Scientific paper
Differences in Unfolding Energetics of CcdB Toxins From V. fischeri and E. coli
Andrej Mernik,1 Uros Andjelkovic,2 Igor Drobnak1 and Jurij Lah1*
1 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva c. 5,
SI-1000 Ljubljana, Slovenia
2 Institute for Chemistry, Technology and Metallurgy, Department of Chemistry, University of Belgrade, Studentski trg 12 - 16, 11000 Belgrade, Serbia
* Corresponding author: E-mail: jurij.lah@fkkt.uni-lj.si
Received: 01-02-2012
Dedicated to Prof. Dr. Gorazd Vesnaver on the occasion of his 70h birthday
Abstract
Ccd system is a toxin-antitoxin module (operon) located on plasmids and chromosomes of bacteria. CcdBF encoded by ccd operon located on Escherichia coli plasmid F and CcdBVf encoded by ccd operon located on Vibrio fischeri chromosome are members of the CcdB family of toxins. Native CcdBs are dimers that bind to gyrase-DNA complexes and inhibit DNA transcription and replication. While thermodynamic stability and unfolding characteristics of the plasmidic CcdBF in denaturant solutions are reported in detail, the corresponding information on the chromosomal CcdBVf is rather scarce. Therefore, we studied urea-induced unfolding of CcdBVf at various temperatures and protein concentrations by circular dichroism spectroscopy. Global model analysis of spectroscopic data suggests that CcdBVf dimer unfolds to the corresponding monomeric components in a reversible two-state manner. Results reveal that at physiological temperatures CcdBVfi exhibits lower thermodynamic stability compared to CcdBF. At high urea concentrations CcdBVfi, similarly to CcdBF, retains a significant amount of secondary structure. Differences in thermodynamic parameters of Ccd-BVf and CcdBF unfolding can reasonably be explained by the differences in their structural features.
Keywords: Toxin-antitoxin module, CcdB, CcdA, protein unfolding, thermodynamics
1. Introduction
Toxin-antitoxin (TA) modules are operons located on plasmids and chromosomes of bacteria and archea. The ccd operon is a TA module encoding the toxin CcdB and the antitoxin CcdA. C-terminal domain of CcdA is intrinsically disordered and vulnerable for proteolytic attack. In the absence of ccd operon expression, CcdA is degraded by proteases faster than CcdB and is thus unable to form CcdA:CcdB complexes. This leads to activation of the toxin CcdB which binds to its cellular target, DNA gyrase, and inhibits DNA transcription and replication. When ccd operon is expressed CcdB action is inhibited by the formation of the CcdA:CcdB complexes. ccd expression is auto-regulated on the level of transcription by binding of the multimeric CcdA:CcdB complex, with CcdA/CcdB molar ratio of about 1:1, to the promoter DNA.1-3
ccd operon located on plasmid F of Escherichia coli encodes CcdBF which has been studied in detail both in terms of structure and thermodynamic stability.3-7 On the other hand, very little thermodynamic information is available on CcdBs encoded by bacterial chromosomes. An example of chromosomal CcdB is CcdBVfi from the marine bacterium Vibrio fischeri, that shows 41% sequence similarity to CcdBF. Both CcdBF and CcdBVfi form dimers in the solid state as well as in solution.5,7-9 The structures of CcdBVfi and CcdBF dimers show similarity of the secondary and tertiary structure (Figure 1). Each CcdBF and CcdBVfi monomer consist of a major N-terminal P-sheet, a few smaller P-sheets and a C-terminal a-helix. In contrast to CcdBVfi, CcdBF has a notable charge separation due to a large number of positively charged amino acids located on P-sheet and the more negatively charged helix,5,8 which may cause differences in thermodynamic stability of CcdBVfi and CcdBF.
In this work an attempt was made to characterize urea-induced unfolding of CcdBVfi by CD spectroscopy. The obtained thermodynamic parameters were compared to the known values reported for CcdBF.4,6 The observed differences in thermodynamic stability of CcdBVfi and CcdBF are discussed in terms of structural differences between the two proteins.
2. Experimental
Proteins were prepared and purified as described elsewhere.7'8 Solutions of CcdBVfi were dialyzed against TRIS buffer (0.02 M TRIS, 0.15 M NaCl and 0.001 M EDTA with pH = 7.5). Their concentrations were determined by measuring the absorbance at 280 nm using the absorption coefficients calculated by the method introduced by Gill and von Hippel.10 CcdBVfi stock solutions were mixed with concentrated urea solutions to prepare solutions with final urea concentration ranging from 0 to 8 M.
Spectropolarimetry measurements were performed using the CD spectropolarimeter AVIV 62A DS (Aviv Associates, Lakewood, NJ, USA). Experimental conditions: (1) urea induced unfolding: temperature, T = 5 °C - 45 °C, wavelength, X = 222 nm; (2) thermally induced unfolding: temperatures T = 20 °C - 85 °C, X = 225 nm with step size 1 °C; (3) CD spectra: X = 260 - 210 nm with step size 1 nm. Slit bandwidth was set to 2 nm. Temperature equilibration time was 2 minutes for thermally induced unfolding and
30 seconds for other measurements, signal averaging time was 10 seconds for thermally and urea induced unfolding and 3 seconds for other measurements. CD measurements were conducted for solutions with protein (monomer) concentration of about 3 pM (1 cm cuvette) and protein monomer concentration of about 30 pM (0.1 cm cuvette). The ellipticies, 6, measured at given wavelength corrected for the corresponding contribution of the buffer, were converted to molar ellipticies, [6], by dividing with optical path-length, l, in cm and protein concentration, c, in pM.
Protein structures needed for surface area calculations were derived from the PDB files 1X75 (CcdBF) and 3KU8 (CcdBVfi). Solvent accessible surface areas (SASA) of native protein structures were estimated using the program Nac-cess 2.1.1.11 SASA of for the unfolded proteins were calculated as the sum of accessibilities of the protein residues X located in the corresponding Ala-X-Ala tripeptides. SASA calculations were performed using the solvent probe size of 1.40 A and Z-slices of 0.05 A. Other parameters needed for SASA calculations were taken as the program default values.
3. Results and Discussion 3. 1. Thermally and Urea Induced Unfolding
of CcdB
Vfi
Thermal denaturation monitored by CD spectros-copy reveals very high thermal stability of CcdBVfi. The
Figure 1: Comparison of crystal structures of CcdBVfi and CcdBF dimers. Elements of secondary structure are colored red (a-helix) and violet (P-sheet). The structures were drawn with the program UCSF Chimera15 from PDB files: 1X75 (CcdBF) and 3KU8 (CcdBVfi). Gray marked regions that represent the difference in protein primary sequences (monomers) were obtained using the program SIM.16
Figure 2: CD spectra and melting curve of CcdBVfi. Rescan at 25 °C was performed after cooling from 85 °C and 10 min incubation at 25 °C. CD spectrum measured in 8 M urea indicates significant fraction of residual secondary structure of CcdBVf in the urea denatured state. Inset: Thermal denaturation followed at 225 nm.
CcdB in the denatured state at given T and u defined as a(T,u) = [D]/c, where c is the total molar concentration of CcdBVfi monomers. According to the suggested model (equation 1), the measured ellipticity at a given wavelength corrected for the corresponding buffer contribution normalized to c = 1 pM and l = 1 cm, [6](Tutj, can be expressed as:
[0k> = (lM^r,) + <*iT,u) [«1

(3)
where [ö]N(j,u) and [6]^^ represent the corresponding molar ellipticities of N2 and D given per mol of CcdBVfi monomer that can be estimated at any measured T as linear functions of urea concentration u (pre- and post-transitional baselines presented in Figure 3). The measured a(T,u) (Figure 4) can be expressed as:


[^LilJlnl [^Iv^r
(4)
<ïj<)
thermally induced denaturation transition is irreversible and occurs at temperatures above 70 °C (Figure 2). Dena-turation was also induced by addition of denaturant urea and monitored by CD spectroscopy at various temperatures (Figures 3 and 4). To test the reversibility of the observed urea induced denaturation transition we prepared solutions with fixed protein and different urea concentrations by dilution of (denatured) protein solutions prepared in 8 M urea. Since the extent of recovery of ellipticity is very high (Figure 3) and independent on the protein concentration between 3 and 30 pM we considered the observed urea denaturation to be a reversible process.
3. 2. Urea Induced Unfolding of CcdBVfi as Two-state Dimer-monomer Transition
Since CcdBVfi exists as a (native) dimer in both, the solid state and in the solution,8,9 we attempted to describe its urea induced unfolding as a reversible two-state process

->2D
(1)
where N2 represents the native CcdBVfi dimer, D the denatured CcdBVfi monomer and K(J>) the apparent equilibrium constant which is a function of temperature (T) and molar urea concentration (u). K(Tu) is defined as:
K(T.ai -
[Nj l-a.
-2c
(2)
{T.ii)
Figure 3: Typical urea-induced CD denaturation curve (red symbols) of CcdBVfi (molar ellipticity,	versus urea concentration) measured at c = 29.6 pM and 5 °C. Black symbols represent molar ellipticites measured for CcdBVfi solutions obtained by dilution of CcdBVf solutions prepared in 8 M urea (reversibility test). Blue and green line represent the molar ellipticities [#]N (^ and [®]d©i) corresponding to the native and denatured state, respectively. Together with the measured molar ellipticies [6](Tu) they were used in calculations of fractions of the protein in the denatured state Œ(T-u) (equation 4).
On the other hand, a(J>) can be connected to the energetics of unfolding through the two-state transition model (equation 1). According to this model the dependence of the apparent standard Gibbs free energy of unfolding (AG^)) on u can be at any T expressed as:
AG°TiU}=AG°T> -m-u
(5)
where [N2] and [D] represent equilibrium molar concentrations of N2 and D. In equation 2 a^ is the fraction of where m is an empirical parameter strongly correlated to
the changes of protein accessible surface upon unfoldingr
ver, unfolding curves measured at about ten times lower
and assumed to be temperature independent. AG°T is the CcdBVfi concentration (Figure 4-inset) show that unfolding at this concentration occurs at lower u suggesting that the transition is not monomolecular. In this light the observation that the model function (equation 7) is able to
standard Gibbs free energy of unfolding in the absence of urea (u = 0) that may be expressed in terms of the corresponding standard Gibbs free energy (AG°T)) and standard enthalpy of unfolding (AH°T)) at a reference temperature To = 25 °C and standard heat capacity of unfolding (AC°) (assumed to be temperature independent) through the Gibbs-Helmholtz relation (integrated form):
AC?°n=r.


I
T T
+ ac;:
T T 1—--In—
T r„
(ó)
It follows from equations 5 and 6 that the model (adjustable) thermodynamic parameters AG(T), AH(T), AC°P and m define AG(Zu) and also the corresponding» K^ (K(Tu) = exp(-AG(T;H)/RT)). Thus, the model function for a,Tu can be derived from equation 2 as:
K
a
I T.«)
<r.">
4c
k
(7\ »)
K
(T. il)
le
4c
(7)
its value at any T and u can be calculated for a given set of adjustable parameters and compared to a(J,u) values estimated experimentally from equation 4. The values of adjustable parameters (Table 1) were obtained by global fitting of the model function (equation 7) to the family of CD unfolding curves measured at various temperatures (Figure 4) using the non-linear Levenberg-Marquardt regression procedure. Global fitting results in a good agreement between the model and experimental data. Moreo-
Figure 4: Global model analysis of urea induced denaturation od CcdBVfi. Fraction of the protein in the denatured state a(I,u) as a function of urea concentration determined at various temperatures, T, and protein concentrations, c (inset). The points represent experimental data and the lines represent the best global fit of the model function (equation 7). The inset shows that the transition monitored at low c occurs at lower urea concentration and is well described by the corresponding model function based on the reversible two-state dimer-monomer model (equation 1).
describe the curves measured at lower CcdBVfi concentration well using the same set of adjustable parameters (Table 1), represents additional support of the proposed model of CcdBVfi dimer denaturation accompanied by dissociation of subunits (equation 1). Therefore, and due to the observed good quality of the global fit we consider the obtained thermodynamic parameters to be reliable and physically sound.
3. 3. Differences in Unfolding Energetics Between CcdBVfi and CcdBF and Their Structural Interpretation
Thermodynamic profile of CcdBVfi unfolding (Figure 4) was obtained from the best global fit values of AG(T), AH(To), ACp (Table 1). They were used to estimate AG(1T) (from equation 6), AHT from the Kirchhoff's law
ah;t)=ah°+&c*p(t-t0)
(8)
and the corresponding entropy contribution, TAS^ from the Gibbs relation
ac;n = ah't} -tas't)
(9)
Table 1: Comparison of the thermodynamic parameters of CcdBVfi and CcdBF unfolding obtained from urea-induced denaturation studies.
AG; AH
(To)
"O
0(To)
ToAS(0To) AC0 o
CcdBVfi	CcdBFa	CcdBF - CcdB,
l8 ± l	2l ± l	3 ± l
3 ± l	25 ± 4	22 ± 4
-l5 ± 2	4 ± 5	l9 ± 5
l.O ± 0.2	2.6 ± 0.3	l.6 ± 0.4
3.0 ± O.l	3 ± 3	0 ± 3
Data taken from ref. ó.
A comparison of AG°T) versus T curves (Figure 5a) for CcdBVfi and CcdBF indicates that at physiological temperatures the thermodynamic stability of CcdBF is higher compared to CcdBVfi. On the other hand, the maximum stability for CcdBF is observed at lower temperature as for CcdBVfi. The obtained AC°° of CcdBVfi unfolding was compared to the corresponding AC° estimated as a function of no. of amino acid residues for a large set of proteins.13 The comparison shows that the measured AC° represents only 42% of the value expected for the protein of the same size. This suggests that a degree of unfolding of CcdBVfi in concentrated urea solutions is significantly lower than the degree of unfolding
m
a)
b)
c)
100
Figure 5. Thermodynamic profile of CcdBVfi and CcdBF unfolding. (a) Standard Gibbs free energy, aG°r); (b) enthalpy, aH^y (c) the corresponding entropy contribution, TAS°r). All thermodynamic quantities extrapolated to urea concentration u = 0 are presented as functions of temperature. The profiles were calculated from the best-fit parameters (Table 1) by using equations 6, 8 and 9.
seen in an average protein of the large data set analyzed in ref. 13. The result is in accordance with those of the CD measurements (Figure 2) suggesting that CcdBVfi in the urea denatured state retains a significant amount of secondary
structure. Similar features of the urea unfolded state were observed also for previously studied CcdBF.6
To correlate the observed differences in thermodynamic parameters of CcdBVfi and CcdBF (Table 1, Figure 4), with differences in CcdBVfi and CcdBF structural features structural characteristics of CcdBVfi and CcdBF native (dimeric) and unfolded (monomeric) state are needed. One of those are solvent accessible surface areas (SASA). SASA of native CcdBVfi and CcdBF were calculated from crystal structures presented in Figure 1. SASA of the unfolded proteins were approximated as the sum of SASA of the protein residues X located in the corresponding Ala-X-Ala tripeptides (the sum runs over all CcdBVfi or CcdBF residues). The values are presented in Table 2.
Table 2: Solvent accessible surface areas (SASA) of CcdBVf and CcdB
CcdBVfi	CcdBF	CcdBF-
		CcdBFVfi
12.09	12.94	0.86
3.69	3.81	0.12
8.39	9.13	0.74
19.33	18.99	-0.33
6.10	5.68	-0.42
13.23	13.31	0.09
Apolar (2D) /10 A2 Apolar (N2V 103 A2
AApolgr (N2 ^ 2D) /103 a2
Anon-polar (2D) /103 A Anon-polar (N2>/ 103 A
Apolar (N2 ^ 2D) /103 A2
It can be seen that the changes of polar SASA accompanying CcdBF unfolding are about 740 A2 higher than for CcdBVfi unfolding. By contrast, the difference in changes of non-polar SASA of unfolding between CcdBF and CcdBVfi is only about 90 A2. The empirical parameterizations correlating SASA of unfolding to the corresponding thermodynamic parameters13,14 suggest that changes of polar SASA have much higher impact on AH°T) while changes of non-polar SASA have much higher impact on AC° and AS°{T). Thus, the observed AAHT > 0 and smaller magnitudes of AAC° and TAAS^) resulting in AAG°{T) > 0 can be reasonably explained by differences in CcdBF and CcdBVfi structural features.
4. Acknowledgment
We thank Prof. R. Loris for providing the purified toxin CcdBVfi used in this work. Ministry of Higher Education, Science and Technology and Agency for Research of Republic of Slovenia are acknowledged for the financial support through the Grant No. P1-0201.
5. References
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Povzetek
Genetski sistem ccd je predstavnik t.i. modulov toksin-antitoksin, ki se nahajajo na plazmidih in kromosomih različnih bakterij. CcdBF, katerega genetski zapis je vsebovan na plazmidu F Escherichie coli in CcdBVfi, katerega genetski zapis je vsebovan na kromosomu Vibrio fischeri, pripadata družini toksinov CcdB. Nativna proteina CcdBF in CcdBVf sta di-mera, ki se vežeta na kompleks giraze z DNA in tako inhibirata prepisovanje in podvojevanje DNA. Medtem ko so ter-modinamska stabilnost in značilnosti razvitja plazmidnega CcdBF v raztopinah denaturantov dobro poznane, so informacije o omenjenih značilnostih CcdBVf zelo redke. Zato smo s spektropolarimetrijo proučevali razvitje CcdBVf v raztopinah sečnine pri različnih temperaturah. Globalna modelska analiza spektroskopskih podatkov pokaže, da je denatu-racija enostopenjski prehod, pri katerem se dimer CcdBVf razvije in hkrati disociira v dva monomera. Rezultati kažejo, da ima pri fizioloških temperaturah CcdBVf nižjo termodinamsko stabilnost od CcdBF. Podobno kot CcdBF pa pri visokih koncentracijah sečnine ohrani znatno količino sekundarne strukture. Razlike v termodinamskih parametrih razvitja CcdBVfi and CcdBF je mogoče smiselno pojasniti z razlikami v njunih strukturnih značilnostih.