Functional dissection of the conjugative coupling protein TrwB

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We desig...

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Authors: De Paz, Héctor D., Larrea, Delfina, Zunzunegui, Sandra, Dehio, Christoph, Cruz, Fernando de la, Llosa, Matxalen
Format: article
Publication Date:2010
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/49815
Online Access:http://hdl.handle.net/10261/49815
Access Level:Open access
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spelling Functional dissection of the conjugative coupling protein TrwBDe Paz, Héctor D.Larrea, DelfinaZunzunegui, SandraDehio, ChristophCruz, Fernando de laLlosa, MatxalenThe conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwBΔN70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.This work was supported by grants BIO2008-00133 from the Spanish Ministry of Science and Innovation and API 07/01 from the Fundación Marqués de Valdecilla to M.L. and grants LSHM-CT-2005_019023 from the European Commission, BFU2008-00995/BMC from the Spanish Ministry of Science and Innovation, and RETICS Research Network RD06/0008/1012, Instituto de Salud Carlos III, Spanish Ministry of Health, to F.C. H.P. and D.L. were the recipients of predoctoral fellowships from the University of Cantabria and JAE-predoc (CSIC), respectively.Peer reviewedAmerican Society for Microbiology201220122010info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501http://hdl.handle.net/10261/49815reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttp://dx.doi.org/10.1128/​JB.01692-09info:eu-repo/semantics/openAccessoai:digital.csic.es:10261/498152026-05-22T06:33:51Z
dc.title.none.fl_str_mv Functional dissection of the conjugative coupling protein TrwB
title Functional dissection of the conjugative coupling protein TrwB
spellingShingle Functional dissection of the conjugative coupling protein TrwB
De Paz, Héctor D.
title_short Functional dissection of the conjugative coupling protein TrwB
title_full Functional dissection of the conjugative coupling protein TrwB
title_fullStr Functional dissection of the conjugative coupling protein TrwB
title_full_unstemmed Functional dissection of the conjugative coupling protein TrwB
title_sort Functional dissection of the conjugative coupling protein TrwB
dc.creator.none.fl_str_mv De Paz, Héctor D.
Larrea, Delfina
Zunzunegui, Sandra
Dehio, Christoph
Cruz, Fernando de la
Llosa, Matxalen
author De Paz, Héctor D.
author_facet De Paz, Héctor D.
Larrea, Delfina
Zunzunegui, Sandra
Dehio, Christoph
Cruz, Fernando de la
Llosa, Matxalen
author_role author
author2 Larrea, Delfina
Zunzunegui, Sandra
Dehio, Christoph
Cruz, Fernando de la
Llosa, Matxalen
author2_role author
author
author
author
author
description The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwBΔN70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.
publishDate 2010
dc.date.none.fl_str_mv 2010
2012
2012
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/49815
url http://hdl.handle.net/10261/49815
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv http://dx.doi.org/10.1128/​JB.01692-09
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv American Society for Microbiology
publisher.none.fl_str_mv American Society for Microbiology
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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