Effect of Mutations at the Monomer-Monomer Interface of cAMP
Receptor Protein on Specific DNA Binding*
Ying
Shi,
Shenglun
Wang,
Susan
Krueger
, and
Frederick P.
Schwarz§
From the Center for Advanced Research in Biotechnology, National
Institute of Standards and Technology, Rockville, Maryland 20850
 |
ABSTRACT |
To determine the thermodynamic role of binding of
an operon to cAMP receptor protein (CRP) in the activation of
transcription, isothermal titration calorimetry measurements were
performed on the binding of three 40-base pair DNA sequences to the
cyclic nucleoside complexes of CRP and its mutants at 296 K. The three 40-base pair sequences consisted of a consensus DNA (conDNA) duplex derived from the CRP-binding site sequences of the operons activated by
CRP and two DNA sequences based on the CRP-binding site sequences of
the lac operon (lacDNA) and of the gal operon (galDNA). The mutants
of CRP consisted of a T127L mutant, a S128A mutant, and a mutant
containing both mutations (CRP*) which not only alter the
transcriptional activity of the CRP complexes but also are involved in
the monomer-monomer interfacial interactions of the CRP dimer. The
binding reactions of the DNA duplexes to the fully cNMP-ligated
CRP-mutant complexes were endothermic with binding constants as high as
6.6 ± 1.1 × 106
M
1 (conDNA·CRP(cAMP)2). ConDNA
binding to the unligated T127L and CRP* mutants was observed as well as
conDNA and lacDNA binding to CRP with cAMP bound to only one
monomer. The reduction of the binding constants with increase in KCl
concentration indicated the formation of two ion pairs for the
cAMP-ligated CRP and S128A complexes and four ion pairs for the
cAMP-ligated T127L and CRP* complexes. Reduction of the DNA binding
constants upon substitution of D2O for H2O in
the buffer, the large heat capacity changes, and the enthalpy-entropy
compensation exhibited by the binding reactions indicate the importance
of dehydration in the binding reaction. Small angle neutron scattering
measurements on the lacDNA·CRP(cAMP)2 complex in
D2O/H2O mixtures show that the DNA is bent
around the cAMP-ligated protein in solution.
 |
INTRODUCTION |
The transcription of over 20 different operons encoded for enzymes
involved in carbohydrate metabolism in Esherichia coli is
activated by the binding of 3',5'-cyclic adenosine monophosphate (cAMP)
receptor protein (CRP)1 to
the operon. CRP is a 45,000 g mol
1 dimer, consisting of a
3',5'-cyclic adenosine monophosphate (cAMP)-binding site in the
amino-terminal domain and an
-helical DNA-binding region in the
carboxyl-terminal domain of each monomer. Upon binding of cAMP to CRP,
CRP undergoes a change to a conformation which binds specifically to
the operon and results in bending of the operon around the RNA
polymerase. The nature of this conformational change is not clear but
may be induced by a stronger monomer-monomer interaction in CRP since
this interaction is enhanced in the presence of cAMP (1). X-ray
crystallographic studies of the CRP(cAMP)2 complex show
that the sequence at the monomer-monomer interface is similar to that
of the leucine zipper motif and that a serine (Ser128) from
the other monomer crosses over the interface and forms a hydrogen bond
with the cAMP (2). More recent x-ray crystallographic studies of the CRP(cAMP)2 complexed with DNA (3, 4) show that the DNA is symmetrically bound across the
-helices of the two
monomers of CRP. Mutations at the monomer-monomer interface of CRP
alter the activity of CRP in the cell as is evident by a doubly mutated
(T127L/S128A) CRP mutant (CRP*) which activates transcription in the
absence of cAMP, a CRP (T127L) mutant which also activates
transcription in the presence of an analog of cAMP, 3',5'-cyclic
guanosine monophosphate (cGMP), and a CRP (S128A) mutant which does not
activate transcription in vivo (5).
Since it has been shown from isothermal titration calorimetry (ITC)
measurements that changes in the in vivo transcriptional activity arising from these interfacial mutations are not dependent on
the binding affinity of the cyclic nucleotide monophosphate (cNMP) to
the CRP-mutant (6), the transcriptional activity of cNMP-ligated
CRP-mutants may depend on the influence of these interfacial mutations
on the binding of the operon to CRP. It is, thus, important to
determine the thermodynamics of DNA binding to CRP and its T127L,
S128A, and CRP* mutants. Heretofore, indirect measurement techniques
based on filter binding assays and shifts in gel electrophoresis bands
have been employed to determine the operon binding affinities for CRP
with often contradictory results. Equilibrium constants for binding of
lac operon fragments to cAMP-ligated CRP range from 2.3 ± 0.1 × 10 9 M
1 for a
216-base pair fragment (7) to 8.4 × 1010
M-1 for a 203-base pair fragment (8) at
ambient temperatures. Since it has been shown that the CRP-binding site
on the lac operon requires a minimum of 28 base pairs for full binding
affinity (9), the binding of shorter lac operon fragments consisting of
40-41 base pairs was determined and, again, the binding constants ranged from 6 × 10 6 M
1 in
gel electrophoresis measurements at 310 K (9) to 3.2 ± 0.1 × 10 8 M
1 from filter binding
assays (10). In addition, the DNA binding constants determined from
these indirect techniques exhibit a large decrease with increase in
salt concentration (11) in contrary to in vivo
transcriptional assays which show high yields even at high salt
concentrations (12).
To investigate the effect of monomeric interfacial mutations on
specific DNA binding, ITC was employed to determine the binding constant (Kb) and the changes in enthalpy
(
Hb0) and entropy
(
Sb0) for the binding of DNA to cAMP,
cGMP, and 3',5'-cyclic inosine monophosphate (cIMP)-ligated CRP, T127L,
S128A, and CRP*,
|
(Eq. 1)
|
in 50 mM potassium phosphate buffer containing 0.2 mM EDTA, 0.2 mM dithiothreitol, and 0.15 M KCl (KPB buffer). The DNA duplexes consisted of 40 base
pairs with the following sequences,
and
conDNA is a consensus sequence based on the sequences of the 20 operons while lacDNA is the CRP-binding site of the lac operon. The
conDNA and lacDNA sequences are from Ebright et al. (10) and are identical from the 1 to 5' terminus and from 23 to the 3'
terminus, using the numbering system from Ebright et al.
(10). For comparison, galDNA was constructed so that bases from 1 to 21 are the same as the CRP-binding site of the gal operon (29). Although
the DNA binding was determined at cyclic nucleoside concentrations where both sites of the CRP-mutants were occupied, the binding of
conDNA and lacDNA to CRP was also investigated at subsaturate concentrations of cAMP where significant concentrations of singly occupied CRP, CRP(cAMP), as calculated from previous measurements (13),
were present to determine if the DNA binds to CRP(cAMP). In addition,
the binding reactions of lacDNA and conDNA with
CRP(cAMP)2 were investigated as a function of salt
concentration and with D2O substituted for water in the KPB
buffer since dehydration has been shown to be involved in the binding
reaction (7). A preliminary analysis of the bending of the
lacDNA bound to the CRP(cAMP)2 complex in water and
D2O was performed by small angle neutron scattering (SANS)
since the neutron scattering from the DNA can be characterized
separately from the protein in H2O/D2O mixtures of the buffer. The results of this study will contribute to a better
understanding of the thermodynamics of DNA binding to proteins since
bending of the DNA, the
helical structure of the DNA-binding site,
and the presence of a leucine zipper between the protein subunits are
characteristic properties of other DNA-protein complexes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The DNA sequences were synthesized by Oligos,
Inc. and purified by high performance liquid chromatography. The DNA
was analyzed by capillary electrophoresis and an analytical ion
exchange column and found to be at a purity level >90%. Gel
electrophoresis of the single strands revealed essentially one intense
band at 12,000 g mol
1, the molecular mass of the strand.
A procedure similar to that described by Ebright et al. (10)
was followed to prepare the duplexes from the single strand DNA
sequences. Briefly, milligram quantities of the lyophilized powdered
DNA strands were dissolved in 4 ml of 10 mM Tris-HCl buffer
containing 1 mM MgCl2 and 0.5 M
NaCl at pH 7.5. Equal amounts of the DNA strand and its complementary strand were mixed together, heated up slowly to 368 K, held at 368 K
for about 10 min, and then cooled very slowly at a rate of 10 K
h
1 to ambient temperature. Gel electrophoresis
measurements on aliquots of the DNA solution showed the presence of
only one band at the molecular mass of the duplex. The DNA duplex
solutions were then concentrated up to 0.3-0.6 mM by
centrifugation, dialyzed in the KPB buffer used for dialysis of the
protein, and stored in a freezer at 253 K. Prior to the ITC
measurements, the DNA duplex concentrations were determined from
optical density measurements at 260 nm using the extinction coefficient
of 1.32 × 104 M
1 cm
-1 per base pair (8). The production from E. coli of CRP and mutants and their purification have been described
previously (5) and their activities were checked by an in
vitro transcription assay as described by Zhang et al.
(14). The concentration of the CRP-mutants was determined from UV
measurements at 280 nm using an extinction coefficient of 3.5 × 104 M
1 cm -1 (15).
The potassium phosphate salts, KCl, Tris, MgCl2, and sodium
salts of cAMP, cGMP, and cIMP were reagent grade from Sigma. The
dithiothreitol was Ultra-pure brand from Life Technologies, Inc. and
the NaEDTA was from Serva Co. The D2O was from Cambridge Isotope Laboratories.
ITC Measurements--
All calorimetric titrations were preformed
according to the methods of Wiseman et al. (16) and Schwarz
et al. (17) using a Microcal Omega titration calorimeter.
The Omega titration calorimeter consists of a matched pair of sample
and reference vessels (1.374 ml) containing the protein solution in the
phosphate buffer and the buffer solution, respectively. Four to 10-µl
aliquots of the DNA solution at concentrations × 10-20 the
protein concentration of 0.01-0.05 mM in the sample vessel
were added 3-4 min apart. A separate titration of the DNA solution
into the buffer was performed to determine any DNA heat of dilution
which was then subtracted from the heats obtained during the titration
of the DNA solution into the protein solution. A nonlinear, least
squares minimization performed by Microcal Origin scientific plotting
software was used to fit the incremental heat of the ith titration
(
Q(i)) of the total heat,
Qt, to the total DNA titrant concentration, Xt, according to the following equations (16,
18),
|
(Eq. 2)
|
|
(Eq. 3)
|
where n is the stoichiometry of the binding reaction,
Ct is the total protein concentration in the sample
vessel, and V is the vessel volume. Binding entropies,
Sb0, were calculated using the
following equation of thermodynamics,
|
(Eq. 4)
|
where
|
(Eq. 5)
|
and r = 8.31451 J mol
1
K
1.
Two methods were employed to determine the dependence of DNA binding on
the subsaturate concentration of cAMP in the solution. In one method,
aliquots of cAMP in KPB buffer were titrated into CRP solutions
containing the DNA duplex and the amount of heat exchanged was
monitored as a function of the relative CRP(cAMP) and
CRP(cAMP)2 concentrations. Separate ITC runs with just CRP in the protein solution showed very little contribution of the heat of
binding of cAMP to CRP under these titration conditions and any
observed heat exchange would be from the binding of DNA to
CRP(cAMP)2 and perhaps to CRP(cAMP). In the second method, the amount of cAMP added to the CRP solution prior to the DNA titration
was well below saturation so that the CRP contained about equal
concentrations of CRP(cAMP) and CRP(cAMP)2. In both methods, the concentrations of CRP(cAMP) and CRP(cAMP)2
were determined by solving the following equations, where
K1 and K2 are the
macroscopic cAMP to CRP binding constants from Gorshkova et
al. (13),
|
(Eq. 6)
|
|
(Eq. 7)
|
So that
|
(Eq. 8)
|
and
|
(Eq. 9)
|
where
|
(Eq. 10)
|
|
(Eq. 11)
|
[cAMP]t is the total cAMP concentration in the sample.
The concentration of CRP(cAMP)2 thus determined was then employed with the total DNA concentration in the sample to determine if
the total observed amount of heat exchanged could be attributed solely
to the CRP(cAMP)2 complex. In this latter calculation, the
binding constant and binding enthalpy for DNA binding to
CRP(cAMP)2 obtained at saturating concentrations of cAMP
were employed. In the alternative procedure where DNA is titrated into
solutions of CRP at subsaturate cAMP concentrations, the DNA binding
constant and binding enthalpy were determined employing Equations 2 and 3 and compared with the DNA binding quantities determined at fully saturated CRP(cAMP)2 concentrations.
The standard uncertainties in the values of Kb and
Hb0 were determined by combining in a
quadrature the standard deviations of several determinations of each
value with estimates of their systematic standard uncertainties. The
systematic standard uncertainties are 0.03 Kb and
0.03
Hb0 resulting from uncertainty
in the solution concentrations, 0.01 Kb and
0.01
Hb0 resulting from uncertainty in
the ITC calibration, and 0.005 Kb and 0.005
Hb0 resulting from uncertainty in the
titrant and cell volumes.
SANS Measurements--
The lacDNA·CRP(cAMP)2
product from an ITC measurement was concentrated up to a concentration
of 0.085 mM and 0.5-ml aliquots of the solution were
dialyzed in 100, 70, 30, and 0% (v/v) D2O KBS buffer. The
neutron scattering intensity from each of the four solutions was
measured as a function of the scattering angle on the NG3 30-m SANS
instrument at the NIST Center for Neutron Research. At high water
concentrations the major component of the scattering intensity is from
the nucleic bases of the DNA and at high D2O concentrations
the major component is from the cAMP-ligated CRP complex. Thus, by
solving a set of simultaneous equations defined by the measured neutron
intensities as a function of water concentration, the neutron
intensities of the CRP and DNA can be separately identified (19).
Details of the subsequent analysis of the data have been described
previously by Krueger et al. (20). The x-ray
crystallographic structure of the DNA complexed to the cAMP-ligated CRP
determined by Schultz et al. (3) was used as the scattering
model for the lacDNA·CRP(cAMP)2.
Solvent-accessible Surface Areas--
The change in the
solvent-accessible surface on formation of the
DNA·CRP(cAMP)2 complex (
As) was calculated
with "NACCESS," a computer program from Hubbard (21), which
conveniently divides the change into polar (
Asp) and non-polar area
changes(
Asn). The
As was calculated as,
|
(Eq. 12)
|
Here As(CRP in complex) refers to the solvent-accessible
surface area of the CRP in the DNA·CRP(cAMP)2 complex and As(CRP(cAMP)2) refers to the solvent-accessible
area of the unligated CRP. Asp(CRP) and Asn(CRP) were calculated from the atomic coordinates from the Protein Data Bank file 3GAP; while
Asp(complex) and Asn(complex) were calculated by using both PDB
file 1CGP and 1RUN. Average solvent-accessible surface area changes
were determined to be
626 (angstrom)2 =
Asn(complex-CRP) and
2008 (angstrom)2 =
Asp(complex-CRP) yielding a total solvent accessible surface area
change of
2633(angstrom)2.
 |
RESULTS |
Thermodynamics of DNA Binding--
Results of a typical ITC
measurement consisting of titrating 5.0-µl aliquots of 0.250 mM conDNA into a solution of 0.020 mM CRP(cAMP)2 complex at 295.6 K along with the binding
isotherm are presented in Fig. 1. Prior
to this titration, a 10 mM cAMP solution was first titrated
into the 0.020 mM CRP solution to completely (99%) ligate
the CRP with cAMP. The stoichiometry of the binding reaction as
determined by the binding results was always close to one (0.90 ± 0.05). Similar results were obtained with the other cNMP-ligated
CRP-mutant complexes and are summarized in Table
I for all 12 complexes including binding
of conDNA to unligated L127 and unligated CRP*. Binding of the conDNA
duplex with unligated CRP and S128A was not observed. The thermodynamic quantities in Table I are average values of at least two ITC scans with
different concentrations of ligand and protein and the average
stoichiometry was again close to one. As shown in Table I, all the
conDNA binding reactions are endothermic with binding enthalpies
ranging from 84.1 ± 0.3 kJ mol
1 for binding to
CRP(cAMP)2 to 166 ± 12 kJ mol
1 for
binding to L127(cGMP)2 and binding constants ranging from 8.1 ± 0.9 × 104 M
1
for binding to S128A(cGMP)2 to 6.6 ± 1.1 × 106 M
1 for binding to
CRP(cAMP)2 at room temperature. The enthalpy change for
binding of conDNA to CRP(cAMP)2 is close to the literature value of 80 ± 17 kJ mol
1 obtained from a van't
Hoff analysis of the binding constants determined from filter binding
assays in a 10 mM MOPS buffer at pH 7.3 containing 0.2 mM NaCl, 0.1 mM dithiothreitol, 50 µg
ml-1 serum albumin, and 0.2 mM cAMP, while the
corresponding binding constant is much higher, 3.9 ± 0.3 × 1010 M
1 (10). The reason for this
large discrepancy is unknown except to emphasize that ITC is a direct
measurement of the binding event. The heat capacity change accompanying
the conDNA to cAMP-CRP binding reaction, determined from the dependence
of the binding enthalpy on the temperature (Fig.
2), is
4.0 ± 0.4 kJ
mol
1 K
1, close to an estimate of
2.85 kJ
mol
1 K
1 (22) from filter binding
assays by Ebright et al. (10).

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Fig. 1.
a, a typical ITC scan consisting of the
addition of 5-µl aliquots of 0.250 mM conDNA into 0.020 mM CRP(cAMP)2 in KPB buffer at 296.0 K. b, the binding isotherm for this titration.
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Table I
Thermodynamic quantities of con DNA binding to cNMP-ligated CRP
complexes
The uncertainties in the tables are standard uncertainties.
|
|
For comparison, the thermodynamics of binding of the lacDNA and
galDNA duplexes to the fully cAMP-ligated CRP, T127L, S128A, and CRP*
complexes and to T127L(cGMP)2 and CRP*(cIMP)2
were determined from ITC measurements and are presented in Table
II. Typical ITC titrations at 296 K of
10-µl aliquots of 0.250 mM lacDNA into 0.024 mM CRP(cAMP)2 and 10-µl aliquots of 0.90 mM galDNA into 0.052 mM L127(cGMP)2
are shown, respectively, in Figs. 3 and
4. The binding constants of lacDNA
and galDNA are at least an order of magnitude lower than their
corresponding conDNA binding constant, the stoichiometries are again
close to one, and the binding reactions are all endothermic. For
lacDNA, the binding constants range from 3.3 ± 1.5 × 104 M
1 for binding to
CRP*(cIMP)2 to 4.0 ± 0.6 × 105
M
1 for binding to CRP(cAMP)2,
while the binding enthalpies range from 98 ± 4 kJ
mol
1 for binding to CRP(cAMP)2 to 163 ± 8 kJ mol
1 for binding to CRP*(cAMP)2. The
binding constant for lacDNA binding to CRP(cAMP)2 as
reported by Ebright et al. (10) is 7.6 ± 0.6 × 107 M
1, about 160 times greater
than the binding constant in Table II. However, the binding constant of
a 41-base pair lacDNA fragment of 6 × 106
M
1 determined at 310 K from gel
electrophoresis measurements (9) is also less by a factor of 50 than
the value of 3.2 ± 0.1 × 108
M
1 determined by Ebright et al.
(10). For galDNA, the binding constants range from 1.7 ± 0.4 × 104 M
1 for binding to
CRP*(cIMP)2 to 1.11 ± 0.21 × 106
M
1 for binding to CRP(cAMP)2,
while the binding enthalpies ranged from 85 ± 3 kJ
mol
1 for binding to CRP(cAMP)2 to 174 ± 4 kJ mol
1 for binding to T127L(cAMP)2. For
lacDNA binding to CRP(cAMP)2, the heat capacity change
of
5.7 ± 1.6 kJ mol
1 K
1 is the same
as the heat capacity change observed for binding of conDNA to
CRP(cAMP)2 (
4.0 ± 0.4 kJ mol
1
K
1). All the DNA binding reactions exhibited
enthalpy-entropy compensation as shown in Fig.
5 where the slopes of the
T
Sb0 versus
Hb0 plots are 0.95 ± 0.5 for
conDNA, 0.95 ± 0.06 for lacDNA, and 0.98 ± 0.06 for
galDNA binding to CRP-mutant(cNMP)2. A comparison of all
the DNA binding constants is shown in Fig.
6 where it is observed that the naturally
occurring sequences exhibit a greater range of values, i.e.
specificity than the synthetic conDNA.

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Fig. 3.
a, an ITC scan consisting of the
addition of 10-µl aliquots of 0.25 mM lacDNA into
0.024 mM CRP(cAMP)2 in KPB buffer at 295.3 K. b, the binding isotherm for this titration.
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Fig. 4.
a, an ITC scan consisting of the
addition of 10-µl aliquots of 0.90 mM galDNA into 0.052 mM T127L(cGMP)2 in KPB buffer at 295.6 K. b, the binding isotherm for this titration.
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Fig. 5.
Enthalpy-entropy compensation plots for the
DNA-CRP/mutant (cNMP)2 binding reactions at 296 K where the
solid points and solid line are
conDNA, triangles are lacDNA, and
circles are galDNA binding. The solid
straight line represented by
T Sb0 (kJ
mol 1) = 37.3 ± 5.6 + (0.94 ± 0.05)
Hb0 (kJ mol 1) is the
best least squares fit to the conDNA data points. The dashed
line represented by
T Sb0 (kJ
mol 1) = 30 ± 6 + (0.95 ± 0.06)
Hb0 (kJ mol 1) is the
best least squares fit to the lacDNA and galDNA data points.
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Fig. 6.
Bar graph comparing the DNA binding
specificities in terms of the binding constant for the different
CRP(cNMP)2 complexes. For each DNA
sequence, the CRP complexes are from left to right
CRP(cAMP)2, T127L(cAMP)2,
T127L(cGMP)2, S128A(cAMP)2,
CRP*(cAMP)2, and CRP*(cIMP)2.
|
|
Thermodynamics of DNA Binding to CRP as a Function of cAMP
Concentration--
The results of titrating 0.435 mM cAMP
in KPB into a solution containing 0.023 mM CRP and 0.023 mM conDNA in KPB are presented in the form of a binding
isotherm in Fig. 7 along with the
calculated concentrations of CRP(cAMP) and CRP(cAMP)2, the
calculated heats absorbed by the CRP(cAMP)2 alone, and the
calculated heats absorbed by both CRP(cAMP) and CRP(cAMP)2
using the binding constant and binding enthalpy values from Table I.
Similar results were obtained with titrating the cAMP solution into a
solution containing lacDNA instead of the conDNA. As shown in Fig.
7, at least 90% of the total amount of heat absorbed with each
addition of the cAMP solution can only be attributed to DNA binding to
both CRP(cAMP)2 and CRP(cAMP). An ITC titration of a 0.2 mM lacDNA solution into a 0.05 mM CRP solution containing 0.064 mM cAMP so that equal
concentrations of CRP(cAMP) and CRP(cAMP)2 were present
yielded a binding constant of 1.9 ± 0.4 × 105
liter mol
1 and a binding enthalpy of 82 ± 4 kJ
mol
1. These values are within two standard uncertainties
of the corresponding averages of 4.0 ± 0.6 × 105 M
1 and 98 ± 4 kJ
mol
1 for binding to the fully cAMP-ligated CRP (Table
II). An ITC measurement under similar concentration conditions with
conDNA yielded a binding constant of 6.3 ± 3.7 × 106 M
1 and a binding enthalpy of
88 ± 2 kJ mol
1 close to the results in Table I. The
ITC results, thus, show that the DNA duplex can also bind to CRP(cAMP)
with the same binding affinity and enthalpy as to the
CRP(cAMP)2 species. Equal binding affinities of specific
DNA binding to both CRP(cAMP) and CRP(cAMP)2 were also
observed earlier by Takahashi et al. (23) in equilibrium dialysis measurements on specific DNA binding to CRP as a function of
cAMP concentration.

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Fig. 7.
a, the total heat absorbed upon the
addition of 5-µl aliquots of 0.435 mM cAMP to a solution
containing 0.023 mM CRP and 0.023 mM conDNA
( -), the calculated total heat absorbed by the CRP(cAMP)2
species alone in solution ( ), and the calculated total heat absorbed
by both species employing the binding enthalpy and constant from Table
I ( ). b, the calculated concentrations of CRP(cAMP) ( )
and CRP(cAMP)2 ( ) generated by the addition of 5-µl
aliquots of 0.435 mM cAMP solution in the ITC scan
described in a.
|
|
Solvent Effects on the Thermodynamics of DNA Binding--
Since
DNA binding to CRP involves electrostatic complementarity (24), ITC
measurements were performed on the binding of conDNA to CRP-mutants as
a function of KCl concentration from 0.15 to 0.60 M and are
summarized in Table III. A typical ITC
scan of titrating 5.0 µl of 0.71 mM conDNA into 0.12 mM CRP(cAMP)2 at 0.5 M KCl in KPB
buffer is shown in Fig. 8. An increase in the KCl concentration reduces the binding constant through an increase
in the endothermic binding enthalpy and only a partial compensating
increase in the binding entropy. The decrease in the binding constant
could result from competitive anion binding to the DNA-binding site on
CRP or electrostatic screening of the ion pair interactions between the
DNA bases and the CRP amino acid residues. For B-DNA binding to
proteins based solely on ion pair interactions, it has been shown (25)
that,
|
(Eq. 13)
|
where Kb (1.0 M KCl) is the binding
constant at 1.0 M KCl and m is the number of
CRP-mutant/DNA ion pairs. The results in Table III yield a value of
1.76 ± 0.20 for conDNA binding to CRP(cAMP)2, close
to 2 ion pair interactions between CRP(cAMP)2 and conDNA.
For conDNA binding to the other cNMP-ligated CRP mutants, m
can be estimated from m = 2.173 log(Kb(0.15 M
KCl)/Kb(0.5 M KCl)) which yields close
to 4 ion pairs for T127L(cAMP)2, T127L(cGMP)2, and CRP*(cAMP)2 complexes and again close to 2 ion pairs
for the S128A(cAMP)2 complex. The number of ion pairs of 2 for conDNA binding to CRP(cAMP)2 is not in agreement with
the number of 8 ion pairs determined from the filter binding assays
(11). According to Equation 13, 8 ion pairs would result in a reduction
of the conDNA binding constant by a factor of 4.8 × 103 from 0.15 to 0.50 M KCl in the KPB buffer,
which is clearly not observed by comparing Figs. 1 and 8. In addition,
CRP is able to function in osmotically stressed cells grown at high
salt concentrations (12) and this would not be the case if the DNA
binding constant is reduced by more than 3 orders of magnitude. Thus,
the lower number of ion pairs determined from the ITC measurements is
more consistent with the observations from the osmotically stressed cells. The doubling of the number of ion pairs from 2 to 4 formed between conDNA and the T127L(cNMP)2 and
CRP*(cAMP)2 complexes reflects some alteration of the
topography of the DNA-binding sites in cNMP-ligated T127L and CRP*
complexes.
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Table III
Comparison of thermodynamic quantities of conDNA binding to
cNMP-ligated CRP complexes in buffer at different levels of KCl
concentration
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Fig. 8.
a, an ITC scan consisting of the
addition of 5-µl aliquots of 0.71 mM conDNA into 0.12 mM CRP(cAMP)2 in KPB buffer at 0.5 M KCl concentration and 295.6 K. b, the binding
isotherm for this titration.
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|
Since dehydration has been shown to play an important role in DNA
binding to CRP (7), ITC measurements on the binding of DNA to CRP(cAMP)2 were performed in D2O-buffered
solutions. Substitution of H2O by D2O in the
buffer results in a reduction in the binding constant for both the
conDNA and lacDNA duplexes due to an increase in the endothermicity
of the binding reaction as shown in Table IV. The same results were obtained after
the solutions were left standing for 24 h prior to the ITC
measurement, indicating that the deuterium had completed any exchange
with the hydrogen on the DNA duplex and on the protein surface.
SANS Measurements on the CRP(cAMP)2·lacDNA
Complex--
The radius of gyration (Rg) of the CRP(cAMP)2
component of the complex was 23 ± 1 angstroms in agreement with
recent SANS measurements on CRP(cAMP)2 (20). The radius of
gyration for the lacDNA duplex in the complex was 32 ± 1 angstroms which is close to the value of 29.7 angstroms calculated for
the DNA component in the x-ray crystal structure of the
DNA·CRP(cAMP)2 complex (3). The distance
distribution function, P(r), (20) for the
lacDNA component of the lacDNA·CRP(cAMP)2 complex
in the 0.085 mM solution is shown in Fig.
9 along with the distance distribution
function of the DNA component of the x-ray crystal structure. The shape of the two curves are similar in that they both contain a maximum in
distance distribution function at a radius around 20 angstroms, distance distribution function decreases slowly to a shoulder around 60 angstroms, and then distance distribution function decreases rapidly to
0-95 angstroms. This last rapid decrease occurs when "rigid" rods
are bent into a "U" shape, thereby eliminating the larger distances
in the distribution function. However, the lacDNA in the
lacDNA·CRP(cAMP)2 complex appears to exhibit a
broader distribution around the maximum than the DNA in the
DNA·CRP(cAMP)2 crystal structure. This is probably due to
a globular component in the distance distribution function from
aggregates of the complex in the 70 and 100% (v/v)
D2O-buffered solutions. It can be concluded that the SANS
data show that the DNA in the lacDNA·CRP(cAMP)2 complex is bent, independent of the concentration of D2O in
the sample.

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|
Fig. 9.
Distance distribution function of
lacDNA in lacDNA·CRP(cAMP)2 complex
from SANS data ( ) compared with that calculated for the DNA
components in the crystal structure (line).
|
|
 |
DISCUSSION |
As observed for other protein-DNA associations (26, 27),
the specific binding of DNA to cNMP-ligated CRP-mutant complexes is
endothermic, thus, entropically driven, and involves dehydration. The
binding enthalpy can be attributed to endothermic bending of the DNA
(
Hbn0) and to the formation of ionic
pair interactions, van der Waals interactions, and hydrogen bonding
interactions between the amino acid residues at the CRP-binding site
and the DNA bases (direct interactions) and the ribose-phosphate
backbone (indirect interactions) all of which would be exothermic
(
Hbx0),
|
(Eq. 14)
|
Contributions to
Hbn0 would
principally arise from the energy absorbed in the bending of the DNA
duplex about 90o around the CRP and would expected to be
the same for the different DNA sequences bending around the
CRP(cAMP)2 complex. Differences in the direct and indirect
interactions based on the x-ray crystallographic structure of the
DNA·CRP(cAMP)2 complex by Parkinson et al. (4) are itemized in Table V. Since the number
of A, T, G, and C bases is the same for conDNA, lacDNA, and galDNA,
it can be assumed that the interaction between the isolated DNA
duplexes and water is approximately the same for all three DNA
sequences and, thus, differences in the intermolecular binding
interactions can be conceptualized in terms of differences between the
CRP-binding site sequences of the DNA duplexes.
View this table:
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[in a new window]
|
Table V
Identification of base changes between conDNA, lacDNA, and galDNA
and their possible role in binding to CRP
|
|
Attempts have been made to correlate the binding affinities
(
Gb0) of the catabolite operons
with the 22-base pair sequence of the CRP-binding site in the operon
(28, 29). In all the catabolite operons, the 4 TGTGA sequence is
conserved and there is a center of pseudo 2-fold symmetry between bases
11 and 12 (28). ConDNA with the highest binding affinity
exhibits perfect 2-fold symmetry and this 2-fold symmetry is reduced in
the more naturally occurring lacDNA and galDNA duplexes which also
exhibit weaker binding affinities. By determining the frequency a
specific nucleotide appeared in each of the 1-22 bases of the
CRP-binding site in 26 operons, Berg and von Hippel (29) derived a
quantitative model predicting the binding affinity of an operon from
the difference between its CRP-binding site sequence and that of
conDNA. The greater the deviation of each half of the 22-base sequence
from the perfect 2-fold sequence of conDNA, i.e. the lower
the symmetry, the weaker the CRP binding affinity. However, their model
predicted a weaker binding affinity for galDNA than for lacDNA,
contrary to the ITC results. In addition to a correlation between
sequence and binding affinity, changes in the relative number of direct
and indirect interactions between the sequences, as determined from the
known crystal structures of the DNA·CRP(cAMP)2
complexes (3, 4), would be also expected to contribute to changes in
the binding affinities. As shown in Table V, the number of base changes
between conDNA and lacDNA or galDNA are seven and more of the base
changes in galDNA are involved in direct interactions with CRP, while more of the base changes in lacDNA are involved in indirect
interactions. The lower binding affinity for lacDNA could be
accounted for by the change in more of the indirect interactions
between the phosphate backbone. This implies that the indirect
interactions contribute more than the direct interactions to the
binding affinity.
The variation of the binding affinities of a particular DNA sequence to
the different cNMP·CRP mutant complexes results from differences in
the topography of the DNA-binding site of CRP. Changes in topography
between the two T127L mutants and CRP are evident in the KCl results
which show that two additional ion pairs are formed between the conDNA
and the cAMP-ligated T127L and CRP* complexes. Recent SANS measurements
(20) on unligated CRP and cAMP-ligated CRP showed that CRP changes from
an "open" form, where the DNA binding
-helices of the
carboxyl-terminal are swung away from the cAMP binding amino-terminal
domain, to a "closed" form, where the
-helices are swung in
toward the amino-terminal domain, and this is the conformation observed
in the x-ray crystallographic structure of DNA·CRP(cAMP)2
complexes (3, 4). Since cAMP enhances the monomer-monomer interaction
in CRP (1), the closed form must be favored by a stronger interaction
between the monomers in CRP. The amino acid sequence along the
monomer-monomer helical interface exhibits an almost perfect leucine
zipper motif consisting of a heptad repeat of leucine residues along
one face of the helix with the exception of a Met120 and
Thr127 (6). In the T127L mutant, Thr127 is
replaced by Leu, thereby creating a more perfect leucine zipper which
would lead to stronger association between the monomeric units as is
observed in other leucine zipper proteins (30). Hence, the stronger
association in the T127L and CRP* mutants would alter the topography of
the DNA-binding site more toward the closed form. The tendency toward
the closed form in CRP containing the T127L CRP mutation is further
substantiated by SANS measurements (20) which show, unlike CRP, little
structural difference between the unligated and cAMP-ligated forms of
CRP*. According to a structural model for DNA binding proposed by
Garges and Adhya (31), the closed form is more favorably disposed
toward DNA binding and, thus, conDNA binds to the unligated T127L and
CRP*. The difference in
Gb0 of
30
kJ mol-1 for conDNA binding to T127L and CRP* is about
80% of the average
Gb0 of
37.8 kJ
mol -1 for conDNA bonding to the CRP(cAMP)2
and L127(cAMP)2 complexes. This "tightness" of the
monomer-monomer interaction in T127L and CRP* can account for
contributing at least 80% to the DNA binding affinity. Interestingly,
the S128A mutation in S128A and CRP eliminates the hydrogen bond to
cAMP formed in the crossover of the Ser128 residue from the
other monomer, thereby reducing the monomer-monomer interaction. This
reduction results in a decrease of the DNA binding affinity in S128A
and CRP* by about 4 kJ mol -1. Finally, DNA can also bind
to the singly cAMP occupied CRP and, thus only one cAMP is apparently
enough to strengthen the monomer-monomer association in CRP, perhaps
through formation of the Ser128-cAMP hydrogen bond.
The binding entropy can be attributed to entropy changes arising from
hydrophobic interactions (dehydration)
(
Sbh0), and the conformational change
in the DNA (
Sbc0), as well as smaller
conformational changes in the DNA-binding site. Following the treatment
of Spolar and Record (22), at a temperature (Ts) where the
binding entropy is zero then,
|
(Eq. 15)
|
the entropy loss due to the loss of rotational and translational
degrees of freedom upon binding is the same for protein-ligand interactions,
Sbrt0 =
209 J
mol-1 K
1, and the entropy change due to the
number of ion pairs formed (z = 2) and the salt
concentration [KCl] = 0.15 mol liter-1,
Sbe0, is
0.88 zRln[KCl] = 27 J
mol-1 K
1. The change in the solvent
accessible surface area of the nonpolar residues is
626 (angstroms)
and, thus,
|
(Eq. 16)
|
so that at a Ts = 328 K determined from
Cp =
4.0 ± 0.4 kJ mol
1
K
1 and
Sb0 = 415 J
mol-1 K
1 for conDNA binding to
CRP(cAMP)2, the entropy of bending of the DNA is estimated
to contribute only 139 J mol-1 K
1 to the
binding entropy of 415 J mol-1 K
1. Thus, the
main contribution to the binding entropy must be from the desolvation
process involving the removal of water between the DNA and the
polar residues at the CRP interface upon binding which had
not been taken into account in Equation 15.
Dehydration upon DNA binding to cAMP-ligated CRP has been observed by
Vossen et al. (7) and is evident in DNA binding to the other
cNMP-ligated CRP-mutants by the reduction of the binding constant in
D2O due to an increase in the endothermicity of the binding
reaction. The increase in binding enthalpy of 43 ± 2 kJ mol-1 for conDNA and 34 ± 9 kJ mol-1
for lacDNA arises from enhanced stabilization of the DNA and CRP in
D2O relative to the DNA·CRP(cAMP)2 complex in
D2O. If
8.8 J mol
1
(angstrom)-1 accompanies the transfer of nonpolar amino
acid groups from H2O to D2O (32, 33), then the
enhanced stabilization of the nonpolar binding surface area (626 (angstrom)2 on the protein alone in D2O
contributes about 5.5 kJ mol
1 to the difference between
the binding enthalpies in D2O and H2O. Apparently, as shown in the entropy calculation, the larger buried polar surface area on the protein of 2008 (angstrom)2 must
also contribute to the enhanced interaction between the protein and
solvent in D2O. Also, if the effect of D2O on
the solvent-reactant interaction involved solely solvent
reorganization, the binding constant would be unaffected (34) because
of enthalpy-entropy compensation. The release of water upon complex
formation is also evident in the enthalpy-entropy compensation
exhibited by the binding reaction at ambient temperatures and by the
temperature dependence of the DNA-binding reaction where the large
negative heat capacity changes minimize changes in
Gb0 as the temperature is increased
from 288 K to 303 K.
 |
FOOTNOTES |
*
This work was supported by National Science Foundation Grant
MCB-9722884 (to F. P. S.).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.
Present address: National Institute of Standards and Technology
Center for Neutron Research, National Institute of Standards and
Technology, Gaithersburg, MD 20899.
§
To whom correspondence should be sent.
 |
ABBREVIATIONS |
The abbreviations used are:
CRP, cAMP
receptor protein;
cIMP, 3',5'-cyclic inosine monophosphate;
CRP*, T127L
and S128A mutant of CRP;
ITC, isothermal titration calorimetry;
SANS, small angle neutron scattering;
MOPS, 4-morpholineethanesulfonic
acid.
 |
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