Nucleotide-dependent dimerization of the C-terminal domain of the ABC transporter CvaB in colicin V secretion.
The cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC constitute the bacteriocin colicin V secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter, and its C-terminal domain (CTD) contains typical motifs for the nucleotide-binding and Walker A and B sites and the ABC signature motif. To study the role of the CvaB CTD in the secretion of colicin V, a truncated construct of this domain was made and overexpressed. Different forms of the CvaB CTD were found during purification and identified as monomer, dimer, and oligomer forms by gel filtration and protein cross-linking. Nucleotide binding was shown to be critical for CvaB CTD dimerization. Oligomers could be converted to dimers by nucleotide triphosphate-Mg, and nucleotide release from dimers resulted in transient formation of monomers, followed by oligomerization and aggregation. Site-directed mutagenesis showed that the ABC signature motif was involved in the nucleotide-dependent dimerization. The spatial proximity of the Walker A site and the signature motif was shown by disulfide cross-linking a mixture of the A530C and L630C mutant proteins, while the A530C or L630C mutant protein did not dimerize on its own. Taken together, these results indicate that the CvaB CTD formed a nucleotide-dependent head-to-tail dimer.
Department of Biology, Georgia State University, 24 Peachtree Center Avenue, 402 Kell Hall, Atlanta, GA 30303, USA.
Xiangxue
X
Guo
Robert W
RW
Harrison
Phang C
PC
Tai
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D003087:Colicins / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D009711:Nucleotides / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D018528:ATP-Binding Cassette Transporters; D003087:Colicins; C102358:CvaB protein, E coli; D029968:Escherichia coli Proteins; D009711:Nucleotides