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 Ca2+-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.

Biosynthesis and secretion of the microbial sulfated peptide RaxX and binding to the rice XA21 immune receptor

“…We found that both RaxB and PctB contain sequences that match the conserved C and H motifs, including the conserved Cys, His, and Asp residues (Cys28/12, His101/85, and Asp117/101 of RaxB/PctB, respectively) predicted to form the active site catalytic triad (Fig. 6A) (18,22). Additionally, they contain the conserved Gln residue (Gln22/6 of RaxB/PctB, respectively) proposed to form the oxyanion hole that stabilizes a negatively charged tetrahedral intermediate generated during amide/ester hydrolysis (Fig.…”

“…Additionally, they contain the conserved Gln residue (Gln22/6 of RaxB/PctB, respectively) proposed to form the oxyanion hole that stabilizes a negatively charged tetrahedral intermediate generated during amide/ester hydrolysis (Fig. 6A) (22,23). In contrast, ABC transporters that have a degenerate C39-like domain (CLD) with no proteolytic activity, such as the E. coli hemolysin transporter HlyB, do not contain the catalytically essential Cys or conserved Gln (Fig.…”

Self‐immunity to antibacterial peptides by ABC transporters

“…The consensus sequence of cleavage of the leader peptide is L(-12)XXXE(-8)L(-7) XXXXG(-2)G(-1), where X could be any amino acids. Protein sequence analyses have shown similarity to the C39 peptidase superfamily, which has the catalytic triad Cys-His-Asp [189,190]. They are termed bifunctional ABC transporters and belong to the ABC transporter maturation secretion (AMS) superfamily, or peptidase-containing ATP-binding transporters (PCAT) [191].…”

“…At the time of this review, there were no structures of either isolated class II microcin C39 peptidase domains or full transporters. Details on the function of the C39 peptidase domain come from the functional characterisation of microcin V (MccV) from E. coli [189,190] (Table 2). MccV is a class IIa microcin, and self-immunity requires a tripartite efflux system CvaA-CvaB-TolC in E. coli [192].…”

Secretome of obligate intracellularRickettsia

“…The ABC transporters of T1SSs have recently been categorized into three groups (Kanonenberg et al ., 2013) . Group 1 transporters contain a C39 cysteine protease domain that processes NT SSs of small substrates (e.g., bacteriocins and microcins) (Havarstein et al ., 1995, Wu & Tai, 2004, Nishie et al ., 2009, Wu et al ., 2012). Group 2 transporters contain a C39-like domain, which is proteolytically inactive due to a conserved Tyr residue replacing Cys within the peptidase active site (Kotake et al ., 2008, Ishii et al ., 2010, Lecher et al ., 2012).…”