Organization of cytochrome P450 enzymes involved in sex steroid synthesis

Protein-protein interactions in lipid membranes

Slavica Praporski, Su May Ng, Ann D. Nguyen, C. Jo Corbin, Adam Mechler, Jie Zheng, Alan J Conley, Lisandra L. Martin

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/ 17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline b cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.

Original languageEnglish (US)
Pages (from-to)33224-33232
Number of pages9
JournalJournal of Biological Chemistry
Volume284
Issue number48
DOIs
StatePublished - Nov 27 2009

Fingerprint

Membrane Lipids
Cytochrome P-450 Enzyme System
Steroids
Organizations
Membranes
Quartz Crystal Microbalance Techniques
Steroid 17-alpha-Hydroxylase
Fluorescence Resonance Energy Transfer
Oxidoreductases
Proteins
Aromatase
Atomic Force Microscopy
Quartz crystal microbalances
Atomic force microscopy
NADPH-Ferrihemoprotein Reductase
Biomimetics
Lipid bilayers
Drug and Narcotic Control
Lipid Bilayers
Mixed Function Oxygenases

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Organization of cytochrome P450 enzymes involved in sex steroid synthesis : Protein-protein interactions in lipid membranes. / Praporski, Slavica; Ng, Su May; Nguyen, Ann D.; Corbin, C. Jo; Mechler, Adam; Zheng, Jie; Conley, Alan J; Martin, Lisandra L.

In: Journal of Biological Chemistry, Vol. 284, No. 48, 27.11.2009, p. 33224-33232.

Research output: Contribution to journalArticle

Praporski, Slavica ; Ng, Su May ; Nguyen, Ann D. ; Corbin, C. Jo ; Mechler, Adam ; Zheng, Jie ; Conley, Alan J ; Martin, Lisandra L. / Organization of cytochrome P450 enzymes involved in sex steroid synthesis : Protein-protein interactions in lipid membranes. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 48. pp. 33224-33232.
@article{f7fd87b81cea45adaa1f8e5d193bf072,
title = "Organization of cytochrome P450 enzymes involved in sex steroid synthesis: Protein-protein interactions in lipid membranes",
abstract = "Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/ 17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline b cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.",
author = "Slavica Praporski and Ng, {Su May} and Nguyen, {Ann D.} and Corbin, {C. Jo} and Adam Mechler and Jie Zheng and Conley, {Alan J} and Martin, {Lisandra L.}",
year = "2009",
month = "11",
day = "27",
doi = "10.1074/jbc.M109.006064",
language = "English (US)",
volume = "284",
pages = "33224--33232",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "48",

}

TY - JOUR

T1 - Organization of cytochrome P450 enzymes involved in sex steroid synthesis

T2 - Protein-protein interactions in lipid membranes

AU - Praporski, Slavica

AU - Ng, Su May

AU - Nguyen, Ann D.

AU - Corbin, C. Jo

AU - Mechler, Adam

AU - Zheng, Jie

AU - Conley, Alan J

AU - Martin, Lisandra L.

PY - 2009/11/27

Y1 - 2009/11/27

N2 - Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/ 17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline b cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.

AB - Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/ 17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline b cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.

UR - http://www.scopus.com/inward/record.url?scp=70450248395&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70450248395&partnerID=8YFLogxK

U2 - 10.1074/jbc.M109.006064

DO - 10.1074/jbc.M109.006064

M3 - Article

VL - 284

SP - 33224

EP - 33232

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 48

ER -