Driving forces of gyrase recognition by the addiction toxin CcdB.
Gyrase, an essential bacterial topoisomerase, is the target of several antibiotics (e.g. quinolones) as well as of bacterial toxin CcdB. This toxin, encoded by Escherichia coli toxin-antitoxin module ccd, poisons gyrase by causing inhibition of both transcription and replication. Because the molecular driving forces of gyrase unfolding and CcdB-gyrase binding were unknown, the nature of the CcdB-gyrase recognition remained elusive. Therefore, we performed a detailed thermodynamic analysis of CcdB binding to several fragments of gyrase A subunit (GyrA) that contain the CcdB-binding site. Binding of CcdB to the shorter fragments was studied directly by isothermal titration calorimetry. Its binding to the longer GyrA59 fragment in solution is kinetically limited and was therefore investigated via urea induced unfolding of the GyrA59-CcdB complex and unbound GyrA59 and CcdB, monitored by circular dichroism spectroscopy. Model analysis of experimental data, in combination with the relevant structural information, indicates that CcdB binding to gyrase is an enthalpic process driven mainly by specific interactions between CcdB and the highly stable dimerization domain of the GyrA. The dissection of binding energetics indicates that CcdB-gyrase recognition is accompanied by opening of the tower and catalytic domain of GyrA. Such extensive structural rearrangements appear to be crucial driving forces for the functioning of the ccd toxin-antitoxin module.
Simic
Mario
M
University of Ljubljana, Faculty of Chemistry and Chemical Technology, 1000 Ljubljana, Slovenia.
De Jonge
Natalie
N
Loris
Remy
R
Vesnaver
Gorazd
G
Lah
Jurij
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D002942:Circular Dichroism; D027081:DNA Gyrase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005144:F Factor / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics; D017186:Titrimetry; D059005:Topoisomerase II Inhibitors; D014508:Urea / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D059005:Topoisomerase II Inhibitors; D014508:Urea; D027081:DNA Gyrase