Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide
Binding Domain of Cyclic AMP Receptor Protein from Escherichia
coli*
Ran
Chen and
J. Ching
Lee
From the Department of Human Biological Chemistry and Genetics,
University of Texas Medical Branch, Galveston, Texas 77555-1055
Received for publication, November 13, 2002, and in revised form, January 15, 2003
 |
ABSTRACT |
Cyclic AMP is a ubiquitous secondary message that
regulates a large variety of functions. The protein structural motif
that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable. The cAMP receptor protein
of Escherichia coli, CRP, was employed as a model system to
elucidate the functional roles of these loops. Based on the sequence
differences between CRP and cyclic nucleotide gated channel, three
mutants of CRP were constructed: deletion (residues 54-56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between
Glu-78 and Gly-79) and double mutants. The effects of these
mutations on the structure and function of CRP were monitored. Results
show that the deletion and insertion mutations do not significantly
change the secondary structure of CRP, although the tertiary and
quaternary structures are perturbed. The functional data indicate that
loop 3 modulates the binding affinities of cAMP and DNA. Although the
lengthened loop 4 may have some fine-tuning functions, the specific
function of the original loop 4 of CRP remains uncertain. The function
consequences of mutation in loop 3 of CRP are similar to that of site A
and site B in the regulatory subunits of cyclic
AMP-dependent protein kinases. Thus, the roles played by
loop 3 in CRP may represent a more common mechanism employed by cyclic
nucleotide binding domain in modulating ligand binding affinity and
intramolecular communication.
 |
INTRODUCTION |
Cyclic AMP serves as an intracellular message in both prokaryotes
and eukaryotes by transmitting information through proteins such as
protein kinase A (PKA)1,
cyclic nucleotide-gated ion channels (CNGC), and cAMP receptor protein
in Escherichia coli (CRP). These proteins are involved in a
very diverse set of cellular functions such as signal transduction, excitability, and gene expression (1-6). These proteins of diverse functions all consist of a cAMP binding motif. The structural motif,
which serves as cAMP receptor, is found to display a high degree of
similarity. X-ray crystallography and homology modeling results show
that, despite obvious divergence of sequence among the receptor domains
and significantly different biological functions of these proteins,
their CNB domains appear to share a common architecture, all consisting
of an
-helix (helix A), an eight-stranded
-roll, and two more
-helices (helices B and C). The body of the CNB pocket is mainly
located in the
-roll, with the C-helix forming the back of the
binding pocket (2, 8). The superimposition of the structures of
CNB domains from CRP and the regulatory subunits of PKA, as shown below
in Fig. 1, indicates that the
-roll basically assumes the same
structure with the exception of loops 3 and 4 between strands 4 and 5, and strands 6 and 7, respectively. In some cases, such as in CNGC and
PKA, loop 3 is shortened whereas loop 4 is lengthened, as shown in Fig.
1. Only six residues (Gly-33, Gly-45,
Gly-71, Glu-72, Arg-82, and Ala-84 using the CRP sequence as reference)
are invariant among all members of the families. It has been suggested
that the invariant residues play important and conserved roles in the
folding and function of the CNB sites of these diverse proteins.
Gly-33, Gly-45, and Gly-71 are involved in turns between strands of the
-roll; Arg-82 and Glu-72 contact the cyclic nucleotide, and the
function of Ala-84 is uncertain (3). Despite large variation of primary
sequences, the sizes of secondary structural elements of the CNB domain
are much conserved among the family. For example, the alignment of CRP
and CNGCs by keeping the six conserve residues at the same positions
shows that the size differences in secondary structural elements are only located at two loops, e.g. loop 3 (between
4 and
5) of CNGCs is shorter than that in CRP by three residues, and loop 4 (between
6 and
7) of CNGCs is 5 residues longer than that of
CRP (see Fig. 2) (6). Similarly, loops 3 and 4 among the various
protein kinase isozymes also show heterogeneity in size, as shown in
Fig. 2.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 1.
Structure of the
-roll. The two -roll structures from
CRP (red and green) are superimposed on each
other and the equivalent structures from the regulatory subunits of PKA
(pink and green). Adopted from Ref. 1.
|
|
Based on the sequence alignment, it is interesting to note that loops 3 and 4 are the only structural elements that are different in size among
these various sources of CNB domain. To elucidate the roles of these
loops the cAMP receptor protein, CRP, from E. coli is
employed as a model system for investigation. CRP of Escherichia
coli, also referred to as the catabolite gene activator protein,
is a 47,238-Da homodimer. Each subunit has two domains: the large
N-terminal domain is a cyclic nucleotide binding domain, and the small
C-terminal domain is a DNA binding domain. ApoCRP has very low
affinity for DNA and cannot differentiate between specific and
nonspecific DNA sequences, whereas holoCRP exhibits high affinity for
specific DNA sequences. It is known that binding of cAMP allosterically
induces CRP to assume conformations that exhibit high affinity for
specific DNA sequences (9-13). It has been suggested that allosteric
conformational change, which includes subunit realignment and domain
rearrangement, occurs upon the binding of cAMP to CRP. These changes
are mediated by interactions involving the subunit and domain
interfaces. The C-helix and the hinge region between the domains have
been found to play key roles in transmitting the allosteric signal.
Although earlier spectroscopic comparison between the holo- and apoCRP
showed no apparent secondary structural changes (14-16), the results
of protein footprinting experiments and recent NMR studies indicate
that there are wide-ranging structural differences between apoCRP and
holoCRP. cAMP binding, while perturbing the
-roll that forms the
cAMP binding pocket, has little effect on the secondary structure
elements contained in either the N- or the C-terminal domains. There
does, however, appear to be a significant difference around the C
terminus of C-helix, the hinge region, and loop 3 (1, 17-20). These
studies identified the location of structural changes induced by cAMP binding but do not provide information on the role of these structures in this
-roll motif in binding cyclic nucleotides.
It was postulated that loop 3 in CRP is involved in both interdomain
and intersubunit interactions, whereas loop 4 contacts the coiled-coil
C-helices and forms part of the dimer interface (1), although the
effects of these interactions in CRP function were not predictable by
structural analysis alone. In recent studies, it was shown that a D53H
mutation in loop 3 leads to enhancements of the magnitude of positive
cooperativity in cAMP binding and affinity for specific DNA (21, 22).
These solution biophysical data are consistent with the proposal that
loop 3 plays a role in interdomain and intersubunit communications,
although the specific nature of this role is unknown. In this study,
three mutants of CRP are constructed according to the difference in
sequence alignment between CRP and CNGC, namely, a deletion of residues
54-56 in loop 3 and insertion of 5 residues between residues 78 and
79, respectively. The choices for specific sequences for deletion and
insertion are based on the availability of information on CNGC.
Consequently, it is possible to compare and contrast the data acquired
in this study with the literature. These mutants are the subjects of
investigation to elucidate the roles of these loops in the normal
function of CRP.
 |
MATERIALS AND METHODS |
All in vitro experiments were conducted in TEK (100)
buffer (50 mM Tris-HCl, 100 mM KCl, and 1 mM EDTA at pH 7.8 and 22.5 °C). The concentrations of
protein, cyclic nucleotides, and fluorescence probes were determined by
absorption spectroscopy using the following absorption coefficients:
40,800 M
1cm
1 at 278 nm for CRP
and its mutants, 14,650 M
1cm
1
at 271 nm for cAMP, 12,950 M
1
cm
1 at 254 nm for cGMP, 33,000 M
1cm
1 at 385 nm for CPM, and
6,240 M
1cm
1 at 351 nm for ANS.
All the solutions were made with reagent grade or higher grade
chemicals and filtered prior to use.
Site-directed Mutagenesis--
The sites and nature of mutations
in loops 3 and 4 are based on the sequence differences between CRP and
CNGC, namely, a deletion of Glu-54, Glu-55, and Gly-56 in loop 3 and an
insertion of the sequence KGSKM between Glu-78 and Gly-79 in loop 4, as
shown in Fig. 2. An overlap extension PCR
method was used (23). The outer pairs of primers include an
NdeI site and an HindIII site, respectively. The
sequences were: TAA CCG CATATG GTG CTT GG and CCA CTC CGA C
AAGCTT AA CGA GTG CCG. The sequences of mutagenesis primers for insertion were ACG TTC CTG GCC CAT CTT
AGA GCC CTT CTC TTC AAA CAG GCC CAG
and GTT TGA AGA G AAG GGC TCT
AAG ATG GGC CAG GAA CGT AGC GCA; those for
deletion were AGG AGA GGA TCA TTT CTT T GTC TTT GAT CAG CAC TGC C and
GGC AGT GCT GAT CAA AGA C AAA GAA ATG ATC CTC TCC T. The products of
the second round of amplification were inserted into the pET30a
plasmid, and the constructs were sequenced after cloning.

View larger version (66K):
[in this window]
[in a new window]
|
Fig. 2.
Amino acid sequence alignment of CRP and
other cyclic nucleotide binding domains. See Refs. 6 and 8 for
citations of specific protein sequences.
|
|
Protein Purification--
Wild-type and mutant CRPs were
purified from E. coli strain HMS174(DE3) using a previously
described protocol (21, 24). All purified CRP proteins were >99%
homogeneous as judged by SDS-PAGE stained by Coomassie Blue; 50-60
µg of CRP was routinely loaded onto each lane. Furthermore, the
ratios of the absorbance at 280 nm to that at 260 nm were >1.86,
indicating the absence of nucleic acid contamination. The mass of
proteins was further checked by mass spectrometry.
Analytical Ultracentrifugation--
Experiments were conducted
at appropriate speeds in a Beckman Optima XLA analytical
ultracentrifuge equipped with absorbance optics and an An60Ti rotor.
Sedimentation velocity experiments were performed at 42K rpm. Velocity
data were collected at 280 nm at a spacing of 0.002 cm with no
averaging in a continuous scan mode and were analyzed using DCDT+
version 1.12. The reported weight-average sedimentation coefficient
values (
20,w) obtained from DCDT+ were calculated by a weighted integration over the
entire range of sedimentation coefficients covered by the g(s)
distribution and corrected for the solution density and viscosity.
The apparent weight-average molecular weights were obtained by fitting
the sedimentation equilibrium data with the following equation,
|
(Eq. 1)
|
where C is the observed CRP concentration in
absorbance at radial position r, E is the
baseline offset, C1 and
C2 are the CRP concentrations of monomeric and
dimeric CRP, respectively, at the meniscus r0,
is the partial specific volume,
is the solvent density,
is the angular velocity, M is the apparent weight-average molecular weight, and R and T are
the gas constant and temperature in degrees kelvin, respectively.
Ka is the apparent association constant. The value
of
of CRP in Tris buffer is 0.744 and was derived from the
amino acid composition of CRP using the method of Cohn and Edsall (25).
The apparent partial specific volumes of wild-type and mutant CRPs in 6 M GdnHCl were calculated using the procedure of Lee
and Timasheff (26). The corresponding values in lower concentrations of
GdnHCl were interpolated by assuming a linear relationship between
GdnHCl bound and denaturant concentration.
The quaternary structure of CRP mutant was monitored by sedimentation
equilibrium using a published procedure (27). In most cases, the
subunit-subunit interaction of CRP was strong and could not be
estimated directly by sedimentation equilibrium. The CRP dimerization
was weakened by increasing amounts of GdnHCl, and the quaternary
structure of CRP was monitored under each GdnHCl concentration. The
loading CRP concentrations were between 0.2 and 0.4 mg/ml. Usually,
200-µl samples were loaded in a 12-mm Epon charcoal-filled
centerpiece. The high speed, meniscus depletion procedure was employed
(28).
Having determined the value of Ka by Equation 1,
Ga values, the free energy changes for
subunit assembly at different concentrations of GdnHCl, were calculated
and fitted by a linear least-squares analysis to Equation 2,
|
(Eq. 2)
|
where R and T are the gas constant and
absolute temperatures, respectively,
G
is the extrapolated free energy changes of subunit assembly of CRP in
buffer, and ma is the dependence of
Ga on denaturant concentration.
Circular Dichroism Data Acquisition and Analysis--
CD
measurements were performed on an AVIV 62DS CD spectropolarimeter
using a 0.1-cm (for far-UV region) or 1.0-cm (for near-UV region) path
length microcuvette (200-µl capacity). The protein concentration used
was 7 µM. CD spectra were measured over the range of
200-320 nm. Each spectrum was recorded in 0.5-nm wavelength increments, and signal was acquired for 1 s at each wavelength. Each measurement was performed in triplicate. Deviations between scans
were negligible. Baseline subtraction and smoothing of spectra curves
were performed using the AVIV CDS program.
Fluorescence Data Acquisition and Analysis--
Fluorescence
measurements were carried out in 1-cm quartz cuvettes at 22.5 °C
using a PerkinElmer Life Sciences LS50B luminescence spectrometer.
Protein concentration was 5 µM. Samples were excited at
295 nm, and tryptophan emission was monitored from 310 to 400 nm.
Acrylamide quenching measurements were carried out on samples containing acrylamide (0-0.7 M), either without or with
200 µM cAMP. Quenching data were plotted using the
Stern-Volmer equation,
|
(Eq. 3)
|
where Fo/F is the
fractional decrease in fluorescence due to the quencher
([Q]), and Ksv and V are
the collisional and static quenching constants, respectively.
Cyclic Nucleotide Binding Assay--
Cyclic nucleotide binding
to CRP and mutants were measured by the quenching of ANS-CRP
fluorescence according to the protocol described previously with minor
modification (12). Protein concentration was 12.5 µM. The
binding data fitted with a three-site model (for cAMP binding to
wild-type CRP) and a two-site model (for cAMP binding to mutants and
all cGMP binding), respectively, in accordance to a previous
observation (22),
|
(Eq. 4)
|
where Ki is the association constant
for the binding of the ith ligand. The observed fluorescence
parameter is related to Ki by,
|
(Eq. 5)
|
where Fobs is the normalized value of
observed fluorescence intensity at 480 nm, n is the total
number of L molecules bound to a CRP molecule, i
is the number of bound L molecules, and
Xi and Fi are the fractions of
CRP sub-conformation with different numbers of L bound and
its fluorescence property, respectively. Xi is
related to the fraction distribution parameter,
i,
which corresponds to the number of L bound: for
i = 0,
0 = 1; i = 1,
1 = 2k1[L];
i = 2,
2 = k1k2[L]2;
and i = 3,
3 = 2k1k2k3[L]3.
DNA Binding--
Fluorescence anisotropy measurements, by the
SLM 8000C spectrofluorometer, were employed for quantitative evaluation
of the CRP-DNA interaction. The DNA binding site was the 26-bp fragment of the lac PI promoter with the sequence
5'-ATTAATGTGAGTTAGCTCACTCATTA-3'. The
underlined sequence is the primary binding site for CRP. The reaction
mixture of 1300-1350 µl contained 12 nM of the
CPM-labeled 26-bp fragment of lac promoter DNA and 230 µM cyclic nucleotide. At 230 µM, the high
affinity sites for cyclic nucleotides are occupied in all CRPs employed
in this study (22). The detailed experimental and data analysis
protocols have been previously described (21). Briefly, the data were
fitted to the following equation by non-linear least-squares to
determine the apparent association constant for CRP-DNA interaction,
K,
|
(Eq. 6)
|
where A is the measured value of the anisotropy,
AD and APD are values of
anisotropy with free DNA and CRP·DNA complex, respectively, DT and PT are the total
molar concentrations of DNA and dimeric protein, respectively.
GdnHCl Denaturation--
Stock solutions of 6.9 M
GdnHCl were prepared in TEK (100), and the concentrations were
determined with a Mettler-Paar Precision density meter. Proteins at 5 µM were unfolded in various concentrations of GdnHCl for
1 h at room temperature. Protein unfolding was monitored by CD,
and the data were expressed as SD, the measured
CD signal was normalized to SD at 0 M GdnHCl.
 |
RESULTS |
Quaternary Structure and Hydrodynamic Properties--
The mass of
the purified protein was determined by mass spectrometry. In general,
the observed mass did not deviate more than 5 Da from the calculated
mass. Thus, the identities of these proteins were confirmed by
both mass and DNA sequence. The molecular weight of the proteins in TEK
(100) was monitored by sedimentation equilibrium. A set of typical data
is shown in Fig. 3A. The
molecular weights calculated from fitting a single-species model to the
sedimentation equilibrium data were 4.5 ± 0.9 × 104, 4.4 ± 0.7 × 104, 4.7 ± 0.9 × 104, and 4.5 ± 0.7 × 104 for wild-type, deletion, insertion, and double-mutant
CRP, respectively. The observed molecular weights correspond well to
twice the calculated molecular weights of polypeptides, indicating that
there was no change in the state of oligomerization, i.e.
the smallest kinetic units in solution of all these CRPs were dimers.
The hydrodynamic property of these proteins was monitored by
sedimentation velocity. The weight-average sedimentation coefficient
(
20,w) was
determined by fitting the sedimentation velocity data to a single
species with the DCDT+ analysis, as shown in Fig. 3B. The results show that the data fit well to a single species, and the values
for
20,w were
3.67 (3.65, 3.70), 3.51 (3.48, 3.55), 3.67 (3.65, 3.71), and 3.48 (3.45, 3.52) for wild-type, deletion, insertion, and double-mutant CRP,
respectively. The numbers in parentheses represent the distribution of
a 68% confidence interval. The decrease in
20,w for deletion and
double mutants indicates either an increase in asymmetry or that these molecules assume a less compact conformation.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Analytical ultracentrifugation data of CRPs
in TEK (100) buffer at pH 7.8 and 22.5 °C. The loading protein
concentrations were 15 µM for wild-type ( ), 9 µM for deletion ( ), 13 µM for insertion
( ), and 16 µM for double mutant ( ), respectively.
Dots represent data, and the solid lines
represent the best fits of the data. A, sedimentation
equilibrium; B, sedimentation velocity.
|
|
Secondary and Tertiary Structures--
The secondary structures of
the mutant CRP were monitored by CD, as shown in Fig.
4A. All mutants have far-UV CD
spectra similar to that of wild-type CRP, so it can be concluded that
these loop mutations do not significantly change the secondary
structural content of CRP. Significant differences, however, are
observed in the near-UV CD spectra, as shown in Fig. 4B. The
spectra for all samples showed fine features between 250 and 300 nm,
although the magnitudes of ellipticity for mutants were greater than
that of the wild-type CRP. The 255- and 265-nm peaks are most likely reflective of Phe residues; the 275-nm peak of Tyr residues and the
285- and 295-nm peaks of Trp residues. These results indicate that
these loops mutations lead to a perturbation of the microenvironments of the aromatic residues of CRP. Because all aromatic residues (5 Phe,
6 Tyr, and 2 Trp per CRP subunit) with the exception of Tyr-206 and
Phe-136 are located in or near the CNB domain, these CD results
indicate that the local environments of the CNB domain of CRP are
perturbed by these mutations without significantly altering the net
content of the secondary structures.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
CD spectra of CRPs in TEK (100) buffer at pH
7.8 and 22.5 °C. Protein concentrations were 5 µM
( ) wild-type, ( ) deletion, ( ) insertion, ( ) double mutant.
A, far-UV region; B, near-UV region.
|
|
Solvent Accessibility of Trp Residues--
Each CRP subunit has 2 Trp residues, both of which are located in the CNB domain. Trp 85 is
located inside the
-roll and close to the dimer interface. Thus,
intrinsic Trp fluorescence is a useful probe of the microenvironments
of the CNB domain. The fluorescence emission spectra of the deletion
and double mutants show a red-shift as compared with that of wild-type
CRP, as shown in Fig. 5A. The
intensities of the emission spectra of the deletion and double mutants
were significantly higher than that of wild-type CRP. The difference
between the insertion mutant and wild-type was much smaller (Fig.
5A). These results indicate that deletion of loop 3 leads to
a more polar environment around the Trp residues. Insertion of extra
residues in loop 4 had no apparent effect and did not compensate for
the effect of deletion of loop 3. In the presence of cAMP, significant
fluorescence blue-shifts and quenching occurred in the emission spectra
of the deletion and double mutants, as shown in Fig. 5B. But
the spectra of the insertion mutant and wild-type CRP did not change
much. The blue shifts in spectra indicate that cAMP binding induces a
change in the microenvironments of Trp so that they are less polar in
deletion and double mutants, whereas there are no such detectable
changes in the insertion mutant and wild-type CRP. These differences
indicate that cAMP binding induces much more significant conformational
changes in the CNB domains due to the deletion at loop 3.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Fluorescence emission spectra of CRPs in TEK
(100) buffer at pH 7.8 and 22.5 °C. Protein concentrations were
5 µM. ( ) wild-type, ( ) deletion, ( ) insertion,
( ) double mutant. A, in the absence of cAMP;
B, in the presence of 200 µM cAMP.
|
|
The accessibility of Trp residues is quantitatively assessed using
fluorescence collisional quenching by acrylamide. Results of these
measurements are shown in Fig. 6. The
data were fitted to Equation 3, and the parameters are summarized in
Table I. In the apo-state, the magnitude
of collisional quenching constants (Ksv) was in
the increasing order of insertion < wild-type < double < deletion. These results imply that the Trp residues of the mutants that consist of a deletion in loop 3 are more solvent-assessable than
the rest. In the presence of cAMP, the value of
Ksv increased by 15% for wild-type, decreased
by 44% for deletion, remained the same for insertion, and decreased by
30% for double mutant, as summarized in Table I. These results are
consistent with those of fluorescence experiments, which monitored the
emission spectra of CRP, confirming that the deletion apparently
exposes the Trp residues to solvent, and, on the contrary, the
insertion slightly buries the Trp residues into the protein matrix.
Furthermore, two more important points can be inferred from the
environmental change of the Trp residues accompanying cAMP binding.
First, the increased accessibility of Trp induced by deletion can be
reversed by cAMP binding. Second, the insertion mutation modulates this conformational change.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Acrylamide quenching of Trp fluorescence of
CRPs in TEK (100) buffer at pH 7.8 and 22.5 °C. Protein
concentrations were 5 µM ( ) wild-type, ( ) deletion,
( ) insertion, ( ) double mutant. The solid lines
represent the best fits of the data. A, in the absence of
cAMP; B, in the presence of 200 µM cAMP.
|
|
Energetics of Subunit Assembly--
The effect of mutations on the
energetics of subunit assembly was probed by monitoring the
dissociation of the dimeric protein in the presence of GdnHCl. The
apparent molecular weights of these proteins were monitored by
sedimentation equilibrium. The data were fitted by Equation 1 to obtain
the apparent dimerization constant, Ka. The free
energies of subunit assembly of these proteins in buffer,
G
, were determined by the linear
extrapolation method, as shown in Fig. 7.
The extrapolated values of
G
are
summarized in Table I. These results show that mutations in these loops
induce a destabilization of the formation of CRP dimers. Interestingly,
simultaneous mutations in both loops did not further decrease the
stability of intersubunit interface. In fact, the effect of deletion
seemed to be dominant.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7.
Free energy changes for dimer association of
CRP as a function of GdnHCl concentration. The observed
Ga values at different GdnHCl concentrations
were fitted by linear least squares analysis in accordance to Equation 2. The recovered parameters are listed in Table II. The
symbols are the same as in previous figures.
|
|
Protein Stability--
Unfolding of wild-type CRP and mutants was
monitored by CD in the presence of increasing concentrations of GdnHCl.
Ellipticity at 222 nm was employed as the parameter to reflect on the
changes in secondary structure. The unfolding curves of wild-type and mutant CRP are shown in Fig. 8. The
detectable differences in the unfolding curves are localized between 0 to 3 M GdnHCl, after which the curves seem to merge into
the same curve. The similarity among curves at high GdnHCl
concentration implies that these data points most likely reflect the
cooperative global unfolding of the proteins. The differences observed
at low GdnHCl concentration must reflect the differences in stability
of some secondary structures. Although the observed energetics of
subunit assembly are very similar for all the mutants, the
spectroscopically detected unfolding curves at low GdnHCl
concentrations are not identical for these mutants, as shown in Fig. 8.
This implies that there are likely differences in structural stability
among wild-type, insertion, and deletion mutants while there is no
observable differences between the deletion and double mutants. The
specific nature of the differences in stability has yet to be defined.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 8.
GdnHCl-induced unfolding of CRPs in TEK (100)
buffer. Protein concentrations used were 5 µM ( )
wild-type, ( ) deletion, ( ) insertion, ( ) double mutant. The
solid lines represent the best fits to show the trend of the
data.
|
|
Cyclic Nucleotide Binding--
Each cAMP molecule interacts with
one
-roll and both C-helices from adjacent subunits (1). It is not
surprising that the affinity to cAMP and the cooperativity of cAMP
binding are related to arrangements both within a CNB domain and
between the two CNB domains. The binding of cyclic nucleotide to CRP
was monitored by fluorescence, and the results are shown in Fig.
9. The binding isotherm for wild-type CRP
exhibits the biphasic behavior as reported before (22), representing
the binding of cAMP to the high and low affinity sites. The binding
isotherms for the deletion and double mutants are almost identical but
significantly different from that of wild-type CRP. They show a
significant degree of positive cooperativity, and the second binding
phase seems to be absent. The binding isotherm for the insertion mutant
coincides with the initial phase of that of the wild-type, but there is no evidence of another binding event at high concentrations of cAMP.
These results imply that the mutants have either lost their ability to
bind cAMP at the low affinity site or that the surfaces of these
mutants have been altered so significantly that the binding is not
reflected by ANS fluorescence. The binding of cAMP to the high affinity
sites in the insertion mutant exhibits similar affinity as that of
wild-type CRP. The difference is that wild-type CRP shows moderate
positive cooperativity of cAMP binding, whereas the insertion mutant
shows very weak negative cooperativity. On the contrary, the deletion
and double mutants show very strong positive cooperativity of cAMP
binding. The degree of positive cooperativity is in the following
decreasing order: deletion > double > wild-type > insertion, as reflected by the value of
k2/k1 summarized in Table
II. k1 values
measured for the deletion and double mutants are lower than that for
wild-type CRP, but k2 values of the deletion and
double mutants are significantly higher than that of wild-type CRP
(Table II). Meanwhile, the data for cGMP binding show that all the
mutations exert only insignificant effects (data not shown).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 9.
Binding of cAMP to CRPs in TEK (100) buffer
at pH 7.8 and 22.5 °C. Protein concentrations were 12.5 µM. ( ) wild-type, ( ) deletion, ( ) insertion,
( ) double mutant. The solid lines represent the best fits
of the data in accordance to Equation 5.
|
|
DNA Binding Assay--
Under the described experimental condition,
in the apo-state or in the presence of 230 µM cGMP, all
mutants and wild-type CRP showed only weak affinities for specific DNA:
the observed binding constants Kapp were
<1 × 104 M
1 for wild-type
CRP and insertion mutant and between 1 × 104 and
1 × 103 M
1 for the deletion
and double mutants. In the presence of 230 µM cAMP, all
these proteins exhibited a large increase in affinities for specific
DNA; the binding constants (Kapp, 1 × 10
7 M
1) are 5.6 ± 0.3 for wild-type CRP, 2.0 ± 0.1 for deletion, 3.5 ± 0.1 for insertion, and 2.4 ± 0.1 for double mutants, as shown in Fig.
10 and summarized in Table II. The
affinity of wild-type CRP for DNA is about 2- to 3-fold higher than all
of the mutants. These results indicate that these mutations do not
change the specific requirement of activation by cAMP. The mutations do
not qualitatively but quantitatively change the
cAMP-dependent DNA affinity of CRP, which responds only
specifically to cAMP and not to cGMP. In other words, these results
imply that these mutations perturb but do not change the mechanism of
allosteric communication between the CNB and DNA binding domains.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 10.
Binding of CRP to lac26 DNA
in TEK (100) buffer at pH 7.8 and 22.5 °C. ( ) wild-type,
( ) deletion, ( ) insertion, ( ) double mutant. The reaction
mixture contained 18 nM DNA and 230 µM cAMP.
The solid lines represent the best fits of the data in
accordance to Equation 6.
|
|
 |
DISCUSSION |
Some loops in proteins seem to serve as no more than connectors
between secondary structural elements, others play much more important
functional roles, such as in defining stability (29, 30). On the basis
of structural information and results of mutagenesis analyses, it was
proposed that loop 3 in CRP is involved in both interdomain and
intersubunit interactions, whereas loop 4 contacts the C-helices and is
proposed to form part of the dimer interface (1). Some point mutations,
either within or just outside of these loops, have been reported
to significantly affect the function of CRP, e.g. K52N,
D53H, and S62F (beside loop 3) and E72A, K82Q, and S83K (beside loop 4)
(21, 22, 31-34). Results from protein footprinting and NMR experiments
indicate that the regions, including these loops exhibit significant
environmental changes upon cAMP binding (18, 19). All these results
imply that these loops are involved in the functioning of CRP. This
conjecture on structure-function correlation is further supported by an
alignment analysis of the protein sequence of CNB (Fig. 2) that reveals
an intriguing pattern. The sizes of loops 3 and 4 are the only ones of
the loop structures that are varied. In cyclic nucleotide-gated
channels there are deletion and extension in the sequences of loop 3 and 4, respectively. In cyclic nucleotide-dependent protein
kinases, the sequence variation mostly resides in loop 3. Thus, it is
of interest to probe for the roles of these loops in CRP.
CRP is a transcription factor that exhibits allosteric behavior. There
is homotropic effects in the biding of cAMP and heterotropic effect
between cAMP and DNA bindings. The deletion of residues 54-56 (EEG) in
loop 3 leads to the most significant perturbations in the normal
functional properties of CRP. The binding affinity of the first cAMP
molecule is significantly weaker than that of the wild-type CRP,
however, the mutation also enhances the binding affinity for the second
cAMP molecule, leading to a significant enhancement of positive
cooperativity as indicated by the steeper binding isotherm (Fig. 9 and
Table II). These results indicate that the deletion of residues 54-56
alters the site-site interactions in cAMP binding, leading to an
enhancement of the homotropic effect. In addition, the deletion
mutation leads to a detectable, albeit small, decrease in DNA affinity,
i.e. a negative impact on the heterotropic effect between
cAMP and DNA binding sites (Fig. 10). This mutation also lowers the
energetics of subunit assembly. Thus, a perturbation of this loop
amplifies its impact on functional sites that are located at different
parts of CRP and spatially quite a few angstroms away. The specific
nature of functional impacts by loop 3 mutation on CRP is apparently
also observed in the various isozymes of protein kinases. As shown in
Figs. 1 and 2, the sequence differences between sites A and B of the regulatory subunit are often localized in loop 3, namely, in general there is a deletion of a few residues in loop 3 of site A as compared with site B. It has long been established that the binding affinity of
site A is lower than site B, an observation parallel that of the
deletion and wild-type CRP, respectively. It is not surprising that
cooperativity of ligand binding is different between these two type of
sites (35-42).
One might speculate on the mechanism that enables loop 3 to exert these
homotropic and heterotropic effects. Residue 136 of the adjacent
subunit forms a complex with loop 3. This might be one of the paths of
intersubunit and interdomain interactions. Any mutation that leads to a
perturbation of this interaction could be expected to affect the
allosteric behavior, as shown in this study. Furthermore, the nature of
the perturbation could be manifested to yield different functional
consequences. For example, mutations at residues 52 and 62 lead to
decreases in both homotropic and heterotropic effects, but mutation at
residues 53 leads to an opposite effect (21, 22). Thus, apparently, perturbations of residues in loop 3 can modulate the allosteric effects
either positively or negatively. Therefore, the role of loop 3 is a
modulator in the true sense.
The insertion of five residues in loop 4 seems to have only marginal
effects on the functional properties of CRP. There is no significant
perturbation in cAMP binding, and the effect on DNA binding is small,
although the energetics of subunit assembly is reduced. Thus, these
results are consistent with the proposal that loop 4 forms part of the
dimer interface (1). Results of the double mutant show that the
functional impact of loop 3 is dominant over that of loop 4, which only
modulates marginally the effects exerted by the deletion of loop 3.
Results of the double mutant of CRP can be compared with the report on
the chimeric protein that was constructed by linking the CNB domain
from Br
CNGC and the DNA binding domain from CRP (43). Although
there are significant differences in the primary sequences between the
CNB domains, the chimera reveals structure similar to that of wild-type
CRP. Loop 3 and loop 4 of the chimera are of the same sizes as the
double mutant of this study, thus it is valid to compare and contrast
these proteins. The affinity of the chimera to DNA is about 2-fold
lower than that of wild-type CRP (43) and is of the same level as the
double mutant of this study. This similarity strongly implies that it
is the two loops rather than the primary sequences that fine-tune the
DNA affinity of CRP. Based on the crystal structure of the CRP·cAMP
complex, there are two possible pathways through which these loops
exert their functional roles. Pathway one: they join in the pathway of
allosteric signal communication and are much involved in interfacial interactions between domains and subunits as it has been postulated (1,
9); pathway two: they are involved in ligand binding due to their
proximity to the ligand binding pocket (1, 5). Although the
non-conserved sequences of these loops within the CNB protein family
suggest that they are unimportant to the basic structure and function
of the CNB domain, results of this study indicate that these loops do
play a significant role in defining the affinity and cooperativity of
cAMP binding. However, the mutations in loop size quantitatively change
the cAMP-dependent DNA affinity of CRP without changing the
specificity of requiring cAMP but not cGMP for activation. Thus, it is
clear that these loops do not alter the basic mechanism of activity but
only modulate these activities. They may exert their effects via
regulating interfacial interactions and consequently regulating the
subunit realignment, such as that observed in this study.
The deletion of three residues in loop 3 not only affects the
functional properties of CRP, it significantly perturbs the structure.
The sedimentation velocity data shows that deletion causes a decrease
of 0.2 S, a change that is greater than the distribution for a 68%
confidence interval. Thus, this change indicates a perturbation in the
hydrodynamic properties of CRP. Thus, the deletion causes the CRP dimer
to either assume a more asymmetric or less compact conformation. The
change in the global conformation is associated with a decrease in the
energetics of subunit assembly and perturbations of the local
environments of aromatic residues as indicated by the spectroscopic
data. Results of the fluorescence studies indicate that the
accessibility of Trp residues is increased by the deletion mutation. It
was reported that Trp-13 is substantially solvent-exposed and
contributes to ~80% of the Trp fluorescence, whereas Trp-85 is
buried in the matrix of CRP and contributes to roughly 20% of the Trp
fluorescence (44). Results obtained using single-tryptophan-containing
CRP mutants indicate that Trp-85 is accountable for the total change observed of the intrinsic Trp fluorescence in wild-type CRP (45). The
above-cited reports all strongly suggest that the significant changes
on the tryptophan fluorescence in the deletion and the double mutants
are most likely attributable to Trp-85. Because Trp-85 is sterically
close to the dimer interface, the increased Trp solvent exposure is
consistent with the fact that the dimer interfacial interaction is
perturbed. The weakening of the intersubunit contact might lead to a
change in the subunit realignment induced by cAMP binding, as indicated
by the large shifts in emission spectra in the presence and absence of
cAMP. In contrast, the changes in the Trp fluorescence emission spectra
are small in wild-type CRP
It has been reported that the strong positive cooperativity and the
"functional polarity" are essential properties of the CNB domain of
CNGC. "Functional polarity" is a term employed by these
investigators to describe the phenomenon that the
-roll stabilizes
the ligand in a state-independent manner, whereas the C-helix
selectively stabilizes the ligand in the open state of the channel (46,
47). Just like the cooperativity of ligand binding in CRP, as was
discussed above, the functional polarity is also the result of
the subunit realignment. Recently, the subunit realignment involved in
the cAMP modulation of HCN channels has been reported (48). Therefore,
it is reasonable to postulate that the loosening of the intersubunit
contact caused by the truncated loop 3, which facilitates the subunit
realignment, is important for the modulation of CNGC.
In summary, mutations of loops 3, and to a smaller extent in loop 4, lead to global structural perturbations such as subunit alignment. As a
consequence, the functional properties are quantitatively altered. This
global effect is consistent with the reports from this laboratory on
other mutants (21, 22). All of these mutants affect the dynamic
behavior of CRP. A linear correlation can be established between the
allosteric behavior of these CRP species and protein
dynamics.2 Thus, a picture is
beginning to emerge to indicate that the allosteric behavior of CRP can
be represented by the presence of an ensemble of structures the
distribution of which can be perturbed by mutations, as in the case of
another allosteric system, dihydrofolate reductase (7).
 |
ACKNOWLEDGEMENTS |
We thank Dr. T. Izumi for his advice in
cloning procedures, Dr. E. Czerwinski for Fig. 1, and Drs. Xiaodong
Cheng and Qin Jiang for a critical review of the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM-45579 and by Grants H-0013 and H-1238 from the Robert A. Welch
Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 409-772-2281;
Fax: 409-772-4298; E-mail: jclee@utmb.edu.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M211551200
2
J. Li and J. C. Lee, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
PKA, cAMP-dependent protein kinases;
CRP, cyclic AMP receptor
protein;
CNB, cyclic nucleotide binding;
CNGC, cyclic nucleotide-gated
ion channels;
CD, circular dichroism;
GdnHCl, guanidine
hydrochloride;
CPM, N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl]
maleimide;
ANS, 8-anilino-1-naphthalene sulfonic acid.
 |
REFERENCES |
1.
|
Passner, J. M.,
Schultz, S. C.,
and Steitz, T. A.
(2000)
J. Mol. Biol.
304,
847-859[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Weber, I. T.,
Shabb, J. B.,
and Corbin, J. D.
(1989)
Biochemistry
28,
6122-6127[Medline]
[Order article via Infotrieve]
|
3.
|
Shabb, J. B.,
and Corbin, J. D.
(1992)
J. Biol. Chem.
267,
5723-5726[Free Full Text]
|
4.
|
Su, Y.,
Dostmann, R. G.,
Herberg, F. W.,
Durick, K.,
Xuong, N. H.,
Ten Eyck, L.,
Taylor, S. S.,
and Varughese, K. I.
(1995)
Science
269,
807-813[Medline]
[Order article via Infotrieve]
|
5.
|
Diller, T. C.,
Madhusudan,
Xuong, N. H.,
and Taylor, S. S.
(2001)
Structure
9,
73-82[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Scott, S. P.,
Harrison, R. W.,
Weber, I. T.,
and Tanaka, J. C.
(1996)
Protein Eng.
9,
333-344[Abstract]
|
7.
|
Pan, H.,
Lee, J. C.,
and Hilser, V. J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12020-12025[Abstract/Free Full Text]
|
8.
|
Weber, I. T.,
Steitz, T. A.,
Bubis, J.,
and Taylor, S. S.
(1987)
Biochemistry
26,
343-351[Medline]
[Order article via Infotrieve]
|
9.
|
Harman, J. G.
(2001)
Biochim. Biophys. Acta
1547,
1-17[Medline]
[Order article via Infotrieve]
|
10.
|
Kolb, A.,
Busby, S.,
Buc, H.,
Garges, S.,
and Adhya, S.
(1993)
Annu. Rev. Biochem.
62,
749-795[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Busby, S.,
and Ebright, R. H.
(1999)
J. Mol. Biol.
293,
199-213[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Heyduk, T.,
and Lee, J. C.
(1989)
Biochemistry
28,
6914-6924[Medline]
[Order article via Infotrieve]
|
13.
|
Passner, T. M.,
and Steitz, T. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2843-2847[Abstract/Free Full Text]
|
14.
|
DeGrazia, H.,
Harman, J. G.,
Tan, G. S.,
and Wartell, R. M.
(1990)
Biochemistry
29,
3557-3562[Medline]
[Order article via Infotrieve]
|
15.
|
Tan, G. S.,
Kelly, P.,
Kim, J.,
and Wartell, R. M.
(1991)
Biochemistry
30,
5076-5080[Medline]
[Order article via Infotrieve]
|
16.
|
Heyduk, E.,
Heyduk, T.,
and Lee, J. C.
(1992)
J. Biol. Chem.
267,
3200-3204[Abstract/Free Full Text]
|
17.
|
Baichoo, N.,
and Heyduk, T.
(1997)
Biochemistry
36,
10830-10836[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Baichoo, N.,
and Heyduk, T.
(1999)
Protein Sci.
8,
518-528[Abstract]
|
19.
|
Won, H. S.,
Yamazaki, T.,
Lee, T. W.,
Yoon, M. K.,
Park, S. H.,
Otoma, T.,
Kyogoku, Y.,
and Lee, B. J.
(2000)
Biochemistry
39,
13953-13962[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Won, H. S.,
Lee, T. W.,
Park, S. H.,
and Lee, B. J.
(2002)
J. Biol. Chem.
277,
11450-11455[Abstract/Free Full Text]
|
21.
|
Lin, S. H.,
Kovac, L.,
Chin, A. L.,
Chin, C. C. Q.,
and Lee, J. C.
(2002)
Biochemistry
41,
2946-2955[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Lin, S. H.,
and Lee, J. C.
(2002)
Biochemistry
41,
11857-11867[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Sambrook, J.,
and Russel, D. W.
(2001)
Molecular Cloning: A Laboratory Manual
, 3rd Ed., Vol. 2
, pp. 13.36-13.39, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
|
24.
|
Cheng, X.,
Kovac, L.,
and Lee, J. C.
(1995)
Biochemistry
34,
10816-10826[Medline]
[Order article via Infotrieve]
|
25.
|
Cohn, E. J.,
and Edsall, J. T.
(1943)
Proteins, Amino Acids and Peptides
, p. 372, Van Nostrans-Reinhold, NJ
|
26.
|
Lee, J. C.,
and Timasheff, S. N.
(1979)
Methods Enzymol.
61,
49-57[Medline]
[Order article via Infotrieve]
|
27.
|
Cheng, X.,
and Lee, J. C.
(1998)
Biochemistry
37,
51-60[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Yphantis, D. A.
(1964)
Biochemistry
3,
297-317
|
29.
|
Thompson, M. J.,
and Eisenberg, D.
(1999)
J. Mol. Biol.
290,
595-604[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Minard, P.,
Scalley-Kim, M.,
Watters, A.,
and Baker, D.
(2001)
Protein Sci.
10,
129-134[Abstract/Free Full Text]
|
31.
|
Gronenborn, A. M.,
Sandulache, R.,
Gartner, S.,
and Clore, G. M.
(1988)
Biochem. J.
253,
801-807[Medline]
[Order article via Infotrieve]
|
32.
|
Chu, S. Y.,
Tordova, M.,
Gilliland, G. L.,
Gorshkova, I.,
Shi, Y.,
Wang, S.,
and Schwarz, F. P.
(2001)
J. Biol. Chem.
276,
11230-11236[Abstract/Free Full Text]
|
33.
|
Moore, J.,
Kantorow, M.,
Vanderzwaag, D.,
and McKenney, K.
(1992)
J. Bacteriol.
174,
8030-8035[Abstract]
|
34.
|
Lee, E. J.,
Glasgow, J.,
Leu, S. F.,
Belduz, A. O.,
and Harman, J. G.
(1994)
Nucleic Acids Res.
22,
2894-2901[Abstract]
|
35.
|
Corbin, J. D.,
Sugden, P. H.,
West, L.,
Flockhart, D. A.,
Lincoln, T. M.,
and McCarty, D.
(1978)
J. Biol. Chem.
253,
3997-4003[Medline]
[Order article via Infotrieve]
|
36.
|
Ogreid, D.,
and Doskeland, S. O.
(1981)
FEBS Lett.
129,
287-292[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Ogreid, D.,
and Doskeland, S. O.
(1982)
FEBS Lett.
150,
161-166[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Ogreid, D.,
Doskeland, S. O.,
and Miller, J. P.
(1983)
J. Biol. Chem.
258,
1041-1049[Abstract/Free Full Text]
|
39.
|
Robinson-Steiner, A. M.,
and Corbin, J. D.
(1983)
J. Biol. Chem.
258,
1032-1040[Free Full Text]
|
40.
|
Doskeland, S. O.,
and Ogreid, D.
(1984)
J. Biol. Chem.
259,
2291-2301[Abstract/Free Full Text]
|
41.
|
Bubis, J.,
and Taylor, S. S.
(1987)
Biochemistry
26,
3478-3486[Medline]
[Order article via Infotrieve]
|
42.
|
Herberg, F. W.,
Taylor, S. S.,
and Dostmann, W. R. G.
(1996)
Biochemistry
35,
2934-2942[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Scott, S. P.,
Weber, I. T.,
Harrison, R. W.,
Carey, J.,
and Tanaka, J. C.
(2001)
Biochemistry
40,
7464-7473[Medline]
[Order article via Infotrieve]
|
44.
|
Wasylewski, M.,
Malecki, J.,
and Wasylewski, Z.
(1995)
J. Protein Chem.
14,
299-308[Medline]
[Order article via Infotrieve]
|
45.
|
Malecki, J.,
Polit, A.,
and Wasylewski, Z.
(2000)
J. Biol. Chem.
275,
8480-8486[Abstract/Free Full Text]
|
46.
|
Tibbs, G. R.,
Liu, D. T.,
Leypold, B. G.,
and Siegelbaum, S. A.
(1997)
J. Biol. Chem.
273,
4497-4505[Abstract/Free Full Text]
|
47.
|
Richards, M. J.,
and Gordon, S. E.
(2000)
Biochemistry
39,
14003-14011[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Wainger, B. J.,
DeGennaro, M.,
Santoro, B.,
Slegelbaum, S. A.,
and Tibbs, G. R.
(2001)
Nature
411,
805-809[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.