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Functional dissection of the conjugative coupling protein TrwB

de Paz, Héctor D. and Larrea, Delfina and Zunzunegui, Sandra and Dehio, Christoph and de la Cruz, Fernando and Llosa, Matxalen. (2010) Functional dissection of the conjugative coupling protein TrwB. Journal of bacteriology, 192 (11). pp. 2655-2669.

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Official URL: http://edoc.unibas.ch/dok/A5839844

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Abstract

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 TrwB Delta N70, 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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Infection Biology > Molecular Microbiology (Dehio)
UniBasel Contributors:Dehio, Christoph
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:American Society for Microbiology
ISSN:1098-5530
Note:Publication type according to Uni Basel Research Database: Journal article
Language:English
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Last Modified:21 Sep 2017 12:55
Deposited On:08 Jun 2012 06:49

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