Influence of Transmembrane Helix Mutations on Cytochrome P450-Membrane Interactions and Function.
Journal
Biophysical journal
ISSN: 1542-0086
Titre abrégé: Biophys J
Pays: United States
ID NLM: 0370626
Informations de publication
Date de publication:
05 02 2019
05 02 2019
Historique:
received:
02
08
2018
revised:
01
12
2018
accepted:
17
12
2018
pubmed:
20
1
2019
medline:
21
1
2020
entrez:
20
1
2019
Statut:
ppublish
Résumé
Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids, and xenobiotics. Microsomal CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal transmembrane (TM) helix that is connected to the globular catalytic domain by a flexible linker sequence. However, the structural and functional importance of the TM-helix is unclear because it has been shown that CYPs can still associate with the membrane and have enzymatic activity in reconstituted systems after truncation or modification of the N-terminal sequence. Here, we investigated the effect of mutations in the N-terminal TM-helix residues of two human steroidogenic enzymes, CYP 17A1 and CYP 19A1, that are major drug targets for cancer therapy. These mutations were originally introduced to increase the expression of the proteins in Escherichia coli. To investigate the effect of the mutations on protein-membrane interactions and function, we carried out coarse-grained and all-atom molecular dynamics simulations of the CYPs in a phospholipid bilayer. We confirmed the orientations of the globular domain in the membrane observed in the simulations by linear dichroism measurements in a Nanodisc. Whereas the behavior of CYP 19A1 was rather insensitive to truncation of the TM-helix, mutations in the TM-helix of CYP 17A1, especially W2A and E3L, led to a gradual drifting of the TM-helix out of the hydrophobic core of the membrane. This instability of the TM-helix could affect interactions with the allosteric redox partner, cytochrome b5, required for CYP 17A1's lyase activity. Furthermore, the simulations showed that the mutant TM-helix influenced the membrane interactions of the CYP 17A1 globular domain. In some simulations, the mutated TM-helix obstructed the substrate access tunnel from the membrane to the CYP active site, indicating a possible effect on enzyme function.
Identifiants
pubmed: 30658838
pii: S0006-3495(18)34528-4
doi: 10.1016/j.bpj.2018.12.014
pmc: PMC6369400
pii:
doi:
Substances chimiques
Aromatase
EC 1.14.14.1
CYP19A1 protein, human
EC 1.14.14.1
CYP17A1 protein, human
EC 1.14.14.19
Steroid 17-alpha-Hydroxylase
EC 1.14.14.19
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
419-432Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM118145
Pays : United States
Informations de copyright
Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Références
Proc Natl Acad Sci U S A. 2014 Mar 11;111(10):3865-70
pubmed: 24613931
Proc Natl Acad Sci U S A. 2002 May 14;99(10):6725-30
pubmed: 11997441
Biochemistry (Mosc). 2008 Jul;73(7):806-11
pubmed: 18707589
Biochim Biophys Acta. 2013 Feb;1828(2):851-63
pubmed: 22995244
Biophys J. 2014 May 20;106(10):2126-33
pubmed: 24853741
Biochim Biophys Acta. 2011 Jan;1814(1):223-9
pubmed: 20685623
J Chem Theory Comput. 2014 Feb 11;10(2):676-90
pubmed: 26580045
Nature. 2007 Dec 13;450(7172):1026-30
pubmed: 18075582
Nucleic Acids Res. 2012 Jan;40(Database issue):D370-6
pubmed: 21890895
FEBS Lett. 2014 Aug 25;588(17):3117-22
pubmed: 24997347
J Chem Inf Model. 2013 Aug 26;53(8):2047-56
pubmed: 23927370
Mol Cell. 2000 Jan;5(1):121-31
pubmed: 10678174
Biochemistry. 2006 Apr 11;45(14):4629-37
pubmed: 16584198
PLoS One. 2015 Nov 20;10(11):e0141252
pubmed: 26587646
Endocr Rev. 2004 Dec;25(6):947-70
pubmed: 15583024
Biochim Biophys Acta. 2015 Oct;1848(10 Pt A):2013-21
pubmed: 26025587
PLoS Comput Biol. 2011 Aug;7(8):e1002152
pubmed: 21852944
J Chem Phys. 2015 Dec 28;143(24):243139
pubmed: 26723624
Nature. 2012 Jan 22;482(7383):116-9
pubmed: 22266943
Biochim Biophys Acta. 2004 Nov 3;1666(1-2):62-87
pubmed: 15519309
J Biol Chem. 2000 Jun 23;275(25):19409-15
pubmed: 10781599
Biochim Biophys Acta. 2007 Mar;1770(3):390-401
pubmed: 16920266
Biochemistry. 2013 Aug 27;52(34):5821-9
pubmed: 23899247
J Biol Chem. 2001 Nov 30;276(48):45009-14
pubmed: 11557755
J Mol Biol. 1993 Dec 5;234(3):779-815
pubmed: 8254673
J Endocrinol. 2005 Nov;187(2):267-74
pubmed: 16293774
Chemphyschem. 2018 Oct 19;19(20):2603-2613
pubmed: 29995333
Methods Mol Biol. 2013;987:115-27
pubmed: 23475672
J Chem Theory Comput. 2017 May 9;13(5):2310-2321
pubmed: 28388089
J Comput Chem. 2005 Dec;26(16):1781-802
pubmed: 16222654
J Chem Theory Comput. 2014 Feb 11;10(2):865-879
pubmed: 24803855
J Biol Chem. 1993 Sep 5;268(25):18726-33
pubmed: 8360166
Nucleic Acids Res. 2013 Jul;41(Web Server issue):W349-57
pubmed: 23748958
J Am Chem Soc. 2013 Nov 6;135(44):16245-7
pubmed: 24160919
Steroids. 2004 Apr;69(4):235-43
pubmed: 15183689
Biochem Biophys Res Commun. 2016 Aug 19;477(2):202-8
pubmed: 27297105
Sci Rep. 2013;3:2538
pubmed: 23985776
J Chem Theory Comput. 2015 May 12;11(5):2144-55
pubmed: 26574417
Chem Rev. 2017 Mar 22;117(6):4669-4713
pubmed: 28177242
Arch Biochem Biophys. 1993 Jul;304(1):272-8
pubmed: 8323292
Nucleic Acids Res. 2014 Jul;42(Web Server issue):W337-43
pubmed: 24799431
Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2651-5
pubmed: 8464872
J Mol Recognit. 2015 Feb;28(2):59-73
pubmed: 25601796
Proteins. 2004 Jun 1;55(4):895-914
pubmed: 15146488
Acta Crystallogr D Biol Crystallogr. 2015 Dec 1;71(Pt 12):2412-21
pubmed: 26627649
Sci Rep. 2017 Jun 23;7(1):4116
pubmed: 28646173
J Chem Theory Comput. 2013 Jan 8;9(1):687-97
pubmed: 26589065
Nucleic Acids Res. 2014 Jul;42(Web Server issue):W320-4
pubmed: 24753421
Biochim Biophys Acta. 2003 May 2;1612(1):1-40
pubmed: 12729927
Biol Reprod. 2004 Jul;71(1):83-8
pubmed: 14985252
J Biol Chem. 1993 Sep 15;268(26):19681-9
pubmed: 8396144
J Am Chem Soc. 2013 Jun 12;135(23):8542-51
pubmed: 23697766