English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/49815
Share/Impact:
Statistics
logo share SHARE logo core CORE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:

Title

Functional dissection of the conjugative coupling protein TrwB

AuthorsDe Paz, Héctor D. ; Larrea, Delfina ; Zunzunegui, Sandra ; Dehio, Christoph; Cruz, Fernando de la ; Llosa, Matxalen
Issue Date2-Jun-2010
PublisherAmerican Society for Microbiology
CitationJournal of Bacteriology 192(11): 2655-2669 (2010)
AbstractThe 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.
Publisher version (URL)http://dx.doi.org/10.1128/​JB.01692-09
URIhttp://hdl.handle.net/10261/49815
DOI10.1128/​JB.01692-09
ISSN1098-5530
Appears in Collections:(IBBTEC) Artículos
Files in This Item:
File Description SizeFormat 
Functional dissection of the conjugative.pdf2,32 MBAdobe PDFThumbnail
View/Open
Show full item record
Review this work
 

Related articles:


WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.