Purification and properties of TrwB, a hexameric, ATP-binding integral membrane protein essential for R388 plasmid conjugation.
TrwB is an integral membrane protein linking the relaxosome to the DNA transport apparatus in plasmid R388 conjugation. Native TrwB has been purified in monomeric and hexameric forms, in the presence of dodecylmaltoside from overexpressing bacterial cells. A truncated protein (TrwBDeltaN70) that lacked the transmembrane domain could be purified only in the monomeric form. Electron microscopy images revealed the hexameric structure and were in fact superimposable to the previously published atomic structure for TrwBDeltaN70. In addition, the electron micrographs showed an appendix, approximately 25 A wide, corresponding to the transmembrane region of TrwB. TrwB was located in the bacterial inner membrane in agreement with its proposed coupling role. Purified TrwB hexamers and monomers bound tightly the fluorescent ATP analogue TNP-ATP. A mutant in the Walker A motif, TrwB-K136T, was equally purified and found to bind TNP-ATP with a similar affinity to that of the wild type. However, the TNP-ATP affinity of TrwBDeltaN70 was significantly reduced in comparison with the TrwB hexamers. Competition experiments in which ATP was used to displace TNP-ATP gave an estimate of ATP binding by TrwB (K(d)((ATP)) = 0.48 mm for hexamers). The transmembrane domain appears to be involved in TrwB protein hexamerization and also influences its nucleotide binding properties.
Hormaeche
Itsaso
I
Unidad de Biofisica (Centro Mixto Consejo Superior de Investigaciones Cientificas-Universidad del Pais Vasco and Departamento de Bioquimica, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, Apdo. 644, 48080 Bilbao, Spain.
Alkorta
Itziar
I
Moro
Fernando
F
Valpuesta
Jose M
JM
Goni
Felix M
FM
De La Cruz
Fernando
F
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D001483:Base Sequence; D002853:Chromatography, Liquid; D003227:Conjugation, Genetic; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D008854:Microscopy, Electron; D010957:Plasmids; D011485:Protein Binding
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C403125:trwB protein, E coli; D000255:Adenosine Triphosphate