 |
INTRODUCTION |
The allosteric protein cAMP receptor protein
(CRP)1 is activated by the
binding of cAMP to enhance the transcription of over 25 genes, which
code for enzymes involved in carbohydrate metabolism in
Escherichia coli. The x-ray crystal structure of
cAMP-ligated CRP consists of two 210-amino acid residue subunits, with
the N-terminal domain of each subunit containing a central
-barrel connected to an
/
C-terminal domain via an
-helix, which also is the primary interface between the two subunits (1). The N-terminal
regulatory domain
-strands form an eight-stranded
-barrel that
binds anti-cAMP, and the C-terminal domain contains a
helix-turn-helix motif followed by a small
-sheet that binds to the
promoter site. One subunit (A) has a "closed" form, where the
C-terminal domain is swung in toward the
-helical interface, and the
other subunit (B) has an "open" form, where the C-terminal domain
is swung away from the interface (1). In addition to interactions
between the CRP binding site and the phosphate ribose of cAMP,
Thr127 at the
-helical interface forms a hydrogen bond
with the N6 of the bound cAMP, and Ser128 forms
a hydrogen bond with the bound cAMP in the other subunit (1). The
nature of the conformational change to the allosterically activated
conformation has not been determined because the x-ray crystal
structure of unligated CRP is not known. Small angle neutron scattering
measurements on unligated CRP solutions show that the data best fit the
simulated scattering data from a minimized energy structure of CRP with
both units in the open form (2). Minimum energy calculations starting
with the x-ray structure of both anti-cAMP-ligated subunits
in the closed form and both subunits in the open form in solution show
that the minimum energy conformation of anti-cAMP-ligated
CRP is with both subunits in the closed form (3). In NMR measurements,
five sets of histidine resonances are observed for CRP alone and in its
complexes with cyclic nucleotides, which also implies that both
subunits are in the same conformation in solution (4). The x-ray
structure of the cAMP-ligated complex of CRP bound to a 30-base pair
DNA duplex, which has the same sequence as that of a promoter site,
shows that both subunits are in the closed conformation (5, 6). The
implication of these results is that the allosteric activation of CRP
to specifically bind to the promoter site involves conversion of both
subunits from the open form to the closed form in solution upon cAMP binding.
Mutants of CRP have revealed some interesting alterations of the
allosteric activation of CRP, which should exhibit some correlation with this change to the allosterically active conformation of CRP. The
T127L (7) and T127C (8) CRP mutants remove the specificity of
the activation of CRP by cAMP so that CRP is also activated by the
binding of cGMP. A CRP double mutant containing the T127L mutation and
a S128A mutation (CRP*) activates in vivo and in
vitro transcription in the absence of a cyclic nucleotide monophosphate (7, 9). The Thr127 mutations appear to alter
the conformation of CRP to a conformation close to that of the
allosterically activated CRP so that binding of the cAMP is not
necessary (10). This is supported by small angle neutron scattering
measurements on unligated and cAMP-ligated conformations of CRP* in
solution, which show that there is very little conformational
difference between the two species, in contrast to shrinkage of the
radius of gyration of CRP upon ligation to cAMP in solution (2).
In the present investigation, the x-ray crystal structure of
cAMP-ligated CRP* has been determined at 2.2-Å resolution. Although this structure is close to that of the x-ray crystal structure of
cAMP-ligated CRP with one subunit in the open form and one subunit in
the closed form (1, 11), an additional cAMP (syn-cAMP) is
clearly observed bound on the surface of the C-terminal domain of the
closed subunit. A similar syn-cAMP binding site on the two
closed subunits of a cAMP-ligated CRP-DNA complex was recently reported
by Passner and Steitz (12). Previously, only anti-cAMP bound
to the sites in the N-terminal domains had been observed in the x-ray
crystal structure of cAMP-ligated CRP (1, 11), although an interaction
between syn-cAMP and CRP in solution had been earlier
observed in NMR measurements on the cAMP-ligated CRP complex in
solution (4). The bound syn-AMP in the cAMP-ligated CRP-DNA
crystal structure, which does not appear in the cAMP-ligated CRP
structure alone, indicates that the final state of the CRP-DNA complex
is one where syn-cAMP is bound between the C-terminal domain
and the DNA. Thus, binding of a syn-cAMP to this site on the
closed subunit of CRP* in the absence of bound DNA would imply that
cAMP-ligated CRP* is in a conformation that is more accommodating for
DNA binding than that of cAMP-ligated CRP. Differences between the
conformations of cAMP-ligated CRP* and cAMP-ligated CRP are also
evident in the binding of cAMP. As the pH is decreased from 7.0 to 5.2, the binding mechanism undergoes a change from an exothermic to an
endothermic binding mechanism, an effect similar to that observed by
the T127L mutant but not with CRP (13), which retains its endothermic
cAMP binding mechanism over this pH range. The thermal denaturation of
CRP* exhibits a broad transition similar to the superimposed
multi-transition peaks observed for cAMP-ligated CRP but different than
that of CRP (14) and its S128A mutant (13), which exhibit a relatively
very narrow, single transition peak.
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EXPERIMENTAL PROCEDURES |
Materials--
The production from E. coli of CRP and
mutants and their purification have been described previously (7), and
their activities were checked by an in vitro transcription
assay as described by Zhang et al. (15). The concentrations
of CRP and CRP* were determined from UV measurements at 280 nm using an
extinction coefficient of 3.5 × 104
M
1 cm
1 (16). The glycerol,
potassium phosphate salts, KCl, HEPES, Tris, MgCl2,
and sodium salt of cAMP were reagent grade from
Sigma.2 The dithiothreitol
was Ultra Pure brand from Life Technologies, Inc., and the
NaEDTA was from Serva Co. 4K polyethylene glycol was obtained
from Fluka.
Crystallization--
CRP* was concentrated to 10.8 mg/ml in 10 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 5% (v/v)
glycerol, 0.5 M KCl, and 0.2 mM dithiothreitol. Diffraction quality crystals were grown in hanging drop vapor diffusion
experiments in which the protein solution was mixed with an equal
volume of the reservoir solution containing 10% (w/v) 4K polyethylene
glycol, 7% (v/v) isopropanol, and 10 mM cAMP, 0.1 M HEPES, pH 6.1. The 10-µl hanging droplets were
equilibrated against 1 ml of the reservoir solution. Crystals grew
after ~1 week to a size of 0.2 × 0.3 × 0.1 mm.
X-ray Data Collection and Processing--
The crystal was picked
from a crystallization droplet using a CryoLoop (Hampton
Research) and dipped in a cryostabilizing solution containing 30%
(v/v) glycerol and placed directly in a nitrogen cryostream (Oxford
Cryosystems) operating at 100 K. Crystals were placed on a
Brüker Analytical x-ray systems, Inc. goniostat mounted on
a Brüker Analytical X-ray Systems, Inc. rotating anode x-ray
generator operating at 80 kV and 40 mA. The diffraction images were
collected with the HI-STAR electronic area detector placed 13 cm from
the crystal positioned with 2
angles to intercept diffraction data
from
to ~1.8 Å resolution. The electronic images were collected
using the Brüker FRAMBO program and were processed using the
X-GEN program package (Molecular Simulations Inc.). The crystal was
orthorhombic with space group P212121 and unit cell dimensions
a = 46.1 (Å), b = 93.1 (Å), and c = 104.4 (Å). A total of 177,444 observations were
reduced to 38,054 unique reflections with the
Rmerge = 0.088 with a redundancy of 4.7 at 1.82 Å, the completeness to 97.7% to 2.0 Å resolution. The average
reflection I/
values were 5 and 2 at 2.69 and 2.22 Å resolution, respectively.
Crystallographic Refinement--
The initial phases for the
structure determination were obtained from an automated
molecular replacement (17) molecular replacement solution using a
previously reported CRP structure (11), Protein Data Bank (18)
identifier 3GAP, which has the same space group but different unit cell
parameters, a = 46.5 (Å), b = 96.7 (Å), and c = 105.3 (Å). Rigid body refinement was applied to the molecular replacement solution followed by maximum likelihood torsion angle refinement with a simulated annealing protocol
using Crystallography and NMR System Program version 0.9a (19).
The resulting structure had an r of 0.336 and an Rfree of 0.379. The phases from this model were
used with the automated refinement package ARP/wARP (20) to continue
the maximum likelihood refinement. The resulting model
(r = 20.7 and Rfree = 32.1) was
subsequently refined using Shelx97 (21) to r = 0.227 and Rfree = 0.300. A final refinement was
carried out that included all reflections even the reflections excluded
for Rfree to generate the final model with an
r = 0.228. The same set of reflections was used for
Rfree monitoring for different refinement
packages so that no bias in Rfree values was introduced.
Structure Analysis--
The electron density maps were
visualized, and the model was adjusted/built according to the density
using TURBO-FRODO (22). The stereochemistry of the final model was
checked using PROCHECK (23), and the alignments of the structures used
in comparison were done using ALIGN program (24). Molecular images for
the figures were generated using programs MolScript (25) and Raster3D (26).
Isothermal Titration Calorimetry--
All calorimetric
titrations were performed according to the methods of Wiseman et
al. (27) and Gorshkova et al. (28) using a Microcal
Omega titration calorimeter. The Omega titration calorimeter consists
of a sample vessel (1.374 ml) containing the protein solution and a
matched reference vessel (1.374 ml) containing the buffer solution.
4-10-µl aliquots of the ligand solution at concentrations 10× to
20× the protein concentration of 0.05-0.1 mM in the
sample vessel were added 3-4 min apart. A separate titration of the
ligand solution into the buffer was performed to determine any ligand
heat of dilution, which was then subtracted from the heats obtained
during the titration of the ligand 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 (29)
|
(Eq. 1)
|
where V is the volume of the sample solution. For an
identical independent two-site binding model
|
(Eq. 2)
|
where n = 2, the stoichiometry of the binding
reaction, [Ct] is the total CRP concentration
in the sample vessel,
Hbo is the binding
enthalpy, [Xt] is the total cAMP
concentration, and Kb is the binding constant. For
the interacting two-site model,
|
(Eq. 3)
|
where
|
(Eq. 4)
|
Kb (1) and
Hbo
(1) are the binding constant and the enthalpy for binding to the first
site, and Kb (2) and
Hbo (2) are the binding constant and
enthalpy for binding to the second site. The Origin program yields the
on-site binding constants and, thus, the binding constants reported in
this paper are the on-site binding constants. The macroscopic binding
constants to the first site and to the second site are, respectively,
2Kb (1) and Kb (2)/2 where for
the identical site model, Kb (1) = Kb (2). The binding entropies,
Sbo, were calculated using the following
equation of thermodynamics:
|
(Eq. 5)
|
The combined standard uncertainties in the Kb
and in
Hbo were estimated to be each
1.1% from standard uncertainties in [Ct],
[Xt], Q, and V. This
accounts for the S.D. in the mean value of
Hbo from several titration scans.
However, this combined estimated uncertainty is less than the S.D. in
the mean values of Kb from several titration runs as
shown in Table I.
Differential Scanning Calorimetry--
All DSC measurements were
performed using a Hart 7707 DSC heat conduction scanning
microcalorimeter as described by Schwarz and Kirchhoff (30). The Hart
DSC consists of three removable vessels containing the protein solution
and a fourth removable vessel containing just the buffer solution in an
adiabatic enclosure. The sample size was 0.500 g, and the samples were
scanned at a typical scan rate of 15 K h
1 from 30 to 90 °C. The software program EXAM (31) was used to subtract the
thermal power versus temperature scans of buffer versus buffer from the solution versus buffer
scans and to divide the subtracted scans by the scan rate to obtain the
net heat capacity versus temperature scans. EXAM was used to
extrapolate a sigmoidal base line under the transition peak and to fit
a two-state transition model to the data points to obtain the
transition temperature (Tm, the temperature at
half the peak area), a van't Hoff enthalpy (
trsHv) for the transition and an
area for the transition peak. The transition peak area divided by the
number of moles of protein in the sample yielded the calorimetric
enthalpy,
trsHc.
The standard uncertainty in Tm determined from
imprecision in the temperature readings and imprecision in the
fractional areas under the transition peak is estimated to be ± 0.1 K. The combined estimated uncertainty in the van't Hoff enthalpy
from imprecision in the fractional area under the transition peak is
3%. The combined estimated uncertainty in
trsHc contains uncertainty
contributions from the area under the transition peak, the
concentration of protein, the sample mass, and the heat calibration of
the DSC and is 3.2%. As shown in Table
II, the S.D. of the mean values of
trsHv and
trsHc is greater than the
combined estimated uncertainties of these values.
 |
RESULTS |
Three-dimensional Structure of CRP*--
The final model of the
CRP* structure contains 208 and 205 of the possible 210 residues for
the closed subunit A and the open subunit B, respectively. There are
216 water molecules and three cAMP molecules associated with the two
subunits in the asymmetric unit of the crystal. Each subunit of the
CRP* molecule is composed of two domains, the C-terminal DNA-binding
domain with a helix-turn-helix motif together with a four-strand
-sheet and the N-terminal domain composed of
-strands forming a
-barrel motif that binds anti-cAMP and a long helix
forming the dimer interface. Fig. 1 shows
the overall structure of CRP* with the bound cAMP molecules. The two domains are connected via a flexible hinge comprised of residues Phe136-Thr140. Each subunit has ~10 residues
at both the N and the C termini with weak electron density and
correspondingly high temperature factors. The residues near
Pro155 and Met163 in the closed conformation
(subunit A) have weak electron density; thus, their interpretation is
tenuous, whereas the same regions were clearly interpretable in the
open conformation (subunit B). The density for two anti-cAMP
molecules bound in the N-terminal regulatory domains of the subunits
and an additional syn-cAMP at the hinge region were clearly
observed. From the PROCHECK (23) analysis, the overall geometry of the
polypeptide chain were within acceptable limits, and the Ramachandran
plot (32) shows that 94.1% of the residues are within the most favored
region and the remaining residues in the additionally allowed
regions.

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Fig. 1.
Overall schematic representation of the CRP*
molecule where green is the subunit in the closed and yellow is the
subunit in the open conformation. A and B
are orthogonal views of each other. The cAMP ligands are shown as
ball-and-stick models. N and C denote the
termini, and h indicates the hinge region.
|
|
In the comparison of the two subunits of the CRP* in Fig.
2, only the N-terminal domain of each
subunit was used in alignment, because the N-terminal domains of both
subunits are nearly identical, and the resulting rotation and
translation matrixes were applied to the entire subunit. The apparent
difference between the open and closed conformations is observed in the
C-terminal domain and seems to result from rotation of the domains
around residue 128. Alignment of just the C-terminal domains of each
CRP* subunit showed that the two C-terminal structures alone were
nearly identical.

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Fig. 2.
Comparison of the two subunits of CRP* where
green is the closed subunit and yellow
is the open subunit. Two relative positions of the
cAMP molecules are shown and the CA atom of the residue
Ala128 is shown (CA 128).
|
|
Comparison with the Wild Type--
The structure reported here for
CRP* is the first CRP structure that includes the additional binding
site occupied by syn-cAMP in the closed subunit in the
absence of DNA. The initial concentration of cAMP is 5.0 mM, which is higher than the concentration of 0.5 mM used in the crystallization of CRP (11). Fig.
3 shows a comparison between the subunit
structures of the 3GAP wild type (11) and the CRP* structures using the
N-terminal domains as the reference because the structures of the
N-terminal domains of wild type CRP and CRP* subunits are nearly
identical. A comparison of the structure of the C-terminal domain of
the CRP* closed subunit with the structure of the C-terminal domain of
the 3GAP closed subunit does not show any significant differences
between the two domains. The regions showing any small differences
between the C-terminal domains of the closed subunits of CRP* and 3GAP contain residues with poor electron density. The largest difference between the overall CRP* and the 3GAP structures is observed in the
open subunits near the anti-cAMP binding site at residue 128. This difference, caused by the presence of a water molecule (discussed below) in the binding pocket at residue 128, swings the
C-terminal domain of the open subunit in CRP* closer in to the
N-terminal domain.

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Fig. 3.
Coordination of anti-cAMP by
CRP and CRP*. The yellow trace is the 3GAP structure
(11), and the blue trace is the CRP* structure. The
Ala128 residue responsible for the different orientation of
the open subunit of the CRP* structure is indicated by the
sphere in the C -helix. The syn-cAMP observed
in only the CRP* structure is shown at the top of the
structures.
|
|
Cyclic AMP Binding Sites--
In a comparison of the two
anti-cAMP binding sites in the regulatory N-terminal domains
with other structures (5, 6, 11) in Fig.
4, the only observable differences in the
anti-cAMP binding site are at the mutation sites. In the
wild type CRP molecule, the N6 atom of the purine ring of
cAMP is coordinated by OG1 atom of Thr127 and OG
atom of Ser128 residues. However, in the CRP* structure,
these residues are changed to leucine and alanine, respectively,
resulting in a loss of the hydrogen bonding interactions between the
protein and the cAMP. Partially compensating for the loss of these
interactions is a water molecule bound at the position of the OG atom
of the wild type Thr128 residue coordinating the
N6 atom of cAMP. To accommodate this water molecule, the
residues near Ala128 of the mutant protein have shifted
resulting in flexibility of the C
-helix that is responsible for the
difference in the C helices between the wild type and the mutant shown
in Fig. 3.

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Fig. 4.
Comparison of the anti-cAMP
binding sites of the wild type CRP 1BER (6), 2CGP (12), and 3GAP (11)
structures in light gray backbone tracings with that
of CRP* in the dark opaque backbone tracing. The
two cAMP molecules and the water molecules in red are from
the CRP* structure.
|
|
In CRP*, the syn-AMP binding site is only observed in the
closed subunit and is composed of the residues Gly56 and
Lys57 to Met59 and Gln170,
Gln174, and Gly177 to Arg180 (Fig.
5). The nitrogen atoms, N and NE, from
Arg180 coordinate the two axial oxygen atoms of the
phosphate group of the syn-cAMP. Additional interactions are
observed between an axial oxygen atom of the ligand and the NE2 atom of
Gln170. The N6 atom of the purine ring of the
syn-cAMP interacts with the backbone carbonyl O atoms from
the residues Gly177, Ala135 and
Phe136, where Ala135 and Phe136 are
from the hinge region of the other subunit. The O2* atom of
the ribose of syn-cAMP interacts with the N atom of
Phe58. The same region in the open subunit could not
accommodate a syn-cAMP molecule because residues
Gly177 and Cys178 would sterically hinder
binding. In a comparison of the closed subunit of the CRP* structure
with the 2CGP structure from Passner and Steitz (12), very few
differences were observed in the N-terminal regulatory domains and only
small differences were observed in the four
-strands in the
C-terminal DNA-binding domains. The same residues interacting with the
syn-cAMP in the two closed subunits of the 2CGP structure
are also observed to interact with the syn-cAMP in the
closed subunit of CRP*. When DNA is bound to the CRP molecule, the
syn-cAMP molecule is positioned to interact with the
backbone of the DNA in 2CGP (12).

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Fig. 5.
Comparison of the residues of the
syn-cAMP binding site with the lighter tracing
for the 2CGP (12) structure and the darker tracing for the CRP*
structure. The atoms involved in the interaction between the
protein and the ligand are as indicated, and the bound DNA in the 2CGP
structure is not shown.
|
|
Isothermal Titration Calorimetry Scans--
A comparison of the
binding isotherms in Fig. 6 shows that
the cAMP binding reaction to CRP* changes from an exothermic binding process at pH 7.0 to an endothermic binding process at pH 5.2. This
unique allosteric dependence on pH was also observed for the T127L
mutant but not with the wild type CRP and its S128A mutant (13). The
solid lines in the binding isotherms indicate that the
binding of cAMP to CRP* at pH 7.0 follows an identical, independent
two-site mechanism as observed for S128A at both pH levels,
while at pH 5.2, the binding mechanism follows an interacting two-site
binding mechanism, as observed for CRP at both pH levels (13). Each
solid line generated by the binding mechanism (Equations 1-3) fitted
quite well to the binding isotherms as shown in Fig. 6, although they
were performed without considering the third cAMP binding site in CRP*.
Apparently, there is either a very small contribution of the binding
enthalpy from this third site or the binding affinity is much weaker
than to the cAMP binding sites in the N-terminal domains. The binding
parameters for CRP* and CRP are presented in Table I. At pH 5.2, CRP*,
like that of CRP at pH 7.0 (28), the interacting binding site mechanism exhibits positive cooperativity in that binding to the second site is
enhanced by binding to the first site. At pH 5.2, CRP, however,
exhibits negative cooperativity, where binding to the second site is
reduced by binding to the first site. The change in the cAMP binding
mechanism to CRP* from an exothermic binding mechanism at pH 7.0 to an
endothermic binding mechanism at pH 5.2 undoubtedly results from
protonation of amino acid residues distributed on the surface of CRP*.
The most likely amino acid residues that would protonate in this 5-7
pH range are the six histidine residues distributed on the surface of
each subunit. In contrast, CRP retains its endothermic binding
mechanism, and S128A retains its exothermic binding mechanism at both
pH levels and, thus, protonation of the amino acid residues on the
surface of the protein has little effect on the cAMP binding mechanism. The effect of protonation on CRP* is apparently coupled to the conformational changes on the surface of the C-terminal domain that
also induce binding of the syn-cAMP ligand to the surface of
the closed subunit.

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Fig. 6.
Binding isotherms from isothermal titration
calorimetry measurements at 24 °C on the
binding of cAMP to CRP* at pH 7.0 with 5-µl
aliquots of 2.6 mM cAMP titrated into 0.14 mM
CRP* (a) and at pH 5.2 with5
µl aliquots of 10 mM cAMP titrated into
0.15 mM CRP* (b). The molar ratio is
the number of moles of cAMP per number of moles of CRP* in the sample
vessel.
|
|
DSC Scans--
Typical DSC scans of CRP and CRP* are presented in
Fig. 7, which were scanned at a
concentration of 0.16 mM in 50 mM potassium phosphate buffer containing 0.5 M KCl and a scan rate of 15 K h
1. The transitions did not reappear upon a rescan of
the samples, but the transition temperature and enthalpies were
independent of scan rate from 5 K h
1 to 30 K
h
1. This justified applying a two-state reversible
thermodynamic transition model to the data to obtain the transition
quantities in Table II. Also, presented in Table II are the transition
quantities for the multipeak transitions observed for cAMP-ligated CRP
(14). As shown in Fig. 7, the transition peak observed for CRP* is
about twice as broad as that observed for wild type CRP and is
indicated by the ratio of the van't Hoff enthalpies of 1.6:1 for
CRP:CRP*. The broadness of the transition peak may be due to two
overlapping transitions in this temperature region, and in fact, the
transition could be resolved into a small transition with
trsHv = 800 kJ mol
1 and Tm = 62.1 °C and a
larger transition with
trsHv = 722 kJ mol
1 and Tm = 65.8 °C.
DSC scans of the T127L mutant at pH 5.2 exhibit two overlapping
transitions, whereas CRP exhibits only a single transition peak at this
pH (13). The broad transition of CRP* does confirm that the structural
interactions, such as domain-domain interactions, in CRP* are weaker
than those observed for the unligated wild type, where only a single
narrow transition is observed, but stronger than those observed for
cAMP-ligated CRP, where three transitions are observed. Thus, the
conformational changes induced by the mutations produce structural
interactions within the protein that approach those observed in the
allosteric-activated conformation of cAMP-ligated CRP.

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Fig. 7.
Comparison of the DSC scans for 0.18 mM CRP* and 0.18 mM CRP in phosphate buffer at
pH 7 and 0.5 M KCl. The areas under the transition
peaks are equal for the 0.5-g samples, and the scan rate is 15 K
h 1.
|
|
 |
DISCUSSION |
Because CRP* can be activated at low cAMP concentrations in
vivo and in vitro, the implication is that in solution,
CRP* is close to the allosteric conformation necessary for promoter
binding (13). This is supported by small angle neutron scattering
measurements, which exhibit little difference between the cAMP-ligated
and unligated CRP* conformations in contrast to the difference observed
between the cAMP-ligated and unligated wild type CRP conformations in solution (2). Although the overall x-ray crystal structure of
cAMP-ligated CRP* is very similar to that of the wild type protein (11)
with a root mean square deviation of 0.998 between the two structures
and with one subunit in the open conformation and the other in the
closed conformation, there are two subtle differences: (i) the closed
conformation of CRP* is observed with an additional syn-cAMP
bound in a site on the surface of the C-terminal domain and (ii) the
C-terminal domain in CRP* is shifted closer to the N-terminal domain
than in the corresponding open subunit of the cAMP-ligated wild type
CRP structure. Because the final conformation of CRP in the 2CGP
promoter-bound state (12) consists of both subunits in the closed
conformation with syn-cAMP bound at the same site now
observed in the CRP* structure, the conformation of cAMP-ligated CRP*
in the crystal state is closer to the promoter-bound state than that of
the cAMP-ligated CRP conformation in the crystal state.
The alanine substitution at 128 introduces a flexibility in the C
-helix that is responsible for reorienting the C-terminal domain
toward the N-terminal domain in the open subunit of CRP* (Figs. 2 and
3). There are water-mediated interactions between the N6 of
anti-cAMP and the Leu127 and Ala128
mutations, which replace the hydrogen bonding interactions between N6 and the OH groups of Thr127 and
Ser128. To accommodate the bound water molecule in CRP*,
the residues near Ala128 have shifted, resulting in the
flexibility of the C
-helix that is responsible for the observed
conformational difference between the open subunits of CRP* and CRP.
Because N6 is replaced by a carbonyl group in cGMP and
cIMP, these hydrogen bonding interactions are removed, and, indeed,
cGMP and cIMP do not allosterically activate CRP, which further
substantiates the importance of the interaction between cAMP
N6 and Thr127 and Ser128 in
shifting the conformation of CRP to the allosterically activated form.
Because these water molecules can also accommodate cGMP and cIMP in
CRP*, the specificity requirement for cAMP in the allosteric activation
of CRP can be fulfilled by cGMP and cIMP provided that the allosteric
conformational change is indeed induced by the flexibility in the C
-helix at Ala128. The tendency for water molecules to
maintain the structural integrity of ligated proteins that have been
mutated at the binding site has also been observed in lysozyme
antigen-Fv antibody complexes (33) where replacement of tryptophan in
the Fv binding site by smaller side chain amino acid residues induces
water molecules to fill the void to maintain the structural integrity
of the complex.
In another scenario, the conformational differences between
cAMP-ligated CRP* and cAMP-ligated CRP, which favor the active conformation can result from a shift in the C-terminal domain induced
by the binding of syn-cAMP near the hinge region of CRP*. Observations that mutations at the hinge residue 138 and D-helix residues 141 and 144, which result in activation of CRP in the absence
of cAMP, like that of CRP*, would indicate the direct involvement of
the hinge region and D-helix in the allosteric conformational change
(34-36). A close van der Waal's distance of 4.25 Å is observed
between the C2 atom of the cAMP purine ring and the
O3* atom of the bound DNA backbone at the
syn-cAMP binding site in the wild type cAMP-ligated CRP-DNA
complex (12). In addition, the N6 atom of the
syn-cAMP is tightly coordinated by the carbonyl O atom of
the Ala135 residue in this complex. Additional hydrogen
bonding interactions are provided by the carbonyl O atom of the
Gly177 residue. The absence of allosteric activation of CRP
by cGMP would result from lack of cGMP-binding to this site because the carbonyl oxygen on the C6 atom of the cGMP purine ring
would result in an unfavorable hydrogen bonding interaction between
cGMP and the O atom of the Ala135 residue. Because the
allosteric conformational change entails a shift in the hinge region
and the rest of the C-terminal domain in CRP*, it is possible that such
a large movement may result in the shift of the critical discriminating
Ala135 residue for cAMP specificity far enough away to
accommodate the otherwise unacceptable cGMP ligand in the
syn-cAMP binding site. The additional flexibility at the
Ala128 residue in CRP* would further favor a shift of the
hinge and the C-terminal domain. Therefore, the key discriminatory
interaction between the N6 atom of the cAMP and
Ala135 would be abolished in CRP* and, thus, either a cIMP
or cGMP can be accommodated for activation. In this scenario, the
anti-cAMP bound in the N-terminal domain of the CRP molecule
may not be directly involved in the allosteric conformational change
and thus may not be essential. In support of this scenario, there is
evidence that only a single cAMP ligand is necessary for the allosteric
activation of CRP (10, 37). However, the anti-cAMP ligands
may be essential for subunit assembly because in CRP subunit exchange
assays, Brown and Crothers (38) showed that CRP subunits did not
undergo exchange in the presence of cAMP with or without bound DNA.
Finally, it is possible that both scenarios would contribute to the
allosteric activation of CRP*.
The isothermal titration calorimetry results tend to confirm that the
conformation of CRP* is unique because a decrease in pH from 7.0 to 5.2 substantially alters the cAMP binding mechanism to CRP*, in contrast to
CRP and S128A, which retain their binding mechanisms throughout this pH
range. A similar unique dependence was reported for the T127L mutant
and attributed to conformational changes in the C-terminal domain,
which favor the closed conformation in the mutant (13). For CRP*, which
contains the T127L mutation and an additional S128A mutation, the x-ray
structure shows that the conformational difference is induced by the
flexibility at Ala128 and/or the binding of
syn-cAMP in the hinge region. The DSC results are also
indicative of a conformation for unligated CRP* in solution that is
intermediate between that of unligated CRP and cAMP-ligated CRP,
i.e. the thermal unfolding of CRP* consists of a broad or two transitions, whereas that of CRP and S128A consists of one narrow
transition and that of cAMP-ligated CRP consists of three transitions.
These differences in the thermal unfolding of CRP*, CRP, and
cAMP-ligated CRP would imply that the intramolecular interactions of
CRP* in solution are intermediate between those of CRP and cAMP-ligated
CRP.