Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding

Autores
Blancato, Victor Sebastian; Pagliai, Fernando A.; Magni, Christian; Gonzalez, Claudio Fabricio; Lorca, Graciela L.
Año de publicación
2016
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (KD = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation.
Fil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Pagliai, Fernando A.. University of Florida; Estados Unidos
Fil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Gonzalez, Claudio Fabricio. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Lorca, Graciela L.. University of Florida; Estados Unidos
Materia
CITRATE
ENTEROCOCCUS
FADR FAMILY
FCD DOMAIN
METALLOPROTEIN
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/62579
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/62579
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand bindingBlancato, Victor SebastianPagliai, Fernando A.Magni, ChristianGonzalez, Claudio FabricioLorca, Graciela L.CITRATEENTEROCOCCUSFADR FAMILYFCD DOMAINMETALLOPROTEINhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (KD = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation.Fil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Pagliai, Fernando A.. University of Florida; Estados UnidosFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gonzalez, Claudio Fabricio. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lorca, Graciela L.. University of Florida; Estados UnidosFrontiers Research Foundation2016-02info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/62579Blancato, Victor Sebastian; Pagliai, Fernando A.; Magni, Christian; Gonzalez, Claudio Fabricio; Lorca, Graciela L.; Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding; Frontiers Research Foundation; Frontiers in Microbiology; 7; FEB; 2-2016; 1-121664-302XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.3389/fmicb.2016.00101info:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/articles/10.3389/fmicb.2016.00101/fullinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T14:20:57Zoai:ri.conicet.gov.ar:11336/62579instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-15 14:20:58.076CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
title Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
spellingShingle Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
Blancato, Victor Sebastian
CITRATE
ENTEROCOCCUS
FADR FAMILY
FCD DOMAIN
METALLOPROTEIN
title_short Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
title_full Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
title_fullStr Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
title_full_unstemmed Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
title_sort Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
dc.creator.none.fl_str_mv Blancato, Victor Sebastian
Pagliai, Fernando A.
Magni, Christian
Gonzalez, Claudio Fabricio
Lorca, Graciela L.
author Blancato, Victor Sebastian
author_facet Blancato, Victor Sebastian
Pagliai, Fernando A.
Magni, Christian
Gonzalez, Claudio Fabricio
Lorca, Graciela L.
author_role author
author2 Pagliai, Fernando A.
Magni, Christian
Gonzalez, Claudio Fabricio
Lorca, Graciela L.
author2_role author
author
author
author
dc.subject.none.fl_str_mv CITRATE
ENTEROCOCCUS
FADR FAMILY
FCD DOMAIN
METALLOPROTEIN
topic CITRATE
ENTEROCOCCUS
FADR FAMILY
FCD DOMAIN
METALLOPROTEIN
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (KD = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation.
Fil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Pagliai, Fernando A.. University of Florida; Estados Unidos
Fil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Gonzalez, Claudio Fabricio. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Lorca, Graciela L.. University of Florida; Estados Unidos
description The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (KD = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation.
publishDate 2016
dc.date.none.fl_str_mv 2016-02
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/62579
Blancato, Victor Sebastian; Pagliai, Fernando A.; Magni, Christian; Gonzalez, Claudio Fabricio; Lorca, Graciela L.; Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding; Frontiers Research Foundation; Frontiers in Microbiology; 7; FEB; 2-2016; 1-12
1664-302X
CONICET Digital
CONICET
url http://hdl.handle.net/11336/62579
identifier_str_mv Blancato, Victor Sebastian; Pagliai, Fernando A.; Magni, Christian; Gonzalez, Claudio Fabricio; Lorca, Graciela L.; Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding; Frontiers Research Foundation; Frontiers in Microbiology; 7; FEB; 2-2016; 1-12
1664-302X
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.3389/fmicb.2016.00101
info:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/articles/10.3389/fmicb.2016.00101/full
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Frontiers Research Foundation
publisher.none.fl_str_mv Frontiers Research Foundation
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.22299

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