Driving Forces of Gyrase Recognition by the Addiction Toxin CcdB

Mario Simic,Natalie De Jonge,Remy Loris,Gorazd Vesnaver,Jurij Lah

doi:10.1074/jbc.m109.014035

Abstract

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.

Highlights

DNA gyrase, a type II topoisomerase, is one of the enzymes essential for the modulation of the topological state of DNA in bacteria [1, 2]
The crystal structure of CcdB bound to the dimerization domain of the gyrase A subunit (GyrA) subunit of gyrase suggests that CcdB can bind only to the open conformation of GyrA [7]
Because the thermal denaturation of GyrA59, CcdB, and their complex GyrA59-CcdB is irreversible and because it is known that efficient CcdB binding to GyrA59 at relatively low temperatures necessitates chemical denaturation and renaturation of GyrA59 [11], we attempted to investigate the CcdB binding to GyrA59 via the reversible urea denaturation of GyrA59-CcdB, GyrA59, and CcdB monitored by CD spectroscopy

Summary

Introduction

DNA gyrase, a type II topoisomerase, is one of the enzymes essential for the modulation of the topological state of DNA in bacteria [1, 2]. The crystal structure of CcdB bound to the dimerization domain of the GyrA subunit of gyrase suggests that CcdB can bind only to the open conformation of GyrA [7]. This structural information sets the basis for understanding gyrase poisoning at the molecular level. Because the thermal denaturation of GyrA59, CcdB, and their complex GyrA59-CcdB is irreversible and because it is known that efficient CcdB binding to GyrA59 at relatively low temperatures necessitates chemical denaturation and renaturation of GyrA59 [11], we attempted to investigate the CcdB binding to GyrA59 via the reversible urea denaturation of GyrA59-CcdB, GyrA59, and CcdB monitored by CD spectroscopy. By comparing the CD spectra measured for these diluted solutions with those obtained for the same solutions prepared directly from urea and buffer solutions, we estimated the extent of reversibility of the observed transitions to be higher than 80%

Methods

Results

Conclusion