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(Received for publication, March 22,
1995; and in revised form, July 13, 1995) From the
The thermodynamics of the binding of cyclic adenosine
monophosphate (cAMP) and its non-functional analog, cyclic guanosine
monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its
T127L mutant were investigated by isothermal titration calorimetry
(ITC) in 0.2 and 0.5 M KCl phosphate buffer (pH 7.0) at 24 and
39 °C. Although, the binding of the first cAMP molecule to CRP is
exothermic with an enthalpy change
(
The cyclic AMP receptor protein (CRP) ( CRP exists as a
dimer (molecular mass = 45 kDa) of two identical subunits with
one cyclic adenosine monophosphate (cAMP) binding site per subunit. The
x-ray structure of cAMP ligated CRP refined to 2.5 Å showed each
subunit to be composed of two domains: the carboxyl-terminal domain
which binds to the DNA through a helix-turn-helix motif and a larger
amino-terminal domain which binds to cAMP (Weber and Steitz, 1987).
More recently, Schultz et al.(1991) determined to 3-Å
resolution the structure of the CRP Cyclic guanosine monophosphate (cGMP), a structural analog of
cAMP, also binds to CRP but does not activate specific DNA binding
(Ebright et al., 1985). Eilen and Krakow(1977) showed that
both cAMP and cGMP, although to a lesser extent, protect CRP against
chemical modification by thiol reagents. More recently, Heyduk et
al. (1992) showed that cGMP induced a conformational change in CRP
similar to that induced by cAMP. However, DSC scans of cGMP ligated CRP
can be resolved into only two unfolding transitions (Ghosaini et
al., 1988), implying that the conformation of cGMP saturated CRP
was different than that of cAMP saturated CRP. The binding of cGMP
to the T127L mutant initiates activation of transcription at
approximately 35% of the activity of the cAMP ligated CRP and the
repression of transcription by the T127L mutant is dependent on cGMP
rather than cAMP (Moore, 1993). Although digestion of CRP by subtilisin
occurs only in the presence of cAMP, a similar digestion pattern of the
T127L mutant is evident in the presence cGMP. However, the digestion
pattern for the cAMP ligated mutant is different (Moore, 1993). Thus, a
comparison of the binding properties of CRP and T127L with cAMP and
cGMP would help in understanding the mechanism of events which proceed
specific DNA binding and the activation of transcription. Previous
investigations of the thermodynamics of the interaction of CRP with
cAMP and cGMP showed that the binding exhibited negative cooperativity
at low ionic strength of less than 0.4 m (Takahashi et al.,
1980) and less than 0.1 M (Heyduk and Lee, 1989). However,
Takahashi et al.(1980) observed positive cooperativity at an
ionic strength In this
investigation, the cooperativity and thermodynamics of the binding
reactions of cAMP and cGMP to CRP and the T127L mutant are determined
in terms of the thermodynamic parameters of the binding constants (K
These thermodynamic parameters are determined as a function of
ionic strength and temperature from isothermal titration calorimetry
(ITC) measurements. It is shown that when considering the complete
thermodynamics of the binding reactions, there are, indeed, explicit
differences in the interaction of cGMP and cAMP with CRP.
where n is the stoichiometry, K
where dV Each titration calorimetry scan yields values for n, For ligand binding to two sites (Yang, 1990), the fraction
of protein bound with one ligand, F
and the fraction of protein bound with two ligands, F
Then the heat released per addition of titrant is fitted to using the four parameters K
If the sites interact, the coefficient of cooperativity,
and the energy of interaction,
For the titration scans that exhibited a maximum in the peak
areas after several additions of ligand solution which may be
indicative of a cooperative binding mechanism, the data could only be
fitted to using Q Values for
where R = 8.315 J mol
The cGMP binding results are presented first since they were
used to analyze the more complex cAMP binding data. A typical
calorimetric titration consisting of adding cGMP to CRP at 24 °C
and 0.5 M KCl is shown in Fig. 1a and a plot
of
Figure 1:
a, a calorimetric
titration of 5-µl aliquots of 8.0 mM cGMP into 0.2 mM of CRP in 0.5 M KCl phosphate buffer at 24.1 °C. b, the heat exchanged per mole of titrant versus the
ratio of the total concentration of ligand to the total concentration
of protein and the best least squares fit of the data to and in the text.
A typical calorimetric titration of
adding cAMP to CRP is shown in Fig. 2a along with its
Figure 2:
a, a calorimetric titration of 5-µl
aliquots of 8.0 mM cAMP into 0.15 mM of CRP in 0.5 M KCl phosphate buffer at 25.0 °C. b, the heat
exchanged per mole of titrant versus the ratio of the total
concentration of ligand to the total concentration of protein and the
best least squares fit of the data to and in
the text.
ITC data of the cyclic nucleotide
binding reactions to the T127L mutant are shown in Fig. 3along
with the corresponding fits, indicated by the solid curves, of for Q
Figure 3:
a,
the heat exchanged per mole of titrant versus the ratio of the
total concentration of ligand to the total concentration of protein for
a calorimetric titration of 5-µl aliquots of 5.5 mM cGMP
into 0.14 mM T127L in 0.5 M KCl phosphate buffer at
23.8 °C. The curve is the best least squares fit of the
data to and in the text. b, the heat
exchanged per mole of titrant versus the ratio of the total
concentration of ligand to the total concentration of protein for a
calorimetric titration of 5-µl aliquots of 6.7 mM cAMP
into 0.28 mM of T127L in 0.5 M KCl phosphate buffer
at 24.8 °C. The curve is the best least squares fit of the data to and in the text.
The values for The process by which the binding of cAMP to CRP switches on
specific DNA binding and subsequent activation or repression of
transcription is not completely understood. Previous investigations
(Eilen and Krakow, 1977: Takahashi et al., 1980: Heyduk et
al., 1992), which focused on determining differences between the
binding properties of cAMP and its non-functional analog cGMP to
elucidate the mechanism for specific DNA binding by CRP, were
inconclusive since the binding properties of both cyclic nucleotides
were essentially very similar. However, Ebright et al. (1985)
showed that there are differences between cAMP ligated CRP and cGMP
ligated CRP exhibited by differences in the action of proteases, in the
binding specifically to DNA or poly(dA-dT) sequences, and on the level
of the activation of transcription. Ebright et al. (1985)
proposed that a change in CRP conformation occurs upon cAMP binding
which initiates specific DNA binding. The results presented in this
investigation show that there is indeed a profound difference between
the thermodynamics of cAMP and cGMP binding to CRP. The binding of
the non-activator cGMP to CRP is exothermic, non-cooperative, and
driven by an increase in the entropy of the protein-ligand-solvent
system. Although, the thermodynamic binding parameters of the first
molecule of cAMP to CRP are approximately the same as for cGMP binding
to CRP, the overall binding of cAMP is endothermic and is best
described by an interactive two-site binding model with positive
cooperativity between the two binding sites. This results from the
atypical thermodynamic parameters of the second binding reaction of
cAMP to CRP, Differences in the thermodynamics between cAMP and cGMP binding to
CRP show that the amino group in position 6 of the purine ring of cAMP
is important, not only for specific recognition of this ligand, but for
inducing the cooperativity between the ligand binding sites. The N-6
amino group of cAMP forms a hydrogen bond to the hydroxy group of
Thr-127 of CRP. The point mutation T127L, not only eliminates hydrogen
bond formation between the 6-NH The
point mutation at Thr-127 on CRP also changes the specificity of the
protein: the mutant activates transcription only upon binding of cGMP,
but to a lesser extent than the ligated cAMP Finally, all the
binding reactions of the cyclic nucleotides to CRP and the mutant are
mainly entropically driven at 24 °C. The cyclic nucleotides are
anions at neutral pH and as charged species in water, they increase the
ordered structure of water through the formation of hydration shells.
The observed increase in the entropy would result from a loss of the
ordered water structure of the cyclic nucleotide anion upon binding to
CRP or its mutant. This is expected to be the same for both cyclic
nucleotides and both proteins since differences in their structures are
small.
Volume 270,
Number 37,
Issue of September 15, pp. 21679-21683, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
H) of -6 kJ
mol
, a heat capacity change
(
C) of -0.300 kJ
mol
K
, and an entropy increase
(
S) of 72 J mol
K
, the overall binding of cAMP to CRP is endothermic
and positively cooperative: binding of the first cAMP molecule
increases the affinity for the second one by more than an order of
magnitude at 24 °C. The binding of the second cAMP molecule is
accompanied by large changes of 48.1 kJ mol
in
H, of -1.4 kJ
mol
K
in
C, and of 255 J mol
K
in
S at 24
°C and 0.5 M KCl phosphate buffer. In contrast, the
overall binding of cGMP to CRP is exothermic and non-cooperative with
H,
C, and
S values close to the those values for binding of the first
cAMP molecule to CRP. The point mutation, T127L, switches off the
cooperativity between the cAMP ligated binding sites without affecting
the binding constant of cAMP and changes the specificity of the protein
so that transcription is now activated only upon cGMP binding. All the
binding reactions to CRP and the mutant are mainly entropically driven
at 24 °C.
)from Escherichia coliis a global transcriptional regulatory
protein. In the presence of cAMP, CRP undergoes a conformational change
and binds to specific DNA sequences in the regulatory regions of a
number of operons to activate or repress transcription (de Crombrugghe et al., 1984; Garges and Adhya, 1985). The global alteration
of the CRP structure upon cAMP binding is evident from small angle
x-ray scattering measurements (Kumar et al., 1980), analytical
gel chromatography (Heyduk et al., 1992), proteolysis
digestion studies (Ebright et al., 1985; Moore, 1993), and
differential scanning calorimetry (DSC) where DSC scans of CRP exhibit
one unfolding transition for CRP and three unfolding transitions for
cAMP ligated CRP (Ghosaini et al., 1988).
cAMP
DNA
complex which showed that the fully ligated CRP dimer interacts
directly with 27 of 30 base pairs of duplex DNA. According to the x-ray
structure of the cAMP ligated CRP, hydrogen bonds exist between the
ribose and phosphate groups of cAMP and the Glu-72, Ser-83, and Arg-82
amino acid residues of CRP, and between the N-6 amino group of the
purine ring of cAMP and T127 of the same subunit and Ser-128 of the
other subunit. The hydrogen bonds between the cyclic nucleotide and
Trp-127 and Ser-128 are particularly important since they, potentially,
determine the ligand specificity and participate in intersubunit
communication. Biochemical and genetic studies support this model
(Moore, 1993; Moore et al., 1992; Ebright et al.,
1985).
0.4 M and at low ionic strength in the
presence of DNA. Takahashi et al.(1980) also reported that the
binding constants for cGMP binding to CRP were very similar to those of
cAMP and estimated a binding enthalpy of -6.0 kJ mol for cAMP from the dependence of the binding constant on
temperature. Fried and Crothers(1984) showed that at low ionic
strength, the transfer of CRP from the specific binding site on DNA to
a nonspecific site is accompanied by the net release of one cAMP
molecule. Later, Heyduk and Lee(1989) suggested from CRP-specific DNA
binding studies in the presence of cAMP that CRP bound with only one
molecule of cAMP is active in specific DNA binding.
) and changes in the free energy
(
G
), enthalpy
(H), entropy
(
S), and heat capacity
(
C), for the following
reactions,
Materials
The characterization and mutagenesis
of the wild type and mutant of CRP has been described previously by
Moore(1993). A New Brunswick Fermentor Scientific, Inc. ()SF116 was also used in place of the 5-liter shaker flasks
for preparations of CRP and the T127L mutant. The protein solutions
were dialyzed at 4 °C in 50 mM potassium
phosphate-potassium hydroxide buffer (pH 7.0) containing 0.2 mM sodium EDTA, 0.2 mM dithiothreitol, and 5% glycerol
(phosphate buffer) with either 0.2 or 0.5 M KCl with two
changes in the buffer solution. A Pharmacia Phast SDS electrophoresis
gel with 0.1 M Tris-HCl buffer (pH 7.0) containing 0.2%
bromphenol blue and 20% glycerol was run to determine the purity of the
protein samples. A protein analysis was also performed by comparing the
optical densities at 562 nm of samples of the solution to those of
bovine serum albumin solutions after adding 4% CuSO
and
bicinchoninic acid reagent from the Pierce Chemical Co. to the samples.
The concentrations of the CRP and the mutant were determined by UV
absorption spectroscopy using an extinction coefficient of 3.5 
10 cm M at 280 nm (Ghosaini et al., 1988). A DSC scan from 25 to 100 °C of the protein
solutions performed with a Hart 7707 DSC heat conduction scanning
microcalorimeter exhibited only one thermal transition at 65 °C for
the CRP in agreement with Ghosaini et al.(1988) and one
transition at the same temperature for the mutant. The ligand solutions
were made up by dissolving the sodium salt of the cyclic nucleotide in
the second dialysate solution so that the ligand solvent was the same
as the protein solvent. The concentrations of the ligand solutions were
determined by using an extinction coefficient of 1.23
10 cm M at 260 nm for cAMP and 1.34
10 cm M at 250 nm for
cGMP (Lang, 1966). The sodium salts of cAMP and cGMP, potassium
phosphate, NaOH, EDTA, dithiothreitol, glycerol, Tris, HCl, bromphenol
blue, and KCl were reagent grade from Sigma.
ITC Measurements
The titration calorimetry
measurements were performed with a Microcal Omega titration calorimeter
as described previously by Wiseman et al.(1989) and Schwarz et al.(1991). Briefly, the titration calorimeter consists of a
1.374-ml cell containing the protein solution and a matched reference
cell containing the dialysate in an adiabatic enclosure. Aliquots of
the ligand solution are added via a rotating stirrer-syringe operated
with a plunger driven by a stepping motor. The heat absorbed or
released accompanying the addition of aliquots of the ligand solution
to the protein solution is measured by a thermocouple sensor between
the cells. Samples were centrifuged prior to the titration and were
carefully scrutinized for precipitant after the titration. No
precipitant was observed after the titration even at the lower ionic
strength of 0.2 M KCl. A typical titration consisted of
injecting 5-10 µl aliquots of 2-10 mM of the
ligand solution into 0.05 to 0.4 mM of the protein solution
every 4 min to insure that the titration peak returned to the baseline
prior to the next injection. Aliquots of the more concentrated ligand
solutions were injected into just the dialysate solutions in separate
ITC runs to determine if the more concentrated ligand solutions
exhibited a heat of dilution. At the highest concentrations up to 10
mM, no heat of dilution was observed upon addition to the
buffer solution. The titrations were performed at ionic strengths of
0.2 M and 0.5 M KCl at 24 °C and at 0.5 M KCl at 39 °C in phosphate buffer (pH 7.0).ITC Data Analysis
For analysis of the exothermic
scans which exhibited a continuous monotonic decrease of titration peak
area with addition of titrant, first, a single site data fitting model
was used with a site concentration = 2 CRP
concentration. The total heat content, Q
, is
related to the total ligand concentration,
, via the following equation (Wiseman et al., 1989),
is
an intrinsic binding constant, H is an
intrinsic heat of binding, [CRP] is the total
site concentration, and V is the cell volume. The expression
for the heat released per the ith injection, Q(i), is
then (Yang, 1990),
is the volume of titrant added to
the solution.H, and since the site
concentrationis used, the intrinsic binding constant, K. These values were then used in the two
identical interactive site fitting model described below where the
concentration of sites is replaced by the concentration of
protein., where
[L] is the free ligand concentration is from
Equations 1-3,
, is,
, K,
H, and
H, where H are the enthalpy changes of binding for the two binding reactions (). If the sites do not interact,
the binding constants are related to the intrinsic binding constant, K
, in as
follows;
,
is,
G(),
is,
from .
To facilitate the fitting of the interactive two-site model to the
titration data, the initial fitting parameters of K and
H were chosen to be the same as
for binding of cGMP to CRP.G and S were determined from the fundamental equation of
thermodynamics,
K
and T is the absolute temperature.
The heat capacity changes,
C, were
determined from a linear of
H to T.
Values of the binding constants at 39.5 °C were also determined
using the
H and K
values at 24.0 °C (T
) and the
C values using the van't Hoff
equation.
Q(i) versus the ratio of
[L]/[CRP] in Fig. 1b. A least squares fit of the data to using Q determined from and the site concentration is shown by the solid curve in Fig. 1b. An identical fit was obtained using Q determined from and the protein
concentration, indicating that the two binding sites do not interact.
The thermodynamic parameters for the cGMP-CRP binding reactions are
presented in Table 1. Each parameter for all the binding
reactions in Table 1is an average determined from at least two
different titration runs with different ligand and protein
concentrations. Both cGMP-CRP binding reactions () are exothermic and are mainly
driven by the increase in entropy. Agreement between the binding
enthalpy and entropy at 0.2 and 0.5 M KCl ionic strength
indicates that the binding reaction is independent of ionic strength
over this range of KCl concentration. For cGMP, the binding enthalpy
decreases with increase in temperature and the heat capacity changes,
C and
C, are
-0.300 ± 0.015 kJ mol
K
.
Q(i) versus [L]/[CRP] plot in Fig. 2b. Contrary to the binding of cGMP
to CRP, the cAMP-CRP binding reaction is endothermic and appears to be
cooperative. The ITC data could only be fitted to the interacting
two-site model described by as shown by the
Q(i) versus [L]/[CRP]in Fig. 2b. The thermodynamic parameters
determined from these fits are presented in Table 1. The first
binding reaction of cAMP to CRP is exothermic and very similar to that
for cGMP binding to CRP with, however, twice the intrinsic binding
constant as shown in Table 1. As in the case of cGMP binding, the
intrinsic binding constant is independent of ionic strength from 0.2 to
0.5 M KCl. In contrast to the binding of cGMP to CRP, the
second cAMP binding reaction is strongly endothermic, resulting in the
overall endothermic nature of the total two-step binding reaction. Both
binding reactions are mainly entropically driven with a much greater
S than
S. The binding of the first cAMP
molecule increases the affinity of CRP for the second cAMP molecule by
more than an order of magnitude at 0.2 M KCl and 24 °C,
yielding a cooperativity parameter, of 11.7 (Table 2). Similar
results were obtained at pH 8.0 with Tris as a buffer, conditions
similar to those of Takahashi et al. (1980). This
cooperativity parameter is reduced to 4.8, at 0.5 M KCl ionic
strength but does not depend on temperature from 24 to 39 °C. The
binding enthalpy for both binding reactions decreases with increase in
temperature. The heat capacity change for the first cAMP binding
reaction to CRP, C
, is close to
that for cGMP binding to CRP, while the heat capacity change for the
second cAMP binding reaction to the CRP,
C, is at least a factor of four
more negative (Table 2).
and the site concentration.
Identical fits were obtained using for Q and the protein concentration, indicating that the two binding
sites are independent. The thermodynamic parameters for the cAMP and
cGMP binding reactions to the mutant are presented in Table 1and Table 2. All the binding reactions to the mutant are exothermic,
non-cooperative, and mainly entropically driven. The substitution of
Thr-127 by leucine alters the interaction of cAMP with CRP. In spite of
little change in K and the other thermodynamic
parameters of binding of the first cAMP molecule to CRP, this point
mutation completely switches off the cooperativity of the cAMP-CRP
binding reaction. For both cAMP-mutant binding reactions, the binding
enthalpy decreases with increase in temperature yielding a heat
capacity change of C =
C = -0.47 ±
0.15 kJ mol K
, only slightly more
negative than for binding of the first cAMP molecule to CRP. However,
the heat capacity changes accompanying the cGMP binding reactions are
almost two times more negative,
C = C =
-0.76 ± 0.12 kJ mol K
.
H and K
at 24 °C in Table 1were
used along with the
C values to calculate
the binding constants at 39 °C using . The calculated
values were within experimental error of the K
values at 39 °C in Table 1.
H,
S, and
C, reflect a conformational
change in the fully cAMP-ligated CRP which would cause positive
cooperativity between the two sites. A cooperativity which is dependent
on ionic strength as reflected in the change in from 11.7 at low
ionic strength to 4.8 at high ionic strength but does not depend on
temperature from 24 to 39 °C. A subtle change in the conformation
could also result after binding of the first cAMP molecule to CRP. A
substantial amount of energy, e.g. 48.1 kJ mol at 24 °C and 0.5 M KCl, is absorbed during the
second binding reaction which is almost 10% of the energy needed to
unfold the protein at its denaturation temperature (547 ± 24 kJ
mol
at 66.4 °C and 0.5 M KCl, Ghosaini et al.(1988)). The amount of energy absorbed is less at the
lower ionic strength of 0.2 M KCl implying that electrostatics
have a role in this conformational change. The entropy incease for this
binding reaction is about 130 J mol
K
greater than for the first binding reaction as well as for cGMP
binding to CRP. This increase could result from a change in the
conformational contribution to the entropy. A conformational change
upon binding of the second cAMP is further reflected in the large heat
capacity change observed for this binding reaction of -1.47
± 0.17 kJ mol
K
as
compared to -0.30 kJ mol
K
for the first cAMP binding reaction and for both cGMP binding
reactions. Differences in the heat capacity changes have been related
to differences in the amount of surface area of the protein exposed to
water upon unfolding (Sturtevant, 1977) and upon ligand binding (Spolar
and Record, 1994). Additional evidence for a conformational change in
fully cAMP saturated CRP derives from a Raman spectroscopic study (Tan et al., 1991) on CRP and cAMP ligated CRP as well as from gel
chromatography (Heyduk et al., 1992). It is also apparent in
the multiplicity of the thermal transitions observed in DSC scans of
CRP fully saturated with cAMP (Ghosaini et al., 1989).
on cAMP and the protein,
but also completely switches off cooperativity in cAMP binding without
a large effect on the binding constant of cAMP to the mutant.CRP complex. Although
the binding reaction of cGMP to the mutant does not exhibit any
cooperativity, there is some evidence for a conformational change in
the presence of cGMP. The heat capacity changes accompanying both cGMP
binding reactions (-0.76 ± 0.12 kJ mol K
) are almost twice as great as for both cAMP
binding reactions to the mutant (-0.47 ± 0.15 kJ
mol
K
). In the presence of cGMP,
the mutant is digested by subtilisin and produces the same product
pattern as observed for cAMP ligated CRP (Moore, 1993), implying that
cGMP stabilizes the mutant in a conformation similar to that of fully
cAMP ligated CRP. This conformation may be necessary for specific DNA
binding and subsequent activation of transcription.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
