The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength

Detalhes bibliográficos
Autor(a) principal: Campelo, Diana
Data de Publicação: 2017
Outros Autores: Lautier, Thomas, Urban, Philippe, Esteves, Francisco, Bozonnet, Sophie, Truan, Gilles, Kranendonk, Michel
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: https://doi.org/10.3389/fphar.2017.00755
Resumo: This work was in part funded by a joint ANR/FCT program; France: ANR-13-ISV5-0001 (DODYCOEL), and Portuguese national funds, through the Fundacao para a Ciencia e a Tecnologia (Project FCT-ANR/BEX-BCM/0002/2013).
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spelling The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strengthConformational exchangeDiflavin reductaseElectron transferMultidomain proteinsProtein dynamicsProtein-protein interactionPharmacologyPharmacology (medical)This work was in part funded by a joint ANR/FCT program; France: ANR-13-ISV5-0001 (DODYCOEL), and Portuguese national funds, through the Fundacao para a Ciencia e a Tecnologia (Project FCT-ANR/BEX-BCM/0002/2013).NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM)Centre for Toxicogenomics and Human Health (ToxOmics)RUNCampelo, DianaLautier, ThomasUrban, PhilippeEsteves, FranciscoBozonnet, SophieTruan, GillesKranendonk, Michel2017-11-09T23:03:45Z2017-10-302017-10-30T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article13application/pdfhttps://doi.org/10.3389/fphar.2017.00755eng1663-9812PURE: 3295269http://www.scopus.com/inward/record.url?scp=85032575755&partnerID=8YFLogxKhttps://doi.org/10.3389/fphar.2017.00755info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T04:13:13Zoai:run.unl.pt:10362/25195Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:28:13.880009Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
title The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
spellingShingle The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
Campelo, Diana
Conformational exchange
Diflavin reductase
Electron transfer
Multidomain proteins
Protein dynamics
Protein-protein interaction
Pharmacology
Pharmacology (medical)
title_short The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
title_full The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
title_fullStr The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
title_full_unstemmed The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
title_sort The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: The critical role of ionic strength
author Campelo, Diana
author_facet Campelo, Diana
Lautier, Thomas
Urban, Philippe
Esteves, Francisco
Bozonnet, Sophie
Truan, Gilles
Kranendonk, Michel
author_role author
author2 Lautier, Thomas
Urban, Philippe
Esteves, Francisco
Bozonnet, Sophie
Truan, Gilles
Kranendonk, Michel
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM)
Centre for Toxicogenomics and Human Health (ToxOmics)
RUN
dc.contributor.author.fl_str_mv Campelo, Diana
Lautier, Thomas
Urban, Philippe
Esteves, Francisco
Bozonnet, Sophie
Truan, Gilles
Kranendonk, Michel
dc.subject.por.fl_str_mv Conformational exchange
Diflavin reductase
Electron transfer
Multidomain proteins
Protein dynamics
Protein-protein interaction
Pharmacology
Pharmacology (medical)
topic Conformational exchange
Diflavin reductase
Electron transfer
Multidomain proteins
Protein dynamics
Protein-protein interaction
Pharmacology
Pharmacology (medical)
description This work was in part funded by a joint ANR/FCT program; France: ANR-13-ISV5-0001 (DODYCOEL), and Portuguese national funds, through the Fundacao para a Ciencia e a Tecnologia (Project FCT-ANR/BEX-BCM/0002/2013).
publishDate 2017
dc.date.none.fl_str_mv 2017-11-09T23:03:45Z
2017-10-30
2017-10-30T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://doi.org/10.3389/fphar.2017.00755
url https://doi.org/10.3389/fphar.2017.00755
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 1663-9812
PURE: 3295269
http://www.scopus.com/inward/record.url?scp=85032575755&partnerID=8YFLogxK
https://doi.org/10.3389/fphar.2017.00755
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 13
application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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