Recognition of the Intrinsically Flexible Addiction Antidote MazE
by a Dromedary Single Domain Antibody Fragment
STRUCTURE, THERMODYNAMICS OF BINDING, STABILITY, AND INFLUENCE
ON INTERACTIONS WITH DNA*
Jurij
Lah
§¶,
Irina
Marianovsky
,
Gad
Glaser
,
Hanna
Engelberg-Kulka**,
Jörg
Kinne
,
Lode
Wyns
, and
Remy
Loris
§§
From the
Department of Ultrastructure, Vrije
Universiteit Brussel, Paardenstraat 65, B-1640 St. Genesius Rode,
Belgium, the § Faculty of Chemistry and Chemical Technology,
University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia, the
Departments of
Cellular Biochemistry and ** Molecular
Biology, Hebrew University-Hadassah Medical School, Ein Kerem,
Jerusalem, 91120 Israel, and the 
Central
Veterinary Research Laboratories, P.O. Box 591, Dubai, United Arab Emirates
Received for publication, September 25, 2002, and in revised form, January 14, 2003
 |
ABSTRACT |
The Escherichia coli mazEF operon
defines a chromosomal addiction module that programs cell death under
various stress conditions. It encodes the toxic and long-lived MazF and
the labile antidote MazE. The denaturation of MazE is a two-state
reversible dimer-monomer transition. At lower concentrations the
denatured state is significantly populated. This leads to a new aspect
of the regulation of MazE concentration, which may decide about the
life and death of the cell. Interactions of MazE with a dromedary
antibody domain, cAbMaz1 (previously used as a crystallization aid), as
well as with promoter DNA were studied using microcalorimetric and
spectroscopic techniques. Unique features of cAbMaz1 enable a specific
enthalpy-driven recognition of MazE and, thus, a significant
stabilization of its dimeric native conformation. The MazE dimer and
the MazE dimer-cAbMaz1 complex show very similar binding
characteristics with promoter DNA, i.e. three binding sites
with apparent affinities in micromolar range and highly exothermic
binding accompanied by large negative entropy contributions. A working
model for the MazE-DNA assembly is proposed on the basis of the
structural and binding data. Both binding and stability studies lead to
a picture of MazE solution structure that is significantly more
unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.
 |
INTRODUCTION |
In general, programmed cell death is required for the elimination
of the superfluous or potentially harmful cells (1, 2). In
Escherichia coli, cell death is programmed by genetic
elements called "addiction modules" (3-8). These consist of two
genes encoding for a stable toxin and its labile antidote. Toxin and antidote are co-expressed. Their expression is auto-regulated at the
level of transcription either by a non-covalent complex formed between
toxin and antidote or by the antidote alone (3-18). In the absence of
co-expression the antidote is rapidly degraded by a specific protease,
enabling the toxin to attack its target. The target of the toxin is
known only in the plasmidic ccdAB and kid-kis
systems. In ccdAB, CcdB on plasmid F attacks the A subunit of gyrase, whereas in the kid-kis system Kid on plasmid R1
targets DnaB (19-21).
Until recently, attention was paid mainly to the extrachromosomal
(plasmid) addiction modules, which are responsible for the death that
occurs upon accidental plasmid loss (3-8). However, the
E. coli chromosome also contains several operons homologous to those found in plasmid addiction systems (22-25). The first discovered regulable prokaryotic chromosomal addiction module is the
mazEF system (or chpA), which encodes
for toxic and long-lived MazF and anti-toxic and short-lived MazE (6,
9, 25). In contrast to the extra-chromosomal addiction modules that are
triggered by plasmid loss, death mediated by the chromosomal
mazEF is achieved under several stress conditions that
prevent mazEF expression. It was initially found that the
mazEF module is under the control of
3',5'-guanosinebispyrophosphate (ppGpp) (25, 26), the amino acid
starvation signal produced by RelA protein (27). Overproduction of
ppGpp leads to inhibition of the expression of mazEF and,
thereby, to cell death (25, 26). However, inhibition of
mazEF expression, and thus induction of cell death, can also
be achieved by using general inhibitors of transcription and/or
translation like antibiotics (rifampicin, chloramphenicol and
spectinomycin) (28, 29) and the toxic protein Doc (30). In each case
(ppGpp (25, 26), antibiotics (28, 29) and the Doc protein (30)), the
inhibition of gene expression leads to a lack of the labile MazE and,
thereby, allows the action of the more stable MazF to kill the cells.
To improve our understanding of addiction systems at the molecular
level, structural and also thermodynamic characterization of the
proteins involved is needed. The latter is difficult to achieve because
of the problems of expression of the toxins and the labile character of
the antidotes. Therefore, for some time only a crystal structure of the
toxin CcdB has been known (31). The thermodynamic information on
addiction proteins is also rather scarce, and often only partial
answers are available (10, 32-34).
Antibodies and their derivative fragments have long been used as tools
in a variety of applications in fundamental research, biotechnology,
diagnosis, and human therapy (35, 36). In contrast to conventional IgG
molecules, one type of the antibodies generated by camels, dromedaries,
and llamas (camelids) is formed by two heavy chains but has no light
chains (37). Particularly interesting are camelid single variable
domain antibody fragments (VHH), which contain the smallest
antigen-binding unit with a molecular size of
15 kDa. They are
characterized by high-yield production, high solubility, and high
thermodynamic stability (38, 39).
As MazE alone has a large fraction of unstructured polypeptide,
a MazE-specific VHH (cAbMaz1) fragment has been used as a crystallization aid, leading to the first crystal structure of an
addiction antidote.1 In the
current paper we focus on the thermodynamics of the MazE-cAbMaz1 and
MazE-DNA (MazE promoter) interacting systems and correlate it with the
structure. The research described here is the first example of a
characterization of MazE binding and denaturation energetics.
 |
EXPERIMENTAL PROCEDURES |
Preparation of the Proteins and MazE Promoter
DNA--
Expression and purification of MazE and the cAbMaz1 fragment
are described elsewhere.1 MazE and cAbMaz1 solutions for
spectroscopic and calorimetric measurements were prepared by extensive
dialysis against 50 mM sodium cacodylic buffer, pH 6.9, containing 150 mM NaCl and a 10-min degassing of the sample
solutions before the measurements. For DNA binding studies, the same
buffer without additional salt was used. Concentrations of a
98-residue-long, His tag-fused MazE protein and a 135-residue-long
cAbMaz1 antibody (Fig. 1) were determined spectrophotometrically by
measuring absorbance at 280 nm. The corresponding extinction
coefficients were obtained from MazE and cAbMaz1 amino acid
compositions by the method introduced by Gill and von Hippel (41)
(www.expasy.ch).
The 50-base pair, double-stranded oligonucleotide
(5'-TGCTCGTATCTACAATGTAGATTGATATATACTGTATCTACATATGATAG-3'
and
3'-ACGAGCATAGATGTTACATCTAACTATATATGACATAGATGTATACTATC-5') containing the MazE promoter sequence (underlined) was purchased from
Invitrogen. To evaluate the specificity of MazE binding to the promoter
DNA, the 107-bp control (not related to mazEF module) DNA
was used. The same DNA was used as a control (not related) DNA in the
studies of the ccdAB addiction module as well (32). Lyophilized single strands were dissolved in 50 mM sodium
cacodylate, pH 6.9, and extensively dialyzed against the same buffer.
The concentrations of single strands were determined by UV absorption spectroscopy at 260 nm by using extinction coefficients calculated on
the basis of the nearest neighbor approximation (42). The duplexes were
obtained by mixing the corresponding single strands in the 1:1 molar ratio.
Isothermal Titration Calorimetry
(ITC)2--
The heat
accompanying MazE + cAbMaz1, DNA + MazE, and DNA + MazE-cAbMaz1
associations was measured by an Omega isothermal titration calorimeter
(MicroCal, Northampton, MA). In MazE + cAbMaz1 experiments at 25, 35, 45, and 55 °C the MazE dimer solution (1.33 ml) was titrated by
cAbMaz1 solution in the same buffer using a motor-driven 250-µl
syringe. cAbMaz1 concentration was about 100 µM, whereas
the MazE dimer concentration in the titration cell was 4.8 µM. DNA + MazE and DNA + MazE-cAbMaz1 experiments were
performed at 25 °C. MazE dimer and MazE-cAbMaz1 concentrations were
around 50 µM, whereas the DNA concentration in the
titration cell was about 50 times lower. Each injection generated a
heat burst, with the area under the curve being proportional to the heat of interaction (Fig. 5a). The titration curves (Figs.
5b and 8) were constructed by subtraction of the heat
effects that accompany the ligand dilution.
Fluorescence Spectroscopy (FL)--
FL spectra were recorded
using an AMINCO-Bowman Series2 luminescence spectrometer (Spectronic
Instruments Rochester, NY) equipped with a thermally controlled cell
holder and a cuvette of 1 cm path length. MazE + cAbMaz1 titrations
were performed at 25 °C and 45 °C by incrementally injecting
4-20-µl aliquots of cAbMaz1 solution into 2 ml of 0.5-1
µM MazE dimer solution in the same buffer. After each
injection, FL emission spectrum was recorded between 290 and 480 nm
(
ext = 280 nm). In the case of DNA titration with MazE
at 25 °C the emission spectrum was recorded between 310 and 380 nm
(
ext = 300 nm). The 1.5 µM DNA oligomer
containing the promoter sequence was titrated by a 21 µM
solution of MazE dimer.
Circular Dichroism (CD) Spectropolarimetry--
CD spectra were
recorded on J-715 spectropolarimeter (JASCO, Tokyo, Japan). MazE + cAbMaz1 titrations were performed at 25 and 45 °C by incrementally
injecting 4-20 µl aliquots of cAbMaz1 solution into 2 ml of ~1
µM MazE dimer solution (1-cm cuvette) in the same buffer.
After each injection, the ellipticity,
, was monitored between 210 and 260 nm (Fig. 3). The denaturation of MazE, cAbMaz1, and their
tetrameric complex was followed by recording the far UV (205-260 nm)
CD spectra at different temperatures with the temperature step of 2 or
1 °C at the transition region (Fig. 7). The temperature of the
sample was controlled by a sensor built into the cuvette holder and
connected to a Haake N3 (Gebrueder Haake, Karlsruhe, Germany)
circulating bath which adjusted the temperature of the sample with an
accuracy of 0.1 °C. The concentrations of MazE, cAbMaz1, and their
tetrameric complex in a 0.1- or 0.2-cm cuvette were about 10 µM.
Differential Scanning Calorimetry (DSC)--
The thermally
induced transitions of MazE, cAbMaz1, and their tetrameric complex were
measured using a Nano-II DSC differential scanning calorimeter
(Calorimetry Sciences, Provo, UT). The heating rate was 1 °C/min,
and the concentration of proteins in the measuring cell (0.33 ml) was
about 10 µM. To obtain the presented thermograms (
CP versus T curves,
Fig. 6a), the heat capacity of the protein in the native
state was subtracted from the raw signal (corrected for buffer
contribution). The transition enthalpies
Hcal
were obtained by integration of
CP
versus T curves. Two consecutive temperature
scans were carried out to observe the extent of reversibility, which
was higher than 0.8 for all the proteins.
FL, CD, and Calorimetric Titrations--
Changes in the spectral
properties (Figs. 3 and 4) suggest that the cAbMaz1-MazE complex
formation is accompanied by structural alterations of MazE or/and
cAbMaz1. Moreover, binding of the first cAbMaz1 molecule to one MazE
dimer binding site influences the binding of the second one to the
other available site. Thus, the spectral changes (CD and FL) and the
heat effects seen during titration experiments may contain
contributions from direct binding and structural changes. To describe
the cAbMaz1-MazE association, a mass action model that includes the
mentioned contributions is proposed, as shown below in Reaction 1,
with K1 and K2
defined in Equations 1 and 2, respectively,
|
(Eq. 1)
|
|
(Eq. 2)
|
and where M2, A, M'2A',
and M"2A"2 represent the MazE
dimer, the cAbMaz1 monomer, and their trimeric and tetrameric complexes in the native state, respectively. Quantities in the square brackets are the equilibrium molar concentrations; K1 and
K2 are the corresponding apparent association
constants. Overall change in the thermodynamic quantity (standard Gibbs
free energy,
G°; standard enthalpy,
H°;
standard entropy,
S°) for the presented process can be
expressed as a sum of contributions of the M"2 + 2A"
association as rigid bodies in the final conformation and other
contributions which involve the conformational changes,
|
(Eq. 3)
|
|
(Eq. 4)
|
|
(Eq. 5)
|
as shown above in Equations 3, 4, and 5. It follows that
the contributions of conformational effects (other) can be
estimated as a difference between experimentally obtained
G°,
H°, and T
S° and the
corresponding
Grb°,
Hrb°, and
T
Srb°, which were estimated by
M''2A''2-structure-based calculations.
By assuming linear dependence of a measured physical property
(F) on the concentration of individual components in ideal
solution, it is possible, by subtracting the contributions of
M2 and A, to obtain the difference in physical property
(
F), which can be expressed as shown below (43) in
Equation 6,
|
(Eq. 6)
|
where fM2,
fA, and
fM'2A'i are
concentration-independent physical properties of M2, A and
M'2A'i, respectively. In each of the
techniques used in this work, the described properties have specific
physical meaning as defined in the following: (i) FL (Fig.
3b), where
F is difference fluorescence and
fi depends on the optical path length,
quantum yields, intensity of incident light, and molar extinction
coefficients of M2, A, and
M'2A'i; (ii) CD, where
F = 
= difference ellipticity and
fi is the product between difference molar
ellipticity of M'2A'i and optical path
length; (iii) ITC (Fig. 8), where
F = Q = cumulative heat effect given per mole of added
ligand at single injection and
fi is the
product between
Hi° (standard enthalpy of
M'2A'i complex formation) and the volume
of solution in the measuring cell divided by the amount of added ligand
per injection. In the ITC, the differential form of Equation 6 is
usually used where the signal is given as
H (enthalpy
change given per mole of added ligand at single injection) and
fi as
Hi° (Fig.
5b).
In the case of DNA + M2 titration, the model that assumes
binding of M2 on identical sites on DNA was used. The model
was derived from Reaction 1 and Equations 1 and 6 for i = 1.
It follows from Reaction 1 and Equation 6 that spectroscopic and
calorimetric titration curves (Figs. 3b, 5b, and
8) can be at given total concentrations of M2 and A
described only in terms of parameters
fi and
Ki. Their values were obtained by fitting the model
functions (Equation 6) to the experimental titration curves using the
non-linear procedure based on Levenberg-Marquardt minimization of the
non-weighted
2 function (44).
Temperature-dependent CD and DSC--
Our
results reveal that all monitored denaturation processes can be
adequately described as reversible two state transitions, as
presented in Reaction 2 and Equation 7,
|
(Eq. 7)
|
where Nn represents
M2 (n = 2), A (n = 1) and
M"2A"2 (n = 4) in
the native state, while D corresponds to the MazE and cAbMaz1 in the
denatured state. K is the equilibrium constant of
denaturation,
G° is the corresponding standard Gibbs
free energy change, n is the number of subunits
to which each protein dissociates upon denaturation,
is the degree
of denaturation, and [P] is the total protein concentration given per
mole of protein in its native state.
G° can also be
expressed by the integrated Gibbs-Helmholtz equation shown below in
Equation 8,
|
(Eq. 8)
|
in which
H°(T1/2) is the
standard enthalpy of denaturation at the reference temperature
T1/2 (transition temperature at
= 0.5), and
CP° is the corresponding standard heat
capacity change assumed to be independent of temperature. According to the model (Reaction 2 and Equation 7),
can be expressed as a function of ellipticity,
(measured at single wavelength), as shown
in Equation 9,
|
(Eq. 9)
|
where
N and
D are the
ellipticities of the native and denatured state, respectively, which
are assumed to be linear functions of temperature. In the case of DSC,
the measured
CP (native state is a reference
state) can be expressed as (45-47) as shown in Equation 10,
|
(Eq. 10)
|
where all quantities are defined by Reaction 2 and Equations 7
and 8. Taking into account Reaction 2 and Equations 7 and 8, the
temperature profile can be described in terms of parameters
H °(T1/2),
CP°, and T1/2. Their
values were obtained by fitting of the model function (CD = Equation 9; DSC = Equation 10) to the experimental temperature
profiles using the previously mentioned non-linear
2
regression procedure (44).
Structure-based Thermodynamic Calculations--
The non-polar
(ASAN) and polar accessible surface areas
(ASAP) of proteins were calculated with the
program NACCESS, version 2.1 (48). The ASA of native proteins was
obtained from the crystal structure of the MazE-cAbMaz1 complex (probe
size = 1.4 Å). ASA of the denatured proteins was estimated as the
sum of the accessibilities of the protein residues in an extended
Ala-X-Ala tripeptide.
CP,rb° values were calculated from changes in non-polar and polar accessible areas from the equation introduced by Murphy and Freire (49),
|
(Eq. 11)
|
which is shown above in Equation 11. For binding and
denaturation of the proteins studied here, the combination of Equation 11 and similar relations introduced by Spolar and Record (50), Myers
et al. (51), and Makhatadze and Privalov (52) results in the
same average
CP,rb° values as those
obtained by Equation 11 alone. The enthalpy change for M"2 + A" (rigid body) association was calculated as (53, 54) as shown in
Equation 12,
|
(Eq. 12)
|
and the entropy change upon rigid body association was
calculated as a sum of three contributions (53-55),
|
(Eq. 13)
|
which are shown above in Equation 13. The solvation
term,
Ssol°, was obtained as:
CP,rb°ln(T/385.15)
(55, 56). The term that reflects the change in side chain
conformational entropy,
Ssc°, was
calculated as the sum over each amino acid in the protein-protein interface (excluding alanine, proline, and disulfide-bonded cysteine), scaling its side chain conformational entropy by its change in ASA
normalized to its ASA in Ala-X-Ala tripeptide (54),
i.e.
(
ASAsc
ssc°/ASAAla-X-Ala).
ssc° was taken from Lee et al.
(57).
Smix° was calculated as a
"cratic" term (58) that reflects the mixing of solvent and
solute molecules and effectively accounts for the entropy changes due
to changes in translational/rotational degrees of freedom upon binding
(54, 55). For the M"2 + A"
M"2A" reaction using a 1 M standard
state, this equals Rln(1/55.5).
 |
RESULTS |
Structure of cAbMaz1 and Its Interaction with MazE
Initially, we raised an immune response against MazE in a
dromedary in order to identify VHH domains that could be
used as aids for the crystallization of MazE. Cloning the
VHH repertoire in a phage display vector and isolating the
MazE binders resulted in the identification of a single gene fragment
that encodes a VHH (cAbMaz1) that interacts with MazE
with high affinity.
The amino acid sequence of cAbMaz1 is shown in Fig.
1 and contains all the
VHH-specific features that distinguish VHH
domains from classic VH domains. We have recently reported
the successful crystallization of MazE in complex with cAbMaz1 and
discussed the structure with emphasis on MazE.1 Here we
analyze the complex in terms of its inter-protein contacts. All three
hypervariable regions (H1, H2, and H3) of cAbMaz1 are involved in the
interaction with MazE. However, H1 plays only a marginal role (60 Å2 buried), whereas H2 (190 Å2 buried) and H3
(270 Å2 buried) have the largest contribution. In contrast
to many other VHH domains, the interactions involve mainly
side chain rather than main chain atoms. It was suggested previously
(59) that the preferential use of main chain atoms by VHH
domains could compensate for the lack of contribution to binding from
H1 as well as the missing VL domain.

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Fig. 1.
Amino acid sequence of cabmaz1 aligned with
types of other camelid VHH domains of known
three-dimensional structure. The limits of the framework regions
were determined based on the superposition of the structures of all
VHH fragments. The C-terminal His tails are not shown.
-strands are indicated by arrows. Residues that make up
the specific signature for a VHH domain are
indicated.
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|
Like most other camelid VHH domains, the H1 conformation is
unique, resembling neither the canonical structures observed in classical VH domains nor any of the H1 conformations of
other camelid VHHs with the known three-dimensional
structure. The H2 conformation, on the other hand, is of canonical type
2A and closely resembles that of cabhcg (60) and cabamb9 (61). This
conformation is the one most commonly observed for camelid
VHH's.
The MazE dimer contains two structurally identical binding sites (Fig.
2a) for the antibody fragment.
cAbMaz1 recognizes a cluster of hydrophobic, mainly aromatic residues
that extend from the hydrophobic core of MazE (Fig. 2b). The
epitope recognized by cAbMaz1 consists of two polypeptide stretches.
Most of the interactions involve the short
-helix (18-24), of which
about 340 Å2 gets buried upon complexation. The remaining
180 Å2 of buried surface comes from residues (35-40). It
should be noted that these two stretches come from two different
monomers forming the MazE dimer. Thus, cAbMaz1 specifically recognizes
the dimer and is, as such, expected to stabilize the dimeric folded
conformation of MazE.

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Fig. 2.
Panel a, overall view of the crystal
structure of the MazE-cAbMaz1 complex. Two cAbMaz1 molecules
(green) are bound to identical sites on opposite sides of
the MazE dimer (red and blue). Panel
b, stereo view of the epitope on MazE recognized by
cAbMaz1. Residues interacting with the antibody are shown as
ball-and-stick figures. The epitope consists of two stretches (18-24
and 35-40) coming from two different MazE monomers. Panel
c, left, Trp-9 (in green) of MazE, which is
involved in creating the MazE dimer via planar stacking with itself.
The MazE dimer consists of two monomers presented in light
and dark tones, respectively. The cAbMaz1 is not shown
because it does not come close to Trp-9. Panel c,
right, Trp-102 (in green) of cAbMaz1 (light
tone) interacting with MazE dimer (dark tone).
It interacts with Pro-18, Leu-21 Ala-24, and Thr-20 of one MazE
monomer and with Leu-37 of the other.
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Thermodynamics of cAbMaz1 Binding to MazE
It can be seen from a crystal structure that MazE exists as a
dimer in a solid state. It was proven by gel filtration in 50 mM sodium cacodylic buffer (pH 6.9) that MazE exists as a
dimer also in solution and that its dimeric state does not change under the experimental conditions applied in the binding studies.
Fluorescence Spectroscopy--
Fig.
3a shows raw FL emission
spectra that correspond to the titration of the MazE dimer
(M2) by cAbMaz1 (A) at 45 °C. It can be seen that the
intensity changes more rapidly when the A/M2 molar ratio
(r) is
1, whereas at r > 1 the changes
are less pronounced. The MazE + cAbMaz1 association is
accompanied by a blue shift of FL maximum. The shift is about 8 nm in
comparison with the corresponding sum of FL spectra of "total MazE"
and "total cAbMaz1" at r = 1 (Fig 3a).
At r = 2 the blue shift is reduced to ~5 nm. The FL
spectral changes (induced intensity, blue shift) are a consequence of
different environments of Trp residues in the bound and free state of
MazE and cAbMaz1. According to the crystal structure, the ordered part
of MazE contains a single tryptophane (Trp-9) that does not interact
with the antibody but is involved in creating the MazE dimer via planar
stacking with itself (Fig. 2c). By titration of MazE to the
buffer solution, we observed linear dependence of FL intensity on MazE
concentration with no spectral shift. This is an additional proof that
the oligomeric state of MazE stays the same in the concentration range
used in FL measurements. cAbMaz1 contains three tryptophanes of which
one, Trp-102, is important for binding MazE. In the complex, it
interacts hydrophobically with Pro-18, Leu-21, Ala-24, and Thr-20 of
one MazE monomer and with Leu-37 of the other (Fig 2c). Therefore, the
blue shift and the intensity changes can be interpreted as arising from
the shielding of Trp-102 from its contact with water. Because of the
fact that FL spectral properties accompanying binding are different at
r < 1 and r > 1 and that both cAbMaz1
binding sites on MazE are structurally identical, the differences are
most probably caused by structural changes in MazE and/or cAbMaz1.

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Fig. 3.
FL spectra accompanying MazE (M2) + cAbMaz1 (A) titration at 45 °C at A/M2 molar ratio
r varying between 0 and 3.5. Dotted
lines represent the spectra up to r = 1 and
full lines at r > 1. The bold
line represents a sum of the spectra of "total"
M2 and "total" A at r = 1 (panel
a). The corresponding titration curves at 350 nm measured at
25 °C ( ) and 45 °C ( ) are presented in panel b.
Lines are graphs of the best fitted model function (Equation 6). Speciation diagrams at 25 °C (dotted line) and
45 °C (full line) calculated from the best fitted values
of apparent binding constants (Reaction 1 and Equations 1 and 2) are
presented in panel c. is the fraction of each MazE
species.
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By subtraction of the contributions of MazE dimer (M2) and
cAbMaz1 (A) at each titration point, the difference spectra and the
corresponding titration curve at 350 nm (Fig. 3b) were
constructed. Observations of spectra and titration curves suggest that
A binds to M2 in two distinctive binding modes. This
hypothesis was tested and proved by fitting of the model function
(Equation 6) to the experimental titration curve. It can be seen in
Fig. 3b that model function with K1
and K2 as adjustable parameters correlates well with the experimental curves measured at 25 and 45 °C. The obtained K1 values are about one order of magnitude
higher than the values of K2 (Table
I), which strongly suggests that the
binding of two A molecules to M2 is
anti-cooperative. This allosteric effect can be seen more clearly in
speciation diagram (Fig. 3c) calculated from the fitted
K1 and K2 values at 25 and 45 °C. Up to r
0.5 lowering of free
M2 concentration is a consequence of M'2A'
formation, whereas at r > 0.5 the
M"2A"2 complex starts to form and
finally becomes the predominating species in solution at
r > 1.5 (Fig. 3c). In our FL titration
experiments the initial concentrations of M2 were in the
0.5-1 µM range, which enabled us to obtain reliable K1 and K2 in cases when
their values were lower than
108
M
1. A significant dependence of the model
function (Equation 6) on K1
(K2) is lost at K1
(K2) >108
M
1 and, therefore, the value of
K1 determined at 25 °C (Table I) should be
considered only as a best lower estimate of the apparent binding
constant.
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Table I
Comparison of the binding parameters K1 and K2
Apparent equilibrium constants K1 and
K2 accompanying binding of the first and the second
cAbMaz1 molecule to MazE dimer obtained by model analysis of
calorimetric (ITC), fluorimetric (FL), and spectropolarimetric (CD)
titration curves.
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CD Spectropolarimetry--
The far UV CD spectra that accompany
the titration of M2 by A at 25 °C are presented in Fig.
4. The intensity of the CD signal around
220 nm decreases with r up to r
1 and
increases at r > 1. This fully supports the
conclusions made in the case of FL spectroscopy that binding of A to
M2 occurs in two different binding modes. The changes in
the far UV CD spectra induced by binding correspond to the changes in
the secondary structure of M2 and/or A upon formation of
M'2A' and
M"2A"2. Theoretically, the
changes of each type of the secondary structure upon binding can be
estimated from the far UV CD spectra. Fig. 4 shows that CD spectrum of
A has an "unusual" shape, which has often been observed in the
antibody family but never explained in terms of secondary structure
(38). Proteins with such unusual CD spectra are not involved in the databases of proteins (with known conformations and CD spectra) used
for estimation of the secondary structure. Therefore, a
comparison of the measured CD spectra with those calculated from the
data base would be meaningless. Raw CD spectra (Fig. 4) were analyzed in the same way as the corresponding FL spectra. From the shapes of the
corresponding titration curves we were able to detect two different
binding modes of A both at 25 and 45 °C. Because of high affinities
of A, the starting concentration of M2 was chosen as low as
possible to get reliable K1 and
K2 values from the model analysis (Equation 6).
The changes in CD signal upon titration were still well measurable at
an M2 concentration of ~1 µM (Fig. 4).
However, the quality of the titration curves obtained by subtraction was too low for trustworthy model analysis. Therefore, we were only
able to estimate K1 and
K2 from CD titration at 45 °C (Table I).

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Fig. 4.
CD spectra accompanying MazE (M2) + cAbMaz1 (A) titration at 25 °C at A/M2 molar ratio
r varying between 0 and 3. Dotted
lines represent the spectra up to r = 1 and
solid lines at r > 1. The
bold line is the spectra of free cAbMaz1 (A) in
its 1 µM solution.
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Isothermal Titration Calorimetry--
ITC experiments presented in
Fig. 5 were performed at 25, 35, 45, and
55 °C and analyzed in terms of the same model as the corresponding
FL and CD titrations (Reaction 1 and Equations 1, 2, and 6). As an
advantage over spectroscopic methods, the model function for
description of the ITC signal besides K1 and
K2 (Table I) also contains two other
thermodynamic parameters,
H1° and
H2° (apparent standard enthalpies of
binding). This enabled us to describe the binding of the first and the
second A molecule to M2 with a thermodynamic profile (Fig.
5c) that includes apparent standard Gibbs free energies of
binding
G1° and
G2° (Equation 8) and the corresponding
apparent standard entropies of binding
S1°
and
S2° calculated from Gibbs equation
(
Gi° =
Hi°
T
Si°; i = 1, 2). From the temperature
dependence of
H1° and
H2° (Fig. 5c) the standard heat
capacity changes
CP,1° and
CP,2° were calculated as the slopes of the
linear regression
Hi° versus T lines (
CP,1° = (
Hi°/
T)P;
i = 1, 2). Because of the very high affinity binding of the first
A molecule to M2 at 25, 35, and 45 °C and the applied
concentration conditions, almost all added A is bound at
r < 1 and, therefore, the evaluation of
K1 by a straightforward fitting procedure was
not possible (see also FL spectroscopy). At all applied temperatures
the fitting was successful in obtaining all other model parameters,
i.e. K2,
H1°, and
H2°.
At 55 °C the value of K1 (Table I) was low
enough to be accurately determined by the model analysis. The obtained
K1 and
H1° at
55 °C in combination with
CP,1° enabled
us to calculate K1 values at 25, 35, and
45 °C from Equation 8. Table I shows that K1
values are higher than K2. They are in good
agreement with the values determined by the fitting of FL and CD
titration curves with the same model. This supports the conclusion made in the case of FL and CD that the binding of A to M2 is
anti-cooperative. Furthermore, the anti-cooperative binding effect is
lowered when the temperature rises (the difference between
K1 and K2 is reduced; Table I). By extrapolation of K1 and
K2 via Equation 8 it can be shown that this
allosteric effect is lost above 80 °C. The binding of both A
molecules is highly exothermic with
H1°
being 1-2 kcal/mol lower than
H2°. The
H1° and
H2°
values become more exothermic at higher temperatures, resulting in
negative
CP,1° and
CP,2° values of
0.25 ± 0.04 and
0.18 ± 0.01 kcal/mol, respectively.

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Fig. 5.
Typical calorimetric MazE (M2) + cAbMaz1 (A) titration (panel a) and the corresponding
binding isotherm at 55 °C ( ) (panel b),
where the solid line represents the best fitted
model function (Equation 6). H values are expressed in
kcal/mol of added A. The corresponding thermodynamic profile of
M2 + A association is presented in panel c,
where the symbols ( G1°), ( H1°), and (T S1°) correspond to the binding
of the first A molecule, and ( G2°), ( H2°) and (T S2°) correspond to the binding
of the second one. In the case of Hi°
solid lines are linear regression lines from
which CP,i° values were calculated, whereas
in the cases of Gi° and
T Si° the lines serve just as a
guide to the eye. The thermodynamic parameters of M2 + A
association as rigid-bodies ( Grb°,
Hrb°, and
T Srb°) obtained from the
structure-based calculations (Equations 11-13) are presented as
dotted lines.
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Stability of MazE, cAbMaz1, and Their Complexes
The thermodynamic stability of MazE, cAbMaz1, and their tetrameric
complex was studied by DSC and temperature-dependent CD spectropolarimetry. Usually it is described in terms of the standard Gibbs free energy change,
G°, which corresponds to the
reversible denaturation of a given protein.
G° as a
function of temperature, T, was calculated from Equation 8.
Differential Scanning Calorimetry (DSC)--
Thermograms for
M2, A, and M"2A"2 (Fig.
6a) were described in terms of
the equilibrium two-state model (Reaction 2 and Equation 7). For
M2 and A, the model function (Equation 10) correlates well with the experimental data. Moreover, model-independent transition enthalpy (
Hcal) values for M2 and
A show good agreement with
H°(T1/2),
indicating that the two-state approximation is applicable for
description of their denaturation processes (Table
II). Therefore, the parameters
T1/2,
H °(T1/2),
and
CP° (Table II) derived from model
analysis of DSC data were used in interpretation of the thermodynamic
stability of M2 and A (Fig. 6b). In the case of M"2A"2,
H°(T1/2) is somewhat lower than
Hcal, indicating that the model that takes
into account only native M"2A"2 and
denatured M and A is too simple for description of the DSC thermogram.
Namely, because of high affinities of A to M2, at applied
concentrations and lower T
M"2A"2 is practically the only
protein species in solution. However, from extrapolation of binding
constants by Equation 8, it follows that at T, where the
denaturation occurs (Fig. 6a), the presence of
M'2A' as well as free M2 and A may not be
neglected any more. Because the thermogram is the sum of the
contributions of all the species in solution and only the denaturation
of M"2A"2 involves the interaction
enthalpy of two A molecules, the
H°(T1/2) of
M"2A"2 is higher than predicted by
the described analysis (Equation 7). As the denaturation transitions of
all species occur in the same T interval, the deconvolution of the thermogram based on multiple contributions is not possible. However, because the enthalpy of A binding to M2 was
determined by ITC, we were able to estimate that the integrated
Hcal is <5% (experimental error) different
from the denaturation enthalpy of
M"2A"2. Apparently, the multiple
species contributions affect the shape of the thermogram
(
H°(T1/2)) more than the
corresponding integrated area (
Hcal).
Therefore,
Hcal instead of
H°(T1/2) was used in the calculation
of the thermodynamic stability of M"2A"2 (Fig. 6b).

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Fig. 6.
Panel a, DSC thermograms of MazE
(M2), cAbMaz1 (A) and their tetrameric complex
(M"2A"2). CP is expressed in
kcal/Kmol of monomeric unit. Full lines represent graphs of the best
fitted model function (Equation 10). Panel b,
comparison of the thermodynamic stability of M2, A and
M"2A"2. The standard Gibbs free energy change
upon denaturation versus temperature diagrams is determined
on the basis of parameters obtained by the analysis of the DSC
transition curves (Equations 7, 8 and 10). The dotted
line is a sum of stability curves, M2 + 2A.
Panel c, the transition temperature,
T1/2, as a function of decadic logarithm of protein
concentration, log[P], calculated for reversible two-state
denaturation of M2 and
M"2A"2 (Equation 7).
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Table II
Comparison of thermodynamic parameters of denaturation
Thermodynamic parameters of MazE (M2), cAbMaz1 (A), and their
tetrameric complex (M"2A"2) denaturation obtained by
model analysis of DSC thermograms and spectropolarimetric (CD)
temperature profiles.a
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Temperature-dependent CD Spectropolarimetry--
The
denaturation of M2, A, and
M"2A"2 (Fig.
7a) was monitored by measuring
far UV CD spectra at different temperatures. At the end of the
M"2A"2 transition, the corresponding CD
spectrum (Fig. 7a) is equal to the sum of individual CD
spectra of denatured M and A, suggesting that two M and two A domains
dissociate from M"2A"2 upon
melting. The denaturation temperature profiles (Fig. 7b)
constructed from the corresponding melting curves at a single wavelength were described in terms of an appropriate two state model
(Equations 7-9) to obtain
H°(T1/2)
and T1/2 values. They are very close to those
derived from DSC (Table II). Despite the fact that the fitted model
functions (Equation 9) display good agreement with experimental melting
curves (Fig. 7b) we were able to estimate
CP° only for denaturation of M2
(
CP° = (1.9 ± 0.5) kcal/mol K). It
can be shown by simulation of the model melting curves that their
shapes do not depend significantly on parameter
CP° in the case of sharp transitions (high
H°(T1/2)), so
CP° cannot be obtained by the model
analysis of A and M"2A"2 melting curves.

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Fig. 7.
Panel a, far UV CD spectra of tetrameric
MazE-cAbMaz1 complex (M A ) measured
from 40 to 95 °C. The symbols represent the sum of the CD
spectra of denatured MazE and cAbMaz1 (2 M+ 2A). Inset, the
corresponding melting curve constructed at single wavelength (332 nm).
Panel b, degree of denaturation obtained from CD melting
curves as a function of T for M2 ( ), A ( ),
and M"2A"2 ( ). Full lines are graphs of the
best fitted model function (Equation 9).
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Binding of MazE to Its Promoter DNA and the Influence of
cAbMaz1
To characterize the binding of M2 to its promoter DNA
at 25 °C, FL spectroscopic titrations and ITC were employed. In
addition, a significant difference between the far UV CD spectra of
free M2 and M2 bound to DNA in the 230-220 nm
wavelength range was observed. However, due to high absorption of DNA,
the spectra below 220 nm were not good enough even for qualitative
analysis. The M2 promoter consists two alternating
palindrome sequences (Fig. 9a), which were previously found
to be responsible for binding of a MazE-MazF complex as well as MazE
(M2) alone (9).
Fluorescence Spectroscopy--
M2 binding to promoter
DNA was characterized by a significant quenching of Trp fluorescence
accompanied by a small blue shift of FL maximum (2 nm at
M2/DNA ratio of 3). The emission FL spectra were analyzed
in the same way as in M2 + A titrations (Fig. 3). The
resulting titration curve is presented in Fig.
8. The model function based on
M2 dimer binding to the independent sites on DNA (Equation 6) gave the best agreement for the value of apparent binding constant,
K = (2.8 ± 0.8)·106
M
1, and the number of binding sites on DNA,
n = 3.1 ± 0.1. We have tried to employ more
complicated models (including different types of binding sites and
cooperativity) for description of FL titration curves. However, the
reciprocal correlation between adjustable parameters was too high,
meaning that the data could be equally well fitted by different sets of
model parameters. Because the physical meaning of parameters obtained
by these models is completely lost, we stick to a model of independent
identical sites. The titration of the "control" (not
mazEF related) DNA by M2 resulted in the induced
FL intensities that are negligible in comparison to those resulted from
the binding of M2 to the promoter DNA (Fig. 8).

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Fig. 8.
FL ( ) and calorimetric ( ) titration
curves accompanying DNA + MazE (M2) association at 25 °C
in comparison with corresponding control (unrelated) DNA + M2 FL ( ) and ITC ( ) curves. ITC curve for DNA + MazE-cAbMaz1 (M"2A"2) binding at the same
temperature is presented by black squares ( ).
Dotted lines are graphs of the best fitted model
function (Equation 6). The difference FL at 360 nm
( F360) as a function of M2/DNA molar ratio,
r, was obtained from the corresponding emission spectra.
Q is a cumulative heat effect at each titration point
expressed in kcal/mol of added M2
(M"2A"2) per injection.
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Isothermal Titration Calorimetry--
Calorimetric titration
curves describing the binding of M2 and the complex
M"2A"2 to DNA are presented in Fig.
8. For the same reason as FL data, ITC measurements were analyzed only
in terms of M2 (M"2A"2)
binding to the independent sites on DNA (Equation 6). The model
analysis describes M2 binding as highly exothermic with the
enthalpy of binding
H° =
71 ± 4 kcal/mol,
apparent binding constant K = (2.5 ± 0.6)·106 M
1, and the number of
binding sites for an oligomer, n = 3.0 ± 0.1. Binding of the M"2A"2 complex (Fig.
8) is characterized by
H° =
80 ± 9 kcal/mol,
K = (1.9 ± 0.6)·106
M
1 and n = 3.2 ± 0.3, values which are very similar to those obtained in the case of free
M2. The heat effects accompanying the M2 + "control DNA" titration are negligible in comparison to the
effects that correspond to the M2 + promoter DNA titration
(Fig. 8).
 |
DISCUSSION |
MazE-cAbMaz1 Interactions--
The observed changes in FL and CD
spectra upon MazE + cAbMaz1 titrations strongly suggest that their
association is accompanied also by conformational effects. Thus, the
thermodynamic parameters obtained from model analysis of FL, CD, and
ITC signals contain contributions from both direct binding (rigid body
association) and conformational changes of MazE and/or cAbMaz1. In this
study an attempt was made to separate the two contributions. It was shown that the structure-based thermodynamic calculations give reliable
estimates of thermodynamic parameters of binding in the systems where
the conformational effects are negligible (54, 62). Therefore, they
were used to describe the "rigid body" association of cAbMaz1
with MazE. In the calculations, the conformational states of cAbMaz1
and MazE were defined by the crystal structure of their complex.
Because both binding sites on MazE dimer are structurally identical,
the calculated parameters are the same for binding of the first and the
second cAbMaz1 molecule. Fig. 5c shows that the MazE dimer + cAbMaz1 rigid body association is an entropy-driven process
characterized by a small negative
Hrb° and
a negative
CP,rb°. Such energetics may be
considered as hydrophobic in nature. However, interpretation of rigid
body association solely in terms of hydrophobic effect may be
misleading, because
Hrb°,
Srb°, and
CP,rb° are produced from large compensating effects (Equations 11-13). In MazE, the hydrophobic core is small but
extends to the surface on two sides of the dimer (Fig. 2b). It is exactly this hydrophobic patch that is recognized by two cAbMaz1
molecules. In addition, we have also found several hydrogen-bonding contacts between MazE and cAbMaz1. The small exothermic
Hrb° (Equation 12) value shows that its
part that is associated with the hydrogen-bonding polar groups is only
slightly dominant over the unfavorable (endothermic) non-polar
contribution. The largest contribution that makes
T
Srb° (Equation 13) the major driving force
in stabilizing the rigid body complex comes from hydrophobic effect
(
Ssol°). On the other hand, the
"freezing" of side chains (
Ssc°) and
the lowering of translational/rotational degrees of freedom
(
Smix°) upon binding cause the entropic
loss that reduces the
Srb° to nearly half
of the
Ssol° value. In some studies, much
higher values for the loss of translational/rotational entropy were
taken into account (63, 50); however, it has been suggested that they
are overestimates (54, 64, 65). It was shown that when
Ssol° and
Ssc°
are taken into account the cratic correction (
Smix°) accounts well for the loss of
translational/rotational entropy upon rigid body association (54,
55).
By contrast, the overall thermodynamic profiles obtained from model
analysis of FL, CD, and ITC data (Fig. 5c) indicate that the
binding of both cAbMaz1 molecules to MazE dimer is an enthalpy-driven process accompanied by unfavorable entropic contributions. Fig. 5 shows
that the overall
Gi° is in the same range
with
Grb°. However,
Hi° is much more exothermic than
Hrb°, indicating that additional
energetically favorable contacts (due to structural alternations) are
formed upon cAbMaz1 binding to MazE. The favorable enthalpy difference
is compensated by a large conformational entropy loss
(T
Si° < T
Srb°). Thermodynamic studies of
biomolecular associations and protein unfolding have demonstrated that
CP,i° may be considered as the most
reliable distinctive feature of site-specific binding (49-52, 66-68).
For cAbMaz1 binding to MazE
CP,1° +
CP,2°, the value of
0.43 kcal/mol K
obtained by ITC is significantly lower than the corresponding
2
CP,rb° value of
0.28 kcal/mol K, which is in accordance with the removal of large amounts of nonpolar surface
from water and with the structural alternations MazE or/and cAbMaz1.
The observed binding anti-cooperativity, due to the structural alterations of the involved proteins, is also visible from the thermodynamic parameters. ITC results show that
H2° >
H1°
(more energetically favorable contacts or/and less unfavorable contacts are formed upon binding of the first cAbMaz1 molecule) and
CP,2° >
CP,1°
(larger part of a non-polar surface is buried or/and more polar surface
is exposed upon binding of the first cAbMaz1 molecule).
Thermodynamic Stability of MazE, cAbMaz1, and Their
Complexes--
The stability curves (
G°
versus T) of MazE, cAbMaz1, and their tetrameric
complex are presented in Fig. 6b. The comparison of the
stability curve of MazE to that of another addiction antidote, CcdA
(32), shows that both proteins have very similar thermodynamic stability. Because MazE and the toxin MazF are co-expressed, and their
expression is auto-regulated on the level of transcription (6,
9), the total concentration of MazE in the cell will never be
higher than a certain level set by equilibrium constants of MazE-MazF
promoter and MazE promoter complex formation and the concentration of
promoter DNA. Consequently, the concentration of free MazE (not bound
in the MazE-MazF, MazE-MazF promoter, and MazE promoter complexes) may
be rather low. As lowering the concentration shifts the
T1/2 (Fig. 6c) to lower values, it
follows that, under physiological temperatures, the free MazE might be partially dissociated and unfolded. The native dimer-denatured monomer
equilibrium may therefore play an important role in regulation of the
mazEF system as a whole, because it is known that
fluctuations in the concentrations of the system products may result in
cell death (28). The thermodynamic stability of the cAbMaz1 was also compared with the corresponding
G° values of some other
members of the camelid VHH antibody family determined at
25 °C. The
G° value of our cAbMaz1 at 25 °C is
8.7 kcal/mol, which places it somewhere in the middle of the list of
VHH stabilities (38, 39). However, its transition
temperature T1/2 (Table II) is among the highest
observed for any VHH (38, 39). The high T1/2 and high degree of reversibility (0.85) are the
properties of VHH that may be highly appreciated in
processes where transient heating may take place. Fig. 6b
displays the extent to which MazE dimer is stabilized by the binding of
two cAbMaz1 molecules. The difference between the curve obtained as a
sum of
G° versus T curves for
MazE and cAbMaz1, and the curve of their tetrameric complex is a
measure of apparent binding affinity of the two cAbMaz1 molecules
(
G1° +
G2°).
G1° +
G2°
values obtained from DSC are in good agreement with those obtained by
model analysis of the ITC, FL, and CD titration curves (Table I, and
Fig. 5c). As cAbMaz1 is also relatively small and well
soluble, it proved to be an ideal crystallization and phasing aid for
MazE.1 It follows that VHHs may be applicable
also for stabilization of other proteins with short shelf life and
relatively low thermodynamic stability.
The general architecture of globular proteins is such that a
hydrophobic core is surrounded by hydrophilic shell. This
compartmentalization is the main driving force of the protein folding.
To qualitatively describe to what extent buried hydrophilic groups as
well as exposed hydrophobics destabilize the folded conformation of the
studied proteins, we compared the experimental
CP° values with those calculated from the
changes in the non-polar and polar solvent accessible surface areas
upon denaturation (Equation 11). The calculated
CP° values for the denaturation of the
tetrameric MazE-cAbMaz1 complex and cAbMaz1 agree well with the
experimental ones, whereas in the case of MazE, the calculated
CP° is significantly higher (Table II). As
the opposite effect was observed upon MazE-cAbMaz1 binding, this
discrepancy may be ascribed to the more non-polar (or/and less polar)
residues exposed to solvent in the MazE native state in the solution
than is predicted by MazE-cAbMaz1 crystal structure.
Binding of MazE to the Promoter DNA--
It was reported recently,
that two alternating palindrome sequences in the promoter (Fig.
9) are crucial for DNA binding of the
MazE-MazF complex and thus for the regulation of MazE and MazF
expression (9). Footprint analysis of the promoter revealed protection
of DNA against DNase I by the MazE-MazF complex. On the other hand, it
was suggested that the binding of MazE to DNA is weaker than the
binding of the MazE-MazF complex (9). In the present work, we were able
to estimate thermodynamic parameters of MazE association on the basis
of a simple model (Reaction 1 and Equations 1 and 6), which shows that
up to three MazE dimers are bound at the promoter sequence and that the
binding is highly exothermic (
H° =
71 kcal/mol) with
the apparent binding constant in the micro-molar range
(
G° =
RTlnK =
8.7
kcal/mol). MazE-promoter DNA interactions may be characterized as
specific, because the induced ITC and FL signals upon MazE + control
DNA titration were negligible in comparison to those resulted from the
MazE binding to the promoter DNA. Rather low K can be
ascribed to the highly unfavorable entropic contribution
(T
S° =
H°
G° =
62
kcal/mol). As DNA is relatively rigid and the displacement of water
from interacting surfaces is entropically favorable, this highly
negative value most probably results not just from lowering of degrees of freedom due to association reaction (MazE + DNA = MazE-DNA) but
also from folding of an otherwise unstructured part of the MazE
dimer.1 This makes MazE one of many recently identified
proteins that are intrinsically flexible but become more ordered upon
binding a specific partner (40). We have observed that the binding of the MazE-cAbMaz1 complex to DNA, just like in the case of MazE, is a
highly enthalpy-driven (
H° =
80 kcal/mol) process
accompanied by negative entropy contributions
(T
S° =
69 kcal/mol). The apparent affinity of MazE-cAbMaz1 (
G° =
8.6 kcal/mol) is
practically the same as that of MazE alone. This indicates that the
binding of two cAbMaz1 molecules to the MazE dimer does not
significantly influence its interactions with the promoter DNA. Because
the residues of MazE dimer to which the two cAbMaz1 molecules are bound
cannot interact with DNA, only a limited number of orientations of the
MazE dimer in the complex with DNA are possible.

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Fig. 9.
Panel a, 45 bp of ideal B-DNA showing
the alternating palindrome structure (9) found in the MazE promoter.
Panel b, model for MazE binding to its promoter DNA. Because
of the length of the DNA, large spaces remain between the individually
bound MazE dimers, in agreement with independent binding. Sufficient
room is available for positioning MazF and/or the disordered part of
MazE. Panel c, equivalent model of the binding of the
MazE-cAbMaz1 complex to the mazEF promoter DNA. In this
model, the antibody fragments touch neither each other or the
DNA.
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Based on this observation as well as on results from site-directed
mutagenesis and the observation of residue conservation within the MazE
family of antidotes, we have recently suggested a structural model for
MazE-DNA binding.1 When we consider the 3-fold repeat in
the promoter DNA sequence and dock the MazE dimer into the center of
each repeating unit, we see that all MazE molecules are too far apart
from each other to directly interact. This is in full agreement with
our model of three non-interacting, quasi-equivalent binding sites
(Fig. 9b). Sufficient space is available between the bound
MazE dimers to position the otherwise unstructured MazE domain (which
we assume to become more structured upon DNA binding). The spacing
between the bound MazE dimers is also of the right magnitude to allow them to be bridged by MazF dimers. In the presence of bound cAbMaz1, no
additional protein-DNA contacts or steric hindrance of any sort is
observed in our structural model (Fig. 9c), which is also in
agreement with the experimental results.
The general property of autoregulation of transcription in all
described addiction systems is that the antidote is the main DNA-binding protein of which affinity to promoter DNA is significantly enhanced in the presence of the toxin. On the other hand, the details
such as the number of palindrome DNA sequences involved in binding, the
protection of DNA against DNase I by the complex and also by the
antidote alone, and the length of the protected region differ from
system to system. In the case of phd-doc, under physiological conditions unfolded Phd monomers are stabilized by
binding to promoter DNA, a process that is accompanied by dimer formation (10-11). The additional stabilization of Phd by dimer formation is most probably the reason why Phd binding to each of the
two distinct palindromes is more energetically favorable than the
described binding of MazE, where the dimers already exist in solution.
CcdA and ParD (the antidote from parDE system) are, like
MazE, dimeric proteins in the micromolar concentration range at
physiological temperatures. Under these conditions they bind to DNA as
dimers. However, the binding affinities and other thermodynamic parameters have not been reported (32-34).
 |
ACKNOWLEDGEMENTS |
We acknowledge the use of synchrotron beam
time at the European Molecular Biological Laboratory (EMBL) beamlines
at the DORIS storage ring (Hamburg, Germany) and the European
Synchrotron Radiation Facility (ESRF; Grenoble, France). We thank Joris
Messens and Karolien Van Belle for purifying the MazE-cAbMaz1 complex
and performing quantitative gel filtration experiments with MazE. Professor Gorazd Vesnaver from University of Ljubljana (where the DSC
measurements were performed) and Patrick Van Gelder are gratefully
acknowledged for critical reading of the manuscript and useful suggestions.
 |
FOOTNOTES |
*
This work was supported by the Vlaams Interuniversitair
Instituut voor Biotechnologie (VIB), the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO), and Israel Science Foundation Grants 467/99-19 (to G. G.) and 215/99-2 (to H. E.-K.), which were
administered by the Israel Academy of Science and Humanities.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Recipient of a short-term fellowship as visiting scientist
from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen and to whom
correspondence should be addressed: University of Ljubljana, Faculty of
Chemistry and Chemical Technology, Askerceva 5, 1000 Ljubljana,
Slovenia. Tel.: 386-1-2419-414; Fax: 386-1-2419-437; E-mail:
jurij.lah@uni-lj.si
§§
Postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M209855200
1
R. Loris, I. Marianovsky, J. Lah, T. Laermans,
H. Engelberg-Kulka, G. Glaser, S. Muyldermans, and L. Wyns, submitted
for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
ITC, isothermal
titration calorimetry;
FL, fluorescence spectroscopy;
CD, circular
dichroism;
DSC, diffferential scanning calorimetry;
ASA, accessible
surface area.
 |
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