Toxin and Antitoxin (TA) systems, abundantly found in bacteria, are involved metabolic regulation, growth control, defense against phages and postsegregational killing in plasmid maintenance. One of the TA systems discovered in bacteria is control of cell death (ccd) system. F plasmid-encoded and Vibrio fischeri chromosome-encoded ccd TA modules are the two representatives of the plasmidic and chromosomal ccd TA system. The ccd operon encodes a short-lived antitoxin CcdA and long-lived toxin CcdB. DNA Gyrase, a topoisomerase, is a cellular target of the CcdB protein. The CcdB protein binds to DNA Gyrase, causing Gyrase poisoning which subsequently activates the SOS response and triggers apoptosis. CcdBVfi shares 41% sequence identity with CcdBF. Moreover, the C-terminal α-helix of the CcdB protein is crucial for the interaction of the CcdB with Gyrase. In plasmidic CcdBF, Trp99 is essential for the interaction of CcdBF with Arg462 on GyrA14 (the region from residue 363 to 494 of the Gyrase A subunit). However, the equivalent residue in CcdBVfi is Thr103 and is not crucial for the toxicity of the CcdBVfi protein. Instead, in CcdBVfi, Asp99 (located one turn earlier in the C-terminal helix) interacts with Arg462. These residues (Trp99 and Asn95 in CcdBF or Thr103 and Asp99 in CcdBVfi) vary within the CcdB family, although they seem to be crucial within one specific protein context. This indicates significant epistasis in which the roles of Asn95/Trp99 of CcdBF and Asp99/Thr103 of CcdBVfi are influenced by the other CcdB residues that participate in the CcdB-GyrA14 interface. To elucidate the epistatic network within the GyrA14 binding site of CcdB, possible paths of mutations between CcdBF and CcdBVfi were reconstructed by site-directed mutagenesis and the resulting mutants were tested for CcdB activity in vivo. Single mutations at residue Arg91, Ile95, and Asp99 of the CcdBVfi abolish the activity of the CcdBVfi protein while the double mutation Arg91Glu/Ile95Lys (R91E/I95K) is non-deleterious, indicating reciprocal sign epistasis. An intermediate substitution Arg91Gln (R91Q), allowing the separate existence of the deleterious single mutations R91E and I95K has been found. The double mutation R91E-I95K is allowed in the context of substitution Ala96Asn (A96N) or Ala96Asp (A96D), whereas, only T103W allows the R91E-I95K to be active. Asp99Asn (D99N) becomes non-deleterious in the background of double mutation Ala96Asp/Thr103Trp (A96D/T103W). Based on the mutagenesis data, the possible divergent paths from CcdBVfi to CcdBF are also predicted.
Dissection of a protein-protein interface: the CcdB-gyrase complex
Nguyen, P. A. ((PhD) Student), Loris, R. (Promotor), De Greve, H. ((PhD) Student). 3 sep 2018
Scriptie/Masterproef: Master's Thesis