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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/62579
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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 Técnicas2025-09-29T09:33:01Zoai: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:34982025-09-29 09:33:01.985CONICET 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 |
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CONICET Digital (CONICET) |
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CONICET Digital (CONICET) |
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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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13.070432 |