From the Department of Human Biological Chemistry and Genetics,
University of Texas Medical Branch at Galveston,
Galveston, Texas 77555-1055
The molecular events in the cAMP-induced
allosteric activation of cAMP receptor protein (CRP) involve
interfacial communications between subunits and domains. However, the
roles of intersubunit and interdomain interactions in defining the
selectivity of cAMP against other cyclic nucleotides and cooperativity
in ligand binding are still not known. Natural occurring CRP mutants
with different phenotypes were employed to address these issues.
Thermodynamic analyses of subunit association, protein stability, and
cAMP and DNA binding as well as conformational studies of the mutants
and wild-type CRPs lead to an identification of the apparently dominant roles of residues 128 and 141 in the cAMP-modulated DNA binding activity of CRP. Serine 128 and the C-helix were implicated as playing
a critical role in modulating negative cooperativity of cyclic
nucleotide binding. A correlation was established between a weak
affinity for subunit assembly and the relaxation of cyclic nucleotide
selectivity in the G141Q and S128A/G141Q mutants. These results imply
that intersubunit interaction is important for cyclic nucleotide
discrimination in CRP. The double mutant S128A/G141Q, constructed from
two single mutations of S128A and G141Q, which exhibit opposite
phenotypic characteristics of CRP
and
CRP*, respectively, assumes a CRP* phenotype
and has biochemical properties similar to those of the G141Q mutant.
These observations suggest that mutation G141Q exerts a dominant effect
over mutation S128A and that the subunit realignment induced by the
G141Q mutation can override the local structural disruption created by
mutation S128A.
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INTRODUCTION |
The expression of many genes involved in different cellular
functions in Escherichia coli is regulated by cAMP receptor
protein (CRP)1 and cAMP
(1-3). The molecular events associated with the allosteric activation
of CRP are characterized by negative cooperativity in cAMP binding and
a differentiation among the various cyclic nucleotides present in the
cell. Although the CRP-cAMP system has served as a paradigm of
transcriptional regulation in procaryotes for many years, an
understanding of this allosteric activation event at the structural
level is still missing.
CRP is a 47,238-Da protein made up of two identical subunits, each of
which is composed of two domains connected by a hinge region. The small
carboxyl-terminal domain contains a helix-turn-helix DNA binding motif.
The large amino-terminal domain is responsible for cyclic nucleotide
binding (4). Biochemical and biophysical evidences show that binding of
cAMP allosterically induces CRP to assume a conformation that exhibits
a high affinity for specific DNA sequences (5-13). However, a
comparison between the active, monoliganded CRP and apo-CRP shows no
significant secondary structural changes. The only observable major
structural changes are associated with the doubly liganded CRP
(11-16). These observations imply that conformational changes during
CRP activation most likely involve rigid body movements between
subunits and domains without major structural changes or a change in
the dynamics of various structural elements without any significant
conversion of secondary structures. In CRP these rigid body movements
can be envisioned as subunit realignment and domain rearrangement that
are mediated by interactions involving the subunit and domain
interfaces, respectively. It has been shown that various locations in
the CRP molecule all respond quantitatively to the binding of cAMP
(17). These observations demonstrate that CRP responds to the binding
of cAMP in a global manner. A global conformational switch is
consistent with the proposed mechanisms involving either subunit
realignment and domain rearrangement or a change in structural
dynamics.
There is still no information to identify the structural elements in
CRP that are responsible for imposing negative cooperativity in cAMP
binding. How does CRP differentiate among the various cyclic
nucleotides? What structural element is responsible for establishing a
tight coupling between intersubunit and interdomain interactions? CRP
mutants generated by site-specific mutagenesis are employed to address
these outstanding issues. Studies of point mutations at residue 128 show that serine 128 is not vital for interdomain communication but
plays an important role in mediating the interactions between the two
subunits (18). In a recent study of mutation at residue 141, it was
shown that the Gly
Gln mutation differentially perturbs the two
interfacial interactions (19, 20). Structural and functional studies
reveal that the G141Q mutant assumes a conformation state that has a
realigned subunit interface. The G141Q mutant does not bind to specific DNA sequence without cyclic nucleotide (19). Complete activation of the
mutant requires binding of cyclic nucleotide, which induces the
reorientation of domains (20).
In this study, the role of residue 141 in defining cyclic nucleotide
specificity and the mutual influence of residues 128 and 141 in the
allosteric control of CRP toward binding to specific DNA are addressed.
The in vivo and in vitro functional properties of
mutant CRPs were monitored, with special attention to the subunit and
domain interactions in CRP.
 |
MATERIALS AND METHODS |
Chymotrypsin A and cGMP were purchased from Boehringer Mannheim.
A mutagenesis kit (Altered Sites in vitro Mutagenesis
System) was obtained from Promega, and a sequencing kit (Sequenase
version 2.0) was from U.S. Biochemical Corp. Subtilisin (protease type XXVII) and cAMP were purchased from Sigma. Ultrapure guanidine HCl was
a product of ICN Biochemical. MacConkey agar, bactotryptone, and yeast
extract were obtained from Difco. CPM, fluorescein 5-isothiocyanate, and IAF were purchased from Molecular Probes. Oligonucleotides were
synthesized by Genosys. Restriction endonucleases were from Promega,
Life Technologies, Inc., U.S. Biochemical Corp., or Boehringer Mannheim.
Bacterial Strains and Plasmids--
A detailed description of
the source and identity of the various bacterial strains and plasmids
can be found in Cheng et al. (18).
Methods--
All experiments, except as specifically indicated,
were conducted in buffer A (50 mM Tris, 0.1 M
KCl, and 1 mM EDTA, pH 7.8). The concentration of protein,
cyclic nucleotides, and fluorescence probes was determined by
absorption spectroscopy using the following absorption coefficients:
20,400 M
1 cm
1 at 278 nm for CRP
monomer (21); 14,650 M
1 cm
1 at
259 nm and 12,950 M
1 cm
1 at 254 nm for cAMP and cGMP (22), respectively; 30,000 M
1 cm
1 at 387 nm for CPM (23);
70,800 M
1 cm
1 at 494 nm for
fluorescein (24).
Site-directed Mutagenesis--
The G141Q point mutation was
generated as described previously (18-20). For constructing the double
mutant S128A/G141Q, the G141Q crp gene was subcloned into
the unrepaired pALTER-1 vector. The same protocol described above was
then used for the second round of mutagenesis. With the new Promega
Altered Sites II in vitro Mutagenesis System, multiple
rounds of mutation can be introduced into the same gene without
subcloning.
Lac Operon Activation in Vivo--
To test the effects of
mutation on the lac operon expression, E. coli
CA8445/pPRK248cIts, transformed with plasmid pPLc28 that
contains the appropriate crp mutant gene, was streaked on
MacConkey lactose indicator plates in the absence or presence of 0.5 mM cAMP or cGMP. The plates were incubated at 37 °C
overnight. The fermentation response of the mutant was scored according
to the color of the colonies on the plates. A CRP+
phenotype displays purple colonies only in the presence of cAMP, while
a CRP* phenotype is defined as an amino acid substitution
that yields purple colonies on MacConkey lactose plate
(Lac+) in the absence of external cAMP or in the
presence of cGMP. A CRP
phenotype is a mutant that always
shows white colonies (Lac
) on MacConkey
plates.
Protein Purification--
Wild type CRP was purified from
temperature-induced E. coli K12
H1/pPlcCRP1 cells grown
in LB (17). Mutant CRP was isolated from E. coli
CA8445/PPRK248CIts harboring plasmid PPLC28, which encodes
the appropriate mutant crp gene. All CRP mutants were
purified to more than 95% purity using the same protocol for wild type
CRP purification (18).
Circular Dichroism--
CD spectra of wild type and mutant CRP
were measured with an Aviv 62 DS circular dichroism spectrometer. To
acquire a full range of near- and far-UV CD spectra, fused quartz
cuvettes with path lengths of 0.01 (220-190 nm), 0.1 (270-200 nm),
and 1 cm (360-240 nm) and protein solutions with concentrations of
about 1 mg/ml were used. Each spectrum was recorded with a 0.5-nm
increment and 1-s interval. For each sample, five repetitive scans were obtained and averaged.
Proteolytic Digestion--
Proteolytic digestion of CRP was
employed as a tool to monitor the conformational changes in CRP using a
previously described protocol (17, 18).
Fluorescence Labeling of CRP--
IAF modification of the wild
type and mutant CRPs was carried out in 50 mM Tris, 0.3 M KCl, 1 mM EDTA at pH 7.8 in the dark. 1.1 mg/ml CRP was modified by 0.5 mM IAF at 4 °C overnight
for wild type CRP and 30-45 min at room temperature for G141Q and S128A/G141Q mutants. Labeled CRP was purified by Sephadex G-25 spin
columns and dialyzed extensively against buffer A. The extent of
modification was determined as described previously (16, 25).
Labeling of DNA with Fluorescent Probe--
The DNA used for
studying CRP-DNA interactions is the 40-base pair fragment of the
lac promoter with the sequence of
5
-CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCC-3
. The underlined sequence is the primary binding site for CRP.
Complementary strands of this fragment were synthesized and purified
separately by denaturing polyacrylamide gel electrophoresis. One of the
single-stranded polynucleotides was then labeled with CPM at the 5
-end
as described previously (26, 27). Modified single-strand DNA was
hybridized with an equal amount of its complementary strand to give the
fluorescently labeled double-stranded DNA. A small aliquot of the
hybridization product was analyzed by nondenaturing polyacrylamide gel
electrophoresis to ensure that all of the DNA was in the
double-stranded form.
cAMP Binding Assays--
cAMP binding to CRP was measured by
monitoring the fluorescence signal change of CRP-IAF (
ex = 495 nm,
em = 520 nm). The basic protocol employed has
been previously described (17) with a minor modification (18).
CRP exists as a dimer in solution and can bind to two molecules of cAMP
per dimer. Therefore, all cyclic nucleotide binding data in this study
can be fitted to the following equation by a nonlinear least squares
procedure to yield thermodynamic parameters K1,
K2,
1, and
2 (17),
|
(Eq. 1)
|
where
obs,
1, and
2
are the values of change in fluorescence intensity and the normalized
values of change in going from free CRP to CRP-cAMP1 and
CRP-cAMP2, respectively; K1 and
K2 are Adair constants for the formation of
CRP-cAMP1 and CRP-cAMP2; and [cAMP] is the
free cAMP concentration.
DNA Binding Study--
Fluorescence anisotropy measurements were
used for quantitative measurements of CRP-DNA interactions using a
previously published protocol (17).
The measured anisotropy data were fit to Eq. 2 as described
previously (26) to give the apparent association constant
(K) for CRP-DNA interaction according to a simple reaction
scheme: P + DNA
K P-DNA,
|
(Eq. 2)
|
where A is the measured value of anisotropy;
AD and APD are values of
anisotropy associated with free DNA and CRP-DNA complex, respectively;
and DT and PT are the
total concentration of DNA and protein, respectively.
However, free CRP does not bind specific DNA, and binding of CRP to DNA
in the presence of cAMP follows a more complex scheme (Scheme 1).
Therefore, the apparent DNA binding affinity of CRP
(K) is a function of cAMP concentration, which in accordance
to the above scheme can be expressed as follows.
|
(Eq. 3)
|
K1 and K2 can be
determined independently from the cAMP binding assay. The binding
affinity of CRP-cAMP2 (K4) is very
weak and can not be precisely determined (17). However, the apparent DNA binding affinity of the CRP in the presence of 100-200
µM cAMP, where most of the CRP exists in the monoliganded
form, has been shown to be a good estimation of the intrinsic DNA
binding affinity of the CRP-cAMP1 complex (28). Therefore,
in this study the apparent DNA affinity of CRP in the presence of 200 µM cAMP was assumed to represent
K3.
Sedimentation Equilibrium--
The quaternary structure of CRP
was monitored by sedimentation equilibrium as described previously
(29). Experiments were conducted in a Beckman-Spinco model E analytical
ultracentrifuge equipped with a photoelectric scanner, an electronic
speed control, and an RTIC temperature control. The high speed,
meniscus depletion procedure was employed (30). The loading CRP
concentrations were between 0.2 and 0.4 mg/ml. Sedimentation data were
acquired (10 scans) and then averaged after reaching equilibrium. The
density of the solution was determined with a Mettler-Paar Precision
DMA-02D density meter. Values of the partial specific volume of the
wild type and mutant CRPs in native buffer and 6.0 M GuHCl
were calculated based on the amino acid composition of CRP (31, 32)
using the procedure of Cohn and Edsall (33) and Lee and Timasheff (34),
respectively.
Sedimentation equilibrium data were fit by nonlinear least squares to a
dimer assembly scheme according to the equation,
|
(Eq. 4)
|
where C is the CRP concentration observed at radial
position r,
C is the base-line offset,
C0 is the concentration of CRP at the meniscus
(r0), K is the dimer equilibrium
association constant, and
is the reduced molecular weight given by
= Mr(1

)
2/2RT, where
Mr is the monomer molecular weight,
is
the partial specific volume,
is the solution density,
is the
angular velocity, and R and T are the gas
constant and the temperature in Kelvin, respectively.
Denaturation of CRP--
The effect of amino acid substitutions
on the folding and domain-domain interactions in CRP was probed by
monitoring the unfolding of CRP by GuHCl using fluorescence anisotropy.
A detailed description of the protocol has been published (29).
Briefly, the denaturation of CRP can best be described by a three-state
model,
in which N2, N, and
U are folded dimer, folded monomer, and unfolded monomer of
CRP, respectively. Kd and Ku are
the equilibrium constants of dissociation and unfolding,
respectively.
Unlike wild type CRP, mutant S128A/G141Q exhibits a weakened subunit
association, which leads to a complete uncoupling of dimer dissociation
and monomer unfolding. Therefore, these two processes can be studied
separately. Subunit association of S128A/G141Q was measured by
sedimentation equilibrium as described above. CRP unfolding was
monitored by fluorescence anisotropy at the excitation and emission
wavelengths of 280 and 345 nm, respectively.
To determine the equilibrium constant of unfolding, the fraction of CRP
protein that is in the folded state,
, was calculated by the
equation,
|
(Eq. 5)
|
where
,
folded, and
unfolded
represent the measured fluorescence anisotropy signal of the protein
sample and the values of fluorescence anisotropy of folded and unfolded
monomer CRP at a given GuHCl concentration, respectively.
folded and
unfolded at the unfolding
transition zone were derived by linear extrapolation of the measured
signals at the predenaturation and postdenaturation zones,
respectively.
Once
was determined, the equivalent Ku at
different GuHCl concentrations can be calculated as follows.
|
(Eq. 6)
|
Ku values were converted to
Gu by
Gu =
RTlnKu.
Gu0, the free energy of
unfolding in the absence of denaturant, was subsequently determined by
linear extrapolation of
Gu to zero denaturant
(35).
 |
RESULTS |
In Vivo Genetic Characterization of CRP Mutants--
G141Q
displayed purple colonies on MacConkey plates in the absence of
external cAMP and in the presence of cGMP as reported earlier (15, 19,
36, 37). Thus, this mutant is characterized by a CRP*
phenotype. Interestingly, while the S128A mutant showed a
CRP
phenotype (18), the double mutant S128A/G141Q is a
CRP* mutant in vivo. This in vivo
observation indicates that mutation of G141Q exerts a dominant effect
over mutation S128A.
In Vitro Structural Characterization of the S128A/G141Q
Mutant
Near- and Far-UV CD--
Near- and far-UV CD spectra of the double
mutant, S128A/G141Q are identical to that of the wild type CRP and
S128A and G141Q single mutants (Fig. 1).
These results indicate that no major secondary or tertiary structural
changes have been introduced in CRP by these mutations.

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Fig. 1.
CD spectra of the G141Q ( ), wild type CRP
( ), S128A/G141Q ( ), and S128A ( ) in buffer A. Inset, CD signals of the wild type CRP (long dashed
line), G141Q (solid line), S128A (short dashed
line), and S128A/G141Q (medium dashed line) at the
near-UV region. The axes of the inset and the rest of the
figure are the same.
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Subunit Association--
Like the wild type, S128A, and G141Q
CRPs, the S128A/G141Q mutant exists in solution as an apparent dimer in
the mg/ml concentration regime. However, one way of probing the effect
of mutation on intersubunit communication is by monitoring the
energetics of dimer formation of each mutant. Sedimentation equilibrium
experiments of S128A/G141Q at different concentrations of GuHCl ranging
from 0 to 1.5 M were conducted to determine the dimer
dissociation constant for this double mutant. The sedimentation
equilibrium experiments were performed at multiple speeds of 20,000, 28,000, and 34,000 rpm for each GuHCl concentration. The results are
shown in Fig. 2A. The
experimental data were then analyzed to estimate the apparent dimer
association constant, Ka, app at each
individual GuHCl concentration using the program NONLIN (38). A plot of
LnKa, app versus GuHCl concentration displays an apparent linear relationship. Extrapolation of the data to
zero concentration of GuHCl yields a value of the dimer association
constant for S128A/G141Q in buffer (Fig. 2B). The extrapolated dimer association constant is similar to that of the G141Q
mutant, i.e. weaker dimerization than that of the S128A mutant and wild type CRP. Table I
summarized the results on subunit dissociation for all of the mutants
and wild type CRP. Again, G141Q mutation exerts a dominating effect on
subunit assembly.

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Fig. 2.
Subunit association of S128A/G141Q studied by
sedimentation equilibrium. A, sedimentation equilibrium
profiles of S128A/G141Q at different angular velocity at 0 M GuHCl concentration. , 34,000 rpm; , 28,000 rpm;
, 20,000 rpm. The solid lines represent the best fits of
the experimental data to Eq. 4. B, apparent dimer equilibrium association constants (determined from A) as a
function of GuHCl concentration.
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|
Protein Stability--
Protein stability studies in general can
provide information on the folding stability of protein molecules and
possibly domain-domain interaction. The stability of the double mutant
was monitored by GuHCl denaturation studies using a published procedure
(29). The unfolding curve of the S128A/G141Q mutant is characterized by
a biphasic transition resembling that of the G141Q mutant, as shown in
Fig. 3A. Again this reflects
the uncoupling of the processes of dimer dissociation and monomer
unfolding. Similarly, the unfolding free energy of the S128A/G141Q
monomer was determined to be 7.4 kcal/mol, as shown in Fig.
3B. This value is similar to that of the wild type, S128A,
and G141Q CRPs (Table I).

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Fig. 3.
GuHCl-induced chemical denaturation of
S128A/G141Q. A, denaturation curve of S128A/G141Q (1 µM) measured by fluorescence anisotropy. B,
apparent unfolding free energy of S128A/G141Q monomer as a function of
GuHCl concentration.
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|
Protease Sensitivity--
The proteolytic digestion pattern of
wild type CRP is biphasic as a function of cAMP. An initial increase in
the rate of proteolysis at low cAMP concentration was followed by a
decrease in rate with a further increase in cAMP concentration (17).
The S128A mutant shows a significant decrease in sensitivity to
protease digestion in a wide range of cAMP concentrations (18), while
mutant G141Q is sensitive to protease cleavage in the absence of cAMP.
The digestion pattern of the S128A/G141Q double mutant is different from that of the wild type and S128A mutant but similar to that of the
G141Q mutant (Fig. 4). The S128A/G141Q
mutant is also susceptible to either chymotrypsin or subtilisin
digestion in the absence of cAMP. The digestion rates of S128A/G141Q
and G141Q CRP are dependent on cAMP concentration. Furthermore, the
rate decreases in the order of increasing cAMP concentration. The
effects of cGMP on the proteolytic digestion patterns of CRP and the
mutants were also examined. At a low cGMP concentration (200 µM) no significant effect was observed, while at a high
cGMP concentration (50 mM) the rate of protease digestion
for both G141Q and S128A/G141Q CRP decreased significantly as compared
with the unliganded protein (Fig. 4).

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Fig. 4.
Proteolytic digestion of the wild type CRP,
S128A, G141Q, and S128A/G141Q by chymotrypsin and subtilisin in the
presence or absence of cAMP or cGMP. Lanes 1-5 show results
with chymotrypsin. Lane 1, CRP alone; lanes 2 and
3, with 200 µM and 50 mM cAMP, respectively; lanes 4 and 5, with 200 µM and 50 mM cGMP, respectively. Lanes
6-10 are proteolytic results under the same experimental conditions as lanes 1-5 but in the presence of
subtilisin.
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|
In Vitro Functional Characterization of CRP Mutants
Cyclic Nucleotide Binding--
Unlike wild type CRP, mutant G141Q
CRP can also be activated by cGMP and other cyclic nucleotides for
specific DNA binding (19). Thus, the binding affinities of the G141Q
mutant for cGMP and cAMP were determined by monitoring the fluorescence
quenching of the IAF-labeled G141Q CRP as a function of cGMP
concentration, and the results are shown in Fig.
5. The estimated values of the equilibrium binding constants for cGMP are k1 = 4.7 × 104 M
1 and
k2 = 3.2 × 103
M
1 as listed in Table
II. In comparison, equilibrium binding
constants for cAMP are k1 = 6.4×104
M
1 and k2 = 2.5×103 M
1. Thus, the binding
constants of cAMP and cGMP in the G141Q mutant are essentially
identical.

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Fig. 5.
Binding of cAMP ( ) and cGMP ( ) to the
G141Q mutant as monitored by fluorescence quenching of G141Q-IAF
complex. The solid lines represent the best fits of the
data to Eq. 1.
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|
cAMP binding to S128A/G141Q CRP was monitored by fluorescence quenching
of the S128A/G141Q-IAF complex (Fig. 6).
Values of 1.4×104 M
1 and
1.9×102 M
1 were determined for
k1 and k2, respectively.
The binding constant of the first cAMP is slightly lower than but not
significantly different from that of wild type, S128A, and G141Q CRP,
whereas the binding constant for the second cAMP is about 6 and 14 times lower than that of wild type and G141Q CRP, respectively.

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Fig. 6.
Binding of cAMP to S128A/G141Q as monitored
by fluorescence quenching of S128A/G141Q-IAF. The solid
lines represent the best fits of the data to Eq. 1.
|
|
Negative cooperativity in cAMP binding has been observed in wild type
CRP (17), and the ratio of the microscopic cAMP binding constants,
k1 and k2, can be used to
estimate the degree of cooperativity. For the wild type and G141Q CRPs,
values of about 25 were determined (Table II), while the two binding
constants differed more than 1000-fold in mutant S128A CRP, indicating
that mutation at residue 128 leads to an even higher degree of negative
cooperativity in CRP. Interestingly, a value of 74 for
k1/k2 was observed for
the double mutant. This result suggests that the strong negative
cooperativity for cAMP binding observed in S128A is partially preserved
in S128A/G141Q CRP.
DNA Binding--
cGMP binds to but fails to activate wild type CRP
(21, 39). Interestingly, cGMP not only binds to G141Q CRP with affinity similar to cAMP, but it can also replace cAMP in activating G141Q CRP
to interact with DNA (19). When cGMP was titrated into a solution
containing 13 nM lac-40-CPM DNA and 1 µM of
G141Q CRP, the formation of a protein-DNA complex responded to the
increase of cGMP concentration in a biphasic manner that is similar to the response of wild type CRP to cAMP (Fig.
7A). This biphasic behavior
reflects the strong affinity of CRP-cGMP1 for lac-40 DNA
but a decreased affinity of the CRP-cGMP2 species.
Quantitative determination of the apparent binding constant of G141Q
CRP to DNA in the presence of 100 µM cGMP yields a value
of 3.0 × 107 M
1 (Fig.
7B), while wild type CRP binds to specific DNA with an apparent binding constant of less than 7 × 104
M
1 (Table III).
Such high affinity of G141Q-cGMP1 complex for specific DNA
is close to the affinity of CRP-cAMP1 complex to some
naturally occurring CRP-dependent promoters, indicating a
possible physiological significance, e.g. the activation of
some CRP-dependent promoters in the absence of cAMP.

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Fig. 7.
Interaction of G141Q with lac-40 DNA in the
presence of cGMP. A, G141Q-DNA interaction as a function of
cGMP concentration. B, binding of G141Q to lac-40 DNA in the
presence of 100 µM cGMP. The solid line
represents the best fit of the data to Eq. 2.
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Table III
Apparent DNA binding affinity of the wild type and mutants in the
presence of cAMP or cGMP
The experiments were performed in buffer A and 100-200
µM cAMP or cGMP.
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|
The binding of S128A/G141Q CRP to lac-40-CPM-labeled DNA in the
presence of cAMP was also studied (Fig.
8). The apparent DNA binding constant of
S128A/G141Q CRP in the presence of 200 µM cAMP is
1.7 × 107 M
1, which is
stronger than that of S128A CRP but weaker than that of G141Q CRP. cGMP
can also activate S128A/G141Q CRP for specific DNA binding (Fig. 8).
The estimated apparent DNA binding constant of S128A/G141Q CRP in the
presence of cGMP is about 3.0 × 106
M
1. Under the same conditions, S128A CRP has
no measurable binding affinity to lac-40 DNA.

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Fig. 8.
Binding of the S128A/G141Q to lac-40 DNA in
the presence of 200 µM cAMP ( ) and 200 µM cGMP ( ). The solid lines represent the best fit of the data to Eq. 2.
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|
When the DNA binding constants for CRP in the presence of cAMP or cGMP
are compared, a more than 5,000-fold difference is obtained (Table
III), suggesting that CRP is highly selective for cyclic nucleotides.
Mutation S128A does not seem to affect the selectivity, while mutation
G141Q leads to a dramatic relaxation in cyclic nucleotide selectivity,
decreasing the 5,000-fold difference in binding affinity to only about
8-fold. Moreover, the effect of the G141Q mutation is preserved in the
double mutant (Table III). These results imply that the G141Q mutation
exhibits a dominant effect over the S128A mutation in conferring
recognition to specific DNA sequence and discriminatory ability toward
the various cyclic nucleotides.
 |
DISCUSSION |
In vitro studies of CRP-DNA interaction show that the
species that interacts with specific DNA sequences is
CRP-cAMP1 (17, 18, 20, 26, 28, 40, 41). The
CRP-cAMP2 complex exhibits much lower or no affinity for
these DNA sequences. There is no exception for all of the nine DNA
fragments of natural or artificial sequences studied (28, 41). One
possible rationale for this behavior is the requirement of recognition
between two asymmetric macromolecules, namely CRP-cAMP1 and
CRP-dependent promoter, as proposed by Heyduk and Lee (17).
The natural DNA sequences of CRP binding sites are not palindromic (1,
3), and RNA polymerase seems to recognize a DNA-bound CRP subunit in a
particular orientation (42, 43). Hence, it is conceivable that CRP
binds to the asymmetric DNA site in a specific orientation. Binding of
one cAMP molecule to the dimeric CRP molecule would render the protein
molecule asymmetric. If the functional mode of CRP is the assumption of an asymmetric structure by binding of one cAMP molecule, then it is
imperative that the CRP-cAMP2 species does not form
readily. The negatively cooperative binding of cAMP to CRP is
apparently designed to achieve such a goal. If such an interpretation
is valid, then what are the structural elements in the CRP molecule that impart these functional properties? What is the relation between
these elements? Are they cooperative or antagonistic? Does one element
exert a dominant effect over another? Results of this study indicate
that residues 128 and 141 exhibit dominant effects on different
functional properties of CRP.
Role of Residue 128 and C-helix in the Negative Cooperativity of
cAMP Binding--
An earlier study on cAMP binding to
-CRP, the
ligand binding domain of CRP generated by proteolytic digestion, led to
the observation that the degree of negative cooperativity in cAMP binding is related to the amount of C-helix retained in
-CRP (25).
Retention of the complete C-helix leads to a
-CRP molecule that
binds only one cAMP, i.e. the degree of negative
cooperativity is so great that it prevents occupation of the second
site. Conversely, elimination of the C-helix leads to an abolishment of
negative cooperativity so that both sites can bind cAMP with the same
affinity. These results imply a possible role for C-helix in imparting
negative cooperativity in CRP. Based on the crystallographic data of
CRP, serine 128 occupies an important position in the C-helix (4, 44).
Results show that mutations of serine 128 do not affect the affinity of
the binding of the first cAMP molecule; however, the degree of negative
cooperativity is greater (18). Thus, results of studying site-directed
and deletion mutants implicate the C-helix in CRP in an important role
in imparting negative cooperativity in cAMP binding to CRP.
The linkage between the C-helix and negative cooperativity is further
strengthened by the results for the G141Q mutant. Residue 141 does not
reside in the C-helix. The ligand binding properties of wild type CRP
are quantitatively retained by the G141Q mutant, namely the same degree
of negative cooperativity and same affinities for either cAMP or cGMP.
Thus, mutating residue 141 apparently does not perturb the functional
properties of the cAMP binding domain except to weaken the interaction
along the subunit interface. In addition, the effect of the S128A
mutation on negative cooperativity of cAMP binding is partially
retained in the double mutant S128A/G141Q. The results of this study of
a limited number of mutants indicate that the C-helix apparently plays
an important role in imparting negative cooperativity in cAMP binding
to CRP and that residue 128 apparently exhibits a dominant effect over
residue 141 in maintaining negative cooperativity in ligand
binding.
Role of Residue 141 and Subunit Interface in the Discrimination of
Cyclic Nucleotides--
Another important issue in the mechanism of
CRP function is its ability to bind and be activated only by cAMP.
Although other cyclic nucleotides can bind to CRP, no activation in CRP
toward recognition of specific DNA sequence is observed. Nevertheless, the G141Q mutant provides an opportunity to address the issue of cyclic
nucleotide discrimination. The strict specificity requirement for
cyclic nucleotide observed in wild type CRP is significantly relaxed in
the G141Q mutant (19) as well as in the double mutant S128A/G141Q.
These observations are unexpected. One may speculate that specificity
will be imparted by amino acid residues within the binding site of
cyclic nucleotides. Serine 128 resides in the cAMP domain and is a
prime candidate for discriminating specific cyclic nucleotides, since
the crystallographic data indicate hydrogen bonding between the
hydroxyl group of serine 128 and N-6 of cAMP (4). However, elimination
of the hydroxyl group by a Ser
Ala substitution does not affect the
ability of the S128A mutant to discriminate between cAMP and cGMP. Yet,
a substitution of glycine by glutamine in residue 141, which is neither
located within the cAMP binding domain nor involved in the formation of intersubunit contacts, leads to a breakdown in the ability of CRP to
discriminate cAMP from the other cyclic nucleotides.
Substitution of Gly
Gln does not lead to any change in the
secondary or tertiary structure of CRP as monitored by CD, and this
substitution does not alter the global folding of CRP as monitored by
chemical denaturation (20, 45). Thus, spectroscopically and
energetically, a Gly
Gln mutation does not produce any significant perturbation in the structure of CRP, and the loss of ability to
discriminate cyclic nucleotides is not the result of detectable alteration in the global structure of CRP. Mutation G141Q results in a
weakening in the energetics of intersubunit interaction. The
consequence is an increase in susceptibility to protease digestion along the C-helices, an observation that mimics the effect of cAMP
binding. The net result of this weaker interfacial interaction is a
relaxation of cyclic nucleotide discrimination. Therefore, CRP
maintains its cyclic nucleotide selectivity by imposing an energetic
barrier along the subunit interface that only cAMP can overcome.
Interestingly, the double mutant S128A/G141Q responds to protease
digestion in a manner almost identical to the G141Q. This result can be
interpreted to imply that the structural changes along the subunit
interface introduced by the G141Q mutation can override the local
structural changes created by the S128A mutation.
Role of Domain Interface in CRP Activation--
A combination of
subunit realignment and domain rearrangement activates CRP for specific
DNA binding. The G141Q mutation only leads to a change in realignment
of subunits without inducing the proper domain rearrangement. The
latter structural change is only induced upon binding of cyclic
nucleotide. That is the underlying reason why the G141Q mutant cannot
bind to a specific DNA sequence without cAMP. The supporting evidence
for this interpretation is the response of the IAF-labeled G141Q CRP to
the titration of cAMP or cGMP. The IAF is covalently attached to
cysteine 178, which is located at a position just before the
DNA-binding helix (F-helix). Quenching of fluorescence intensity of the
covalently attached fluorescein probe was observed and must reflect the
domain rearrangement in response to ligand binding. The estimated
values of binding constants are in good agreement with the values
determined by other approaches. This again suggests that the cAMP- or
cGMP-induced fluorescence quenching reflects the intrinsic
conformational changes in the DNA binding domain in response to cAMP or
cGMP binding. The IAF-labeled S128A/G141Q CRP responds to the binding
of cAMP in a manner similar to that of G141Q CRP. This effect further substantiates the involvement of domain-domain rearrangement in CRP
activation by cAMP and the dominating effect of the G141Q mutation.
In summary, the results from this study imply that the cyclic
nucleotide-induced activation process is linked to the energetics of
overcoming an unfavorable intersubunit structure. A weaker interfacial
interaction, such as those observed in the G141Q and S128A/G141Q
mutants, makes it easier to switch over to the appropriate intersubunit
structure and consequently to induce the proper interdomain orientation
for specific DNA sequence recognition. Furthermore, this study reveals
the apparent dominant effects of residues 128 and 141 in conferring
functional characteristics in CRP. The influence of residue 128 is more
significant in ligand binding (in particular, binding of the second
cAMP). Residue 141 exerts more effects on the energetics of
dimerization and all events apparently associated with intersubunit
interactions, e.g. discrimination among various cyclic
nucleotides in their ability to activate CRP for binding to specific
DNA sequence.