Structural and Mechanistic Insights Derived from Saturation Mutagenesis of CcdB

Abstract

CcdB is a 101-residue homodimeric protein, which is part of the toxin-antitoxin (CcdB-CcdA) module important in F-plasmid maintenance in E. coli. We generated a site saturation mutagenesis library of CcdB. This mutant library was subjected to phenotypic screening in a strain with and without the F-plasmid and sequenced using Illumina sequencing. From a comparison of mutational tolerance at each residue in these two strains, we were able to identify the active site residues of CcdB involved in Gyrase binding, solely from mutant phenotypes. Average mutational tolerance at each individual residue further correlated well with residue depth.It is important to understand the mechanism by which binding of antitoxin to toxin in complex with its cellular results in toxin release and target rejuvenation. While an allosteric mechanism has been proposed previously, a transient ternary complex of Gyrase:CcdB:CcdA, which is an essential component for allostery, could not be experimentally detected (De Jonge et al, 2009). We identified CcdB residues important in CcdA binding from the deep sequencing data. This information was used to design SPR experiments to probe for ternary complex formation. We were able to detect a transient ternary complex between the C-terminal residues (61-72) of CcdA and the CcdB-Gyrase complex. The present study led to further insights into the binding mechanism of CcdA to CcdB and the rejuvenation mechanism whereby CcdA extracts CcdB from its complex with Gyrase.Thus saturation mutagenesis combined with deep sequencing is a powerful tool to elucidate the structural and functional determinants of a protein.