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.

and therefore fundamental research on alternative antibiotic leads is necessary.
Certain bacterial toxins also target gyrase. One of them is the CcdB toxin encoded by the ccd operon from E. coli plasmid F (5,6). 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. However, the key question as to the nature of the forces that drive the binding of CcdB to GyrA and how they are connected to the structural alterations of these proteins and to the function of the ccd toxin-antitoxin module (8) remains unanswered. An attempt to answer it requires combination of the structural information with the detailed thermodynamic analysis of CcdB binding to various GyrA fragments and unfolding of proteins involved in the binding process.
Therefore, we studied binding of CcdB to the fragments GyrA59, GyrA14, and GyrA12 (Fig. 1). GyrA59 is the functionally relevant fragment because it is, in the presence of GyrB, able to relax negative supercoils of DNA (9,10). Unfortunately, the binding of CcdB to GyrA59 in solution cannot be followed directly, presumably because of the kinetically limited step of opening of tower and catalytic domains of GyrA59 ( Fig. 1), which enables CcdB to access the binding site located on the GyrA59 dimerization domain (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. Using these data, the thermodynamics of CcdB binding to GyrA59 was successfully described from the thermodynamic cycle in which the thermodynamic functions characterizing the GyrA59-CcdB denatured state were assumed to be equal to the sum of the corresponding functions characterizing the denatured states of free GyrA59 and CcdB.
By contrast, binding of CcdB to the shorter GyrA fragments GyrA12 and GyrA14 (Fig. 1) can be followed directly by isothermal titration calorimetry (ITC), 2 which appears to be the most appropriate method for investigating binding energetics (12)(13)(14)(15)(16)(17)(18). The thermodynamic parameters of binding and unfolding obtained by global analysis of CD and ITC data were dissected into enthalpic and entropic contributions that were further discussed in terms of structural features of the bound (unbound) and folded (unfolded) states of CcdB and GyrA59 and functioning of the ccd toxin-antitoxin module.

Preparation of Protein Solutions
The preparation and purification of CcdB and gyrase fragments GyrA12, GyrA14, and GyrA59 has been described elsewhere (19 -21). Their structure and the corresponding amino acid sequences are given in Fig. 1. The GyrA59-CcdB complex was formed by (urea) denaturation/renaturation and purified as described previously (11). CcdB and all gyrase fragments are dimers in solution at nondenaturing conditions. Therefore, molar concentrations of these proteins are expressed in mol dimer liter Ϫ1 . Prior to calorimetric and spectroscopic measurements, solutions of purified proteins were dialyzed extensively against phosphate buffer (0.02 M sodium phosphate, 0.15 M NaCl, 0.001 M EDTA, pH 7.5). All of the samples for the urea denaturation experiments were prepared by mixing 10 M urea and protein stock solutions to a final urea concentration between 0 and 8 M. The pH level of all solutions was checked and adjusted to 7.5 by the addition of NaOH. The concentrations of protein solutions were determined spectrophotometrically from the absorbance at 280 nm in 6 M GdmHCl at 25°C. using extinction coefficients obtained from the method introduced by Gill and von Hippel (22).

CD Spectroscopy
CD spectroscopy measurements were performed with an AVIV Model 62A DS spectropolarimeter (Aviv Associates) at different temperatures lower than the one at which the proteins start to denature irreversibly. Changes in secondary structure at increasing urea concentrations (0 -8 M) were followed by measuring the ellipticity at 225 nm in a 1-cm cuvette at a protein concentration of ϳ0.7 M (see Fig. 2). The reversibility of the urea-induced transitions was checked by diluting the protein solutions from the post-transition urea concentrations to the pretransition urea concentrations. 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%.

Isothermal Titration Calorimetry
Experiments were performed between 15 and 40°C in a VP-ITC calorimeter from Microcal Inc. (Northampton, MA). Before each experiment the protein solutions were degassed for ϳ20 min. A solution of GyrA12 or GyrA14 (c ϭ ϳ40 M) was titrated into a solution of CcdB (c ϭ ϳ2 M). The enthalpy of interaction (⌬H T ) was obtained by integration of the raw signal, corrected for the corresponding heat of dilution, and expressed per mole of added GyrA12 or GyrA14 per injection (see Fig. 4, a and b). In addition we performed a competition titration of the GyrA59-CcdB complex (c ϭ ϳ2 M) with GyrA14 (c ϭ ϳ80 M) at 25°C (see Fig. 4c).

Thermodynamic Analysis of Experimental Data
Global Model Analysis of Urea-induced Unfolding Curves Monitored by CD-Urea denaturation of GyrA59, CcdB, and the GyrA59-CcdB complex can be successfully described in terms of a two-state or three-state model. The models can be defined as follows,  ellipticity ( (T,u) ) at a given wavelength, T and u can be expressed in terms of the corresponding contributions N(T,u) , I(T,u) , and D(T,u) that characterize pure states N, I, and D as follows, ͑T,u͒ ϭ N͑T,u͒ ␣ N͑T,u͒ ϩ I͑T,u͒ ␣ I͑T,u͒ ϩ D͑T,u͒ ␣ D͑T,u͒ (Eq. 1) where ␣ N(T,u) , ␣ I(T,u) , and ␣ D(T,u) represent fractions of proteins in states N, I, and D, respectively, at given T and u. In the case of A 2 denaturation, the fractions are defined as ␣ N(T,u) ϭ where ( (T,u) Ϫ N(T,u) )/( D(T,u) Ϫ N(T,u) ) is obtained directly from the experiment, whereas the expression F I ␣ I(T,u) ϩ ␣ D(T,u) can be, as shown below, calculated from the model. For twostate denaturation of A 2 and B 2 , the first term in Equation 2 is equal to zero (F I ␣ I ϭ 0). For A 2 B 2 denaturation the quantity F I can be assumed to be temperature-independent and was set as a global model (adjustable) parameter. On the other hand, ⌬ (T,u) /⌬ D(T,u) can be connected to the thermodynamics of unfolding through the proposed models (Models 1-3) according to which the linear dependence of the standard Gibbs free energy of unfolding (⌬G i(T,u) o ) on u for the i-th transition (i ϭ A, B, I, or ID) can be at any T expressed as follows, where m i is an empirical parameter correlated strongly to the amount of protein (A 2 , B 2 , A 2 B 2 , or I) surface area-exposed to the solvent upon denaturation (23) and assumed to be temperature-independent.
It follows from Equations 3 and 4 that the model (adjustable) Consequently, they specify the populations of species in solu- and the corresponding entropy contribution, o . Global Model Analysis of ITC Binding Curves-Fitting of the model function (describing 1:1 GyrA12ϩCcdB and GyrA14ϩCcdB associations) to the sets of ITC curves measured at various temperatures (see Fig. 4, a and b) was performed as described below. The model function at a given temperature T can be defined as (25) follows, is the standard enthalpy of formation of the complex i (GyrA12-CcdB or GyrA14-CcdB), n i is the amount of the complex, and n 2 is the total amount of the added ligand. The derivative in Equation 6 can be expressed as (26,27) Fig. 3).  (Equation 6) at any T and consequently the corresponding thermodynamic profiles (see Fig. 6). The best fit values of the adjustable parameters (Table 1) were obtained using the nonlinear Levenberg-Marquardt regression procedure (24). The competition titration of the GyrA59-CcdB complex with GyrA14 (see Fig. 4c) can be interpreted in terms of a displacement reaction: GyrA59-CcdB ϩ GyrA14 7 GyrA14-CcdB ϩ GyrA59. The general form of the model function for its description is the same as for 1:1 binding (Equation 6), where i represents the formed GyrA14-CcdB complex. In contrast to 1:1 model the derivative in Equation 6 for the displacement model can be expressed as

Process (i) Parameter
is the corresponding apparent displacement constant, and r is the GyrA14/ CcdB molar ratio. The best fit values of the displacement parameters K i(T) and ⌬H i(T) o at T ϭ 25°C were obtained using the nonlinear Levenberg-Marquardt regression procedure (24).
Dissection of Thermodynamic Parameters of Protein Unfolding and Binding-Numerous recent studies of protein unfolding and protein-protein binding processes have shown that the corresponding enthalpy (⌬H i(T) o ) and heat capacity (⌬C P,i o ) changes can be parameterized in terms of changes in solventaccessible polar (⌬A P ) and nonpolar (⌬A N ) surface areas accompanying these processes (23, 28 -31). Such a parameterization is based on the estimation of the nonpolar (A N ) and polar (A P ) solvent-accessible areas of proteins in the initial (folded, unbound) and final (unfolded, bound) states. They were calculated with the program NACCESS version 2.1 using the probe size of 1.4 Å (32). A N and A P of native (folded) GyrA59 (closed conformation) and CcdB dimers were obtained from the known crystal structures (10, 33), whereas the A N and A P values of the completely unfolded GyrA59 dimer and CcdB monomers were estimated as the sum of the accessibilities of the protein residues located in the Ala-X-Ala tripeptides (28,29). A N and A P of the GyrA14-CcdB complex were obtained from the crystal structure, whereas the corresponding values for the GyrA12-CcdB and GyrA59 (open conformation): CcdB complexes were estimated from structural models (7). If not stated otherwise, the atomic coordinates of unbound proteins were extracted from the structures of corresponding complexes by deleting the coordinates of either of the two binding partners. The heat capacity (⌬C P,i o ) and enthalpy changes ( ) accompanying unfolding or binding can be expressed as the sum of nonpolar (subscript N) and polar (subscript P) contributions (28 -31).  . It can be considered as the sum of changes in configurational, translational, rotational, and side chain conformational entropy contributions that accompany unfolding or binding processes (38,40).

RESULTS
Urea Induced Unfolding Monitored by CD-Because the thermal denaturation of GyrA59, CcdB, and GyrA59-CcdB is irreversible, we attempted to investigate the thermodynamics of protein unfolding transitions via the reversible urea denaturation (Fig. 2). The normalized circular dichroism signal (Equation 2) measured as a function of urea concentration (u) at various temperatures (T) can be successfully described in terms of two-state (GyrA59 and CcdB) or three-state (GyrA59-CcdB) models (Fig. 3). To begin with, the appropriateness of the suggested models characterizing CcdB, GyrA59, and GyrA59-CcdB denaturation (Models 1-3) needs some qualitative support. In the case of CcdB, it was well established that its denaturation is accompanied by dissociation of the dimer (41,42). Because this finding was further confirmed by the observed concentration dependence of urea unfolding curves (Fig. 2c), we considered the B 2 7 2B D model (Models 1-3) as an appropriate model for description of CcdB denaturation. By contrast, the transition curves accompanying GyrA59 unfolding ( Fig. 2c; see also supplemental Fig. 1-SM) are concentration-independent, thus indicating that the transition is monomolecular (A 2 7 A 2 D ). Because the measured ellipticity of GyrA14 dimer (Fig. 1) is independent on u (Fig. 2a), we assumed that the GyrA59 transition observed at relatively low u (2-4 M) results only from opening and (partial) unfolding of the tower and/or catalytic domains and not from a complete unfolding to the random coil state. Because the structure of the dimerization domain of GyrA59 ( Fig. 1; CcdB-binding site) is obviously not altered significantly even at high u (Fig. 2a), it follows that the first GyrA59-CcdB denaturation step observed in a similar u interval (2-4 M) may be ascribed to conformational changes in GyrA59, whereas the second step observed at higher u values corresponds to the release of the CcdB dimer from the open

GyrA59-CcdB intermediate and its further dissociation into CcdB monomers.
To rigorously test the appropriateness of the proposed denaturation models, we performed their global fitting to the sets of unfolding curves measured at different temperatures ( Fig. 3; see supplemental Fig. 1-SM). We performed two sets of temperaturedependent denaturation experiments for CcdB, GyrA59, and GyrA59-CcdB. They were all reasonably well described by the proposed models. For CcdB, GyrA59, and the first step (Models 1-3, I step) of GyrA59-CcdB denaturation, the differences between the best fit parameters obtained from fitting the models to the two independent datasets were within errors estimated from the corresponding variance-covarinace matrixes,  Table 1.  meaning that they can be considered as reliable thermodynamic descriptors of these processes. By contrast, the obtained thermodynamic parameters describing the second step (Models  were calculated independently and then used in the fitting procedure as fixed values. Their independent determination was based on the assumption that the ID step in the suggested model (Models 1-3) reflects mainly the release of CcdB dimer from the dimerization domain of GyrA59 followed by its further dissociation into denatured CcdB monomers. Because CcdB-GyrA14 binding can be considered as a process opposite to the release of CcdB dimer from the dimerization domain, we obtained the corresponding thermodynamic parameters as CcdB-GyrA14 binding parameters (Table 1) with the opposite sign. Because the parameters accompanying CcdB denaturation were obtained from analysis of unfolding curves of CcdB alone (Table 1)  The average best fit thermodynamic parameters (Table 1) obtained by global model analysis of the urea unfolding data enable calculation of fractions of protein molecules that populate the predicted thermodynamic states (Models 1-3). It can be seen in Fig. 3d that the denatured states of unbound GyrA59 and CcdB are significantly populated at u Ͼ 3 and Ͼ 4 M, respectively, whereas the fractions of denatured GyrA59 and CcdB resulting from dissociation of the GyrA59-CcdB complex are significant at higher u, indicating a substantial conformational stabilization of the two binding partners upon their association.
Binding of CcdB to GyrA Fragments Monitored by ITC-The calorimetric binding isotherms accompanying association of CcdB to GyrA12 and GyrA14 exhibit the characteristics of a 1:1 association process (Fig. 4, a and b). This observation is confirmed by the global model analysis of ITC data (Equation 6) showing very good agreement of 1:1 binding model with the family of the ITC curves measured at various T (Fig. 4, a and b). To test whether protonation or deprotonation of the proteins is coupled to their binding, we performed the same ITC experiments also in Tris buffer (0.02 M Tris, 0.15 M NaCl, 0.001 M EDTA, pH 7.5, T ϭ 25°C), which has approximately 10 times higher ionization enthalpy than the phosphate buffer (43). Because the titration curves observed in Tris are the same as those observed in phosphate, we concluded that no protonation or deprotonation is involved in association of CcdB with GyrA12 and GyrA14.

Energetics of Unfolding in Correlation with Structural
Features-The thermodynamic parameters presented in Table  1 were used to estimate the corresponding thermodynamic profiles of unfolding of GyrA59, CcdB, and GyrA59-CcdB (Figs. 5 and 6). The observation that folding of all studied proteins in the standard state at physiological temperatures is an enthalpydriven process accompanied by an unfavorable entropy contribution and negative heat capacity change is a general feature of globular proteins (28,29). Thermodynamic stabilities (⌬G i(T) o ) exhibit well defined maxima between T values of ϳ20 and 25°C (Fig. 5a).
Besides the general thermodynamic description we present here the dissection of the measured thermodynamic quantities of unfolding of GyrA59, CcdB, and GyrA59-CcdB at 25°C to various contributions based on Equations 7-9 (Fig. 6). For each protein parsing of the measured enthalpy of unfolding indicates that the contributions caused by the interactions involving polar surfaces that favor protein folding overcompensates the corresponding contributions of nonpolar surfaces that on average favor protein unfolding. Furthermore, parsing of entropic contributions indicates that the contribution caused by the ; schematically presented as insets in panels a and b) from which the corresponding thermodynamic parameters were extracted (Table 1 and Fig. 6). c, competition ITC experiment: The GyrA59-CcdB complex was titrated with GyrA14 at T ϭ 25°C. The line represents the best fit of the displacement model (Equation 6) The obtained best fit thermodynamic parameters ⌬G (T) o ϭ 1 (Ϯ 1) kcal mol Ϫ1 and ⌬H (T) o ϭ 3 (Ϯ 2) kcal mol Ϫ1 corresponding to the GyrA59-CcdB ϩ GyrA14 7 GyrA14-CcdB ϩ GyrA59 process combined with those accompanying CcdB ϩ GyrA14 7 GyrA14-CcdB association (Table 1) result in values of ⌬G (T) o ϭ Ϫ13 (Ϯ1) kcal mol Ϫ1 and ⌬H (T) o ϭ Ϫ10 (Ϯ2) kcal mol Ϫ1 for CcdB ϩ GyrA59 7 CcdB:GyrA59 binding that are in accordance with the corresponding thermodynamic parameters estimated from urea denaturation experiments (Fig. 6). Because of very low heat effects accompanying the titration, we consider this experiment only as an additional support of the physical meaning of the thermodynamic binding parameters presented in Fig. 6. As shown recently (35,36), the correlation of the experimentally determined thermodynamics of protein unfolding with its structural features can be carried out using an approach in which the average number of amino acid residues participating in the unfolding process ͳNʹ is estimated from the combination of experimental thermodynamic data and parametrized Equations 7-9. The value of ͳNʹ can be obtained as ͳNʹ ϭ ͳNʹ ST ⌬A t / ⌬A ST , where ⌬A t refers to the total change in the solvent-accessible surface area calculated from Equations 7 and 8 as ⌬A N ϩ ⌬A P , ⌬A ST (ST ϭ structure) to the corresponding change in accessible area calculated for the complete unfolding of the pro-tein from its structural data, and ͳNʹ ST is the total number of residues contained in the protein. For GyrA59 unfolding the obtained ͳNʹ of ϳ50 is only ϳ5% of its ͳNʹ ST , indicating that the GyrA59 urea-denatured state is far from being completely unfolded, thus suggesting that the observed thermodynamics of unfolding probably results only from opening and partial unfolding of the tower and/or catalytic domains. This conclusion is confirmed by estimation of ͳNʹ from ⌬S i(T),other o (Equation 9) that gives a ͳNʹ value of ϳ60. Namely, in the case of monomolecular unfolding of GyrA59 ⌬S i(T),other o may be attrib-  (mol residue) Ϫ1 is the average overall residual configuration entropy change obtained from the thermodynamic data base for unfolding of monomeric proteins (28). For CcdB unfolding the above approaches give a ͳNʹ value of ϳ140 or 150, respectively, which is ϳ70% of ͳNʹ ST . This indicates that also for CcdB its urea-denatured state cannot be considered as the fully unfolded state. It should be noted that in the case of CcdB the estimate of ͳNʹ of Reasonable agreement between the ͳNʹ values determined from two different relations for both GyrA59 and CcdB suggests that the presented enthalpy and entropy contributions may have a real physical meaning. We are well aware that these contributions determined from the described combination of experimental thermodynamics and structure-based parameterization can be considered only as reasonably good approximations because they comprise errors of the empirical parameterization and those of the measured thermodynamic quantities. Nevertheless, we believe that using this approach one can explain, at least in a semi-quantitative way, the correlation between the thermodynamics of urea-induced unfolding of GyrA59 and CcdB and the structural features of their folded and unfolded states.
As shown in Fig. 6, the thermodynamic profile of the first GyrA59-CcdB unfolding step together with the dissection of its ⌬H i(T) o and T⌬S i(T) o contributions is very similar to those observed with the pure GyrA59, whereas the corresponding thermodynamic characteristics of the second step seem to be very similar to those observed with unfolding of the pure CcdB. This observation is in accordance with the proposed unfolding models. Namely, the first GyrA59-CcdB unfolding step that corresponds to conformational changes of GyrA59 is accompanied by slightly higher Driving Forces of Gyrase Recognition by CcdB-The thermodynamic parameters of binding of CcdB to the GyrA12 and GyrA14 (Table 1) obtained by model analysis of ITC data show that the formation of both complexes is an enthalpy-driven process accompanied by a small favorable entropy contribution (Fig. 6). The significantly negative heat capacities of binding suggest that the hydrophobic effect plays an important role in these binding events. The thermodynamic profiles of CcdB binding to GyrA12 and GyrA14 are nearly the same, indicating that the (connecting) helices (Fig. 1) located on GyrA14 and GyrA59, but not on GyrA12, do not play an important role in CcdB binding to gyrase. The dissection of thermodynamic parameters (Fig. 6) suggests that formation of the GyrA12-CcdB and GyrA14-CcdB complexes is driven mainly by interactions of the polar surfaces of each protein and by the reduced hydrophobic solvation of the complex state. Indeed, according to recent structural (7) and mutagenesis (5) (30) accompanied by the loss of conformational freedom of a side chain (the interacting surface consists almost entirely of the side chain atoms (7); T⌬S i(T),sc o ϷϪ5 kcal mol Ϫ1 (40)).
In contrast to the association of CcdB with GyrA12 and with GyrA14, binding of CcdB to GyrA59 is a kinetically limited process and thus cannot be studied in a real time experiment. Therefore, the thermodynamics of the GyrA59-CcdB complex formation was investigated via the urea unfolding of GyrA59-CcdB, GyrA59, and CcdB (Figs. 2 and 3). The thermodynamic parameters of the GyrA59-CcdB formation (Table 1 and Fig. 6) were obtained from the parameters describing unfolding of GyrA59-CcdB, GyrA59 and CcdB using the thermodynamic cycle in which the thermodynamic functions characterizing the GyrA59-CcdB denatured state were assumed to be equal to the sum of the corresponding functions characterizing the denatured states of free GyrA59 and CcdB (Fig. 2a). The physical meaning of the GyrA59-CcdB binding parameters is supported by the ITC experiment monitoring the displacement of CcdB from the GyrA59-CcdB complex by GyrA14 (Fig. 4c). The obtained thermodynamic parameters of displacement in combination with the known GyrA14-CcdB binding characteristics (Table 1), result in thermodynamic parameters (Fig. 4c) that are in accordance with those presented in Table 1 and Fig. 6. We believe that these results show that our approach gives reasonable estimates of GyrA59-CcdB binding parameters.
The thermodynamic binding profiles (Fig. 6) reveal that the binding of CcdB to GyrA59 (K ϳ 5⅐10 10 M Ϫ1 ) is approximately two orders of magnitude stronger and more enthalpy-driven than its association with GyrA12 or GyrA14 (7,44). This suggests that the tower and/or catalytic domains in the open conformation stabilize the GyrA59-CcdB complex through specific interactions that are predicted by our structural model of the GyrA59-CcdB complex (Fig. 1) and partially confirmed by the site-specific mutagenesis studies on CcdB (5). Similarly to the GyrA12-CcdB and GyrA14-CcdB associations, the dissection of thermodynamic parameters accompanying GyrA59-CcdB binding (Fig. 6) suggests that it is driven mainly by interactions of the polar surfaces of each protein and by the reduced hydrophobic solvation of the complex state. The dissection further suggests that the higher strength of GyrA59-CcdB interac-tions in comparison to those within GyrA14-CcdB comes mainly from additional interactions between polar surfaces of the tower and/or catalytic domains and CcdB. Moreover, the solvation entropy contribution seem to be slightly more favorable than in the case of GyrA14-CcdB. All these findings are in accordance with our structural model of GyrA59-CcdB complex formation (Fig. 1), which predicts burial of additional polar (⌬⌬A P ϭ ϳϪ450 Å 2 ) and nonpolar (⌬⌬A N ϭ ϳϪ200 Å 2 ) solvent-accessible surface areas upon binding of CcdB to the tower and catalytic domains.
Taken together, our results show that gyrase poisoning by CcdB is an enthalpy-driven process, mainly because of specific interactions of CcdB with the GyrA dimerization domain, accompanied by opening of the tower and/or catalytic domains. Furthermore, one can expect that the displacement of CcdB from the gyrase-CcdB complex is accompanied by closing of these domains (cell rejuvenation). In other words, such extensive structural rearrangements appear to be crucial driving forces for the functioning of the ccd toxin-antitoxin module.