(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
(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.
The cyclic AMP receptor protein (CRP) ()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).
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 CRPcAMP
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).
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 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.
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) and changes in the free energy
(
G
), enthalpy
(
H
), entropy
(
S
), and heat capacity
(
C
), for the following
reactions,
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 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),
where dV is the volume of titrant added to
the solution.
Each titration calorimetry scan yields values for n, 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.
For ligand binding to two sites (Yang, 1990), the fraction
of protein bound with one ligand, F, where
[L] is the free ligand concentration is from
Equations 1-3,
and the fraction of protein bound with two ligands, F, is,
Then the heat released per addition of titrant is fitted to using the four parameters K, 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;
If the sites interact, the coefficient of cooperativity, ,
is,
and the energy of interaction, G(
),
is,
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 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.
Values for
G
and
S
were determined from the fundamental equation of
thermodynamics,
where R = 8.315 J mol 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.
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 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
.
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
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).
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 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
.
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 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.
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, 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).
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 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.
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 cAMPCRP 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.
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.