title: Mutational analysis of Cvab, an ABC transporter involved in the secretion of active colicin V.
abstract: CvaB is the central membrane transporter of the colicin V secretion system that belongs to an ATP-binding cassette superfamily. Previous data showed that the N-terminal and C-terminal domains of CvaB are essential for the function of CvaB. N-terminal domain of CvaB possesses Ca(2+)-dependent cysteine proteolytic activity, and two critical residues, Cys32 and His105, have been identified. In this study, we also identify Asp121 as being the third residue of the putative catalytic triad within the active site of the enzyme. The Asp121 mutants lose both their colicin V secretion activity and N-terminal proteolytic activity. The adjacent residue Pro122 also appears to play a critical role in the colicin V secretion. However, the reversal of the two residues D121P - P122D results in loss of activity. Based on molecular modeling and protein sequence alignment, several residues adjacent to the critical residues, Cys32 and His105, were also examined and characterized. Site-directed mutagenesis of Trp101, Asp102, Val108, Leu76, Gly77, and Gln26 indicate that the neighboring residues around the catalytic triad affect colicin V secretion. Several mutated CvaB proteins with defective secretion were also tested, including Asp121 and Pro122, and were found to be structurally stable. These results indicate that the residues surrounding the identified catalytic triad are functionally involved in the secretion of biologically active colicin V.
authors.affiliation: Department of Biology, Georgia State University, Atlanta, Georgia, United States of America.
authors.forename: Kai-Hui
authors.initials: KH
authors.lastname: Wu
authors.forename: Ying-Hsin
authors.initials: YH
authors.lastname: Hsieh
authors.forename: Phang C
authors.initials: PC
authors.lastname: Tai
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003087:Colicins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D018528:ATP-Binding Cassette Transporters; D003087:Colicins; C102358:CvaB protein, E coli; D029968:Escherichia coli Proteins