Intermolecular interactions of CYP3A enzymes and cytosolic sulfotransferases (SULT1E1, SULT2A1)
Year: 2026
Type: preprint
Abstract: ABSTRACT Cytochrome P450 enzymes of the CYP3A subfamily (CYP3A4 and CYP3A5) and cytosolic sulfotransferases (SULT1E1 and SULT2A1) play key roles in the sequential oxidative and conjugative metabolism of steroid hormones and xenobiotics. While their coordinated transcriptional regulation and co-expression patterns are well documented, direct physical interactions between these phase I and phase II enzymes have not previously been demonstrated. In this study, we combined systems biology analysis, SPR biosensing, activity assays, and AlphaFold2 modeling to investigate PPIs between CYP3A4/3A5 and SULT1E1/2A1. STRINGdb and transcriptomic data suggested potential associations, but direct evidence was lacking. SPR confirmed high-affinity binding (Kd 0.29–1.8 nM); the SULT2A1–CYP3A5 pair gave 0.29 nM, while SULT2A1–CYP3A4 gave 1.8 nM. Notably, the presence of the sulfo group donor SULT PAPS, had minimal effect on binding affinity. Functional assays demonstrated that SULTs slightly inhibited CYP3A4 activity while moderately enhancing CYP3A5 activity in the presence of SULT1E1, both with and without PAPS, whereas the addition of CYPs leads to selectively increase in SULT2A1 activity in vitro by 38% (CYP3A4) and 108% (CYP3A5), without affecting SULT1E1. AlphaFold2 models revealed stable interfaces (715–1856 Ų) with 10–16 H-bonds and salt bridges. The results of our in silico tunnel analysis suggested a plausible alignment of substrate exit channels from CYPs with entry pathways to SULTs, supporting a mechanistic basis for substrate channeling in CYP–SULT complexes. These results indicate that the enzymes may form functional multienzyme complexes, offering new insights into coordinated steroid/xenobiotic metabolism and drug clearance. Significance statement This study provides the first direct evidence of high-affinity physical interactions between CYP3A and SULT enzymes, challenging the conventional view of independent metabolic phases. Our findings reveal that these interactions can reciprocally modulate enzymatic activities and suggest a structural basis for substrate channeling, offering a paradigm-shifting perspective on coordinated steroid and drug clearance that may reshape our understanding of hormone homeostasis and pharmacokinetics.
Language: English
Subfield: Pharmacology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7222413967