WT-CYP1A2, thereby demonstrating impaired binding of P1-CYP1A2 to WT-CYP1A2 protein. In contrast, the P1 substitution did not affect its ability to form a heteromeric complex with CYP2B4. P1-CYP1A2 also showed decreased activity as compared with WT-CYP1A2, which was consistent with a decrease in the ability of P1-CYP1A2 to associate with WT-POR, again implicating the P1 region in POR binding. These results indicate that the contact region responsible for the CYP1A2•CYP1A2 homomeric complex resides in the proximal region of the protein.
Identifiants
pubmed: 34032264
pii: 228735
doi: 10.1042/BCJ20210269
pmc: PMC8522333
mid: NIHMS1744732
doi:
Substances chimiques
Recombinant Fusion Proteins
0
CYP1A2 protein, human
EC 1.14.14.1
Cytochrome P-450 CYP1A2
EC 1.14.14.1
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
2163-2178Subventions
Organisme : NIEHS NIH HHS
ID : P42 ES013648
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM123253
Pays : United States
Informations de copyright
© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
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