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(Received for publication, June, 25, 1997, and in revised form, August 1, 1997)
From the ABSTRACT Catabolite repression of a number of catabolic
operons in bacilli is mediated by the catabolite control protein CcpA,
the phosphocarrier protein HPr from the
phosphoenolpyruvate-dependent sugar transport system (PTS),
and a cis-acting DNA sequence termed the
catabolite-responsive element (cre). We present evidence
that CcpA interacts with HPr that is phosphorylated at
Ser46 (Ser(P) HPr) and that these proteins form a specific
ternary complex with cre DNA. Titration experiments
following the circular dichroism signal of the cre DNA
indicate that this complex consists of two molecules of Ser(P) HPr, a
CcpA dimer, and the cre sequence. Limited proteolysis
experiments indicate that the domain structure of CcpA is similar to
other members of the LacI/GalR family of helix-turn-helix proteins,
comprised of a helix-turn-helix DNA domain and a C-terminal effector
domain. NMR titration of Ser(P) HPr demonstrates that the isolated
C-terminal domain of CcpA forms a specific complex with Ser(P) HPr but
not with unphosphorylated HPr. Based upon perturbations to the NMR
spectrum, we propose that the binding site of the C-terminal domain of
CcpA on Ser(P) HPr forms a contiguous surface that encompasses both
Ser(P)46 and His15, the site of phosphorylation
by enzyme I of the PTS. This allows CcpA to recognize the
phosphorylation state of HPr, effectively linking the process of sugar
import via the PTS to catabolite repression in bacilli.
INTRODUCTION Catabolite repression
(CR)1 in Escherichia
coli has provided a general paradigm for understanding the
regulation of the synthesis of various catabolic enzymes in response to
the availability of rapidly metabolizable carbon sources. The mechanism
for CR in bacteria such as E. coli involves the cyclic
AMP-dependent action of the catabolite gene activator
protein, CAP (1). In Bacillus subtilis, however, the
mechanism is completely different, because no cyclic AMP or CAP
homologue is present (2). CR in some bacilli and a few other
Gram-positive organisms has been shown to be dependent on the
catabolite control protein A (CcpA), which is a member of the LacI/GalR
family of regulators (3, 4), and a cis-active operator DNA
sequence, termed the catabolite-responsive element (cre),
which has been identified in the promoter or in the 5 There is growing evidence that CR is mechanistically linked to the
phosphoenolpyruvate-dependent sugar transport system (PTS), which is responsible for the import of various sugars (1). Mutation of
Ser46 to Ala in the PTS phosphocarrier protein HPr results
in resistance to CR for several catabolic genes in B. subtilis (6). Ser46 in HPr is known to be
phosphorylated by an ATP-dependent kinase that is activated
by glycolytic intermediates such as fructose-1,6-diphosphate (7).
Confirming the link between CR and the PTS, recent DNase I protection
experiments have shown that cre sequences are specifically protected by CcpA only in the presence of HPr phosphorylated at Ser46 (Ser(P) HPr) (8). Other mechanisms for CR have been
established; CcpA is known to bind to the cre sequence of
the amyE operon in the absence of any corepressor (9, 10),
and CR of the B. subtilis lev and bgl operons
appear to be regulated by a mechanism involving Ser(P) HPr and the
transcriptional activator LevR or the antiterminator LicT but not CcpA
(11, 12). CcpA-mediated CR is therefore not the only mechanism for CR,
although it is probably the dominant mechanism (13).
Based upon these data, a general model for CR in Gram-positive bacteria
has been proposed. The availability of a rapidly metabolizable carbon
source results in elevated levels of glycolytic intermediates, which
leads to the phosphorylation of Ser46 of HPr. Ser(P) HPr is
proposed to interact with CcpA, resulting in negative regulation at the
level of transcription of a number of catabolic genes. A direct
interaction between CcpA and Ser(P) HPr is suggested based upon the
observation that elution of Ser(P) HPr from a Ni2+-NTA
column loaded with His-tagged CcpA is retarded, whereas elution of
unphosphorylated HPr is not (14). A specific ternary complex involving
CcpA, Ser(P) HPr, and cre DNA is suggested by results from
DNase I footprinting protection studies; specific protection of
cre sequences by CcpA only occurs in the presence of Ser(P) HPr (8, 13). To date, many of the details of how this complex is formed
are unclear.
We have therefore used a variety of biophysical methods to characterize
CcpA and its interactions with DNA and Ser(P) HPr. The results
demonstrate the formation of a ternary complex between CcpA, Ser(P)
HPr, and cre DNA. Binding of the binary CcpA·Ser(P) HPr
complex to cre DNA is clearly distinguishable from the
nonspecific complex formed between CcpA and cre DNA in the
absence of Ser(P) HPr. Additionally, the binding site for CcpA on
Ser(P) HPr allows CcpA to recognize the phosphorylation state of HPr,
effectively linking the process of sugar import via the PTS to
catabolite repression.
EXPERIMENTAL PROCEDURES His6-CcpA from
Bacillus megaterium was purified using a
Ni2+-NTA resin (Qiagen) as described previously (13, 15).
Following elution with imidazole, CcpA was dialyzed in 10 mM Tris-HCl (pH 7.5). Protein was quantified using the
method of Gill and von Hippel (16). B. subtilis HPr was
purified as described previously (17). Ser(P) HPr was generated by
phosphorylation and purified as described previously (18).
15N isotopic labeling of HPr was accomplished by growing
bacteria in minimal medium with [15N]NH4Cl
(Isotec) as the sole nitrogen source.
Synthetic oligonucleotides (Macromolecular Resources, Colorado State
University) were purified as described previously (19). The
single-stranded DNA was annealed in 10 mM MgCl2
by heating to 70 °C and slowly cooling to room temperature. Exact
ratios of each strand for annealing reactions were determined by
polyacrylamide gel analysis of multiple annealing reactions and silver
staining. The double-stranded DNA was quantified assuming
A260 - 1.0 corresponds to 33 µg/ml.
CD experiments were performed on an Aviv
62DS Circular Dichroism Spectrapolarimeter. Typically, experiments were
performed with a 1.5 nm band width, and data points were taken every nm with a 15-s averaging time. DNA concentrations ranged from 5 to 40 µM, and either 1- or 10-mm path length cuvettes were
used. The buffer used in the CD experiments was 10 mM
Tris-HCl (pH 7.5) containing 150 mM NaCl.
Limited proteolysis
experiments were carried out with trypsin, chymotrypsin, or clostripain
(Worthington Enzymes) in 10 mM Tris-HCl (pH 7.5) containing
50 mM NaCl. For clostripain digests, the buffer also
contained 1 mM CaCl2 and 1 mM
dithiothreitol. Typically, the CcpA concentration was 1 mg/ml, and the
ratio of substrate to protease was 100:1 (w/w). Time course reactions
were quenched with 1 mM
1-chloro-3-tosylamido-7-amino-2-heptanone (for trypsin), 1 mM phenylmethylsulfonyl fluoride (for chymotrypsin), or 10 mM EDTA (for clostripain) before analysis by either
SDS-polyacrylamide gel electrophoresis (20) or MALDI-TOF mass
spectrometry. For samples analyzed by mass spectrometry,
trifluoroacetic acid was added to a final concentration of 0.1%.
MALDI-TOF mass spectra were recorded on a Perceptive Biosystems Voyager
Elite (University of Washington Mass Spectrometry Facility) using
3,5-dimethoxy-4-hydroxy-cinnamic acid as the matrix (21).
Purification of CcpA-C was accomplished by limited proteolysis with
clostripain (1 mg/ml CcpA concentration and 100:1 (w/w) ratio of CcpA
to clostripain) for 90 min. The reaction was quenched by the addition
of 10 mM EDTA. Samples were lyophilized, resuspended in
one-tenth the original volume with H2O, and purified by
size exclusion chromatography on a 50 × 2-cm Sephacryl S-100
column (Pharmacia Biotech Inc.).
Titrations of uniformly
15N-labeled HPr (0.5 mM) or Ser(P) HPr (0.4 mM) with CcpA-C were carried out at 30 °C. The
appropriate amounts of protein were dialyzed extensively in 5 mM sodium phosphate buffer (pH 6.9) and lyophilized. The
HPr or Ser(P) HPr was dissolved in 500 µl of 10%
2H2O/90% H2O; the CcpA-C was
dissolved in 100 µl. Titrations were accomplished by the addition of
CcpA, followed by a 15-min equilibration period before recording the
[1H-15N]HSQC spectra. NMR experiments were
performed on a Bruker DMX-500 MHz spectrometer.
[1H-15N]HSQC spectra were recorded with
spectral widths of 7002 Hz (1H) × 2000 Hz
(15N). Data were processed using NMRPipes (22).
RESULTS Near-UV CD has been demonstrated
to be sensitive to changes in either the environment or structure of
DNA (for review see Ref. 23). Therefore, we used CD studies to monitor
a DNA oligonucleotide containing a consensus cre sequence in
the presence of CcpA with and without Ser(P) HPr. Fig.
1 shows various CD spectra of a 26-base pair duplex oligonucleotide in the presence and the absence of CcpA and
Ser(P) HPr. The DNA sequence is
5
We took advantage of the large change in CD signal observed upon the
formation of the protein-DNA complex to determine an apparent binding
constant. The results of titration of cre DNA (5 µM) with CcpA in the presence of excess Ser(P) HPr (40 µM) are shown in Fig. 2.
The CD titration showed a clear isodichroic point (at 278 nm),
indicating that a simple two-state equilibrium can be used to obtain an
approximate affinity. The observation of an isodichroic point, which
occurs at the same wavelength observed in the comparison of the spectra
of DNA alone compared with the ternary complex (Fig. 1) and which is
different from the isodichroic point (~273 nm) for the comparison of
the spectra of DNA in the presence of CcpA and the ternary complex,
strongly suggests that the CD titration data are monitoring the
formation of the ternary complex from the DNA and a CcpA·Ser(P) HPr
complex. The change in CD signal at 267 nm is well fit to a single
binding event with an apparent Kd of 4.5 ± 0.5 µM. The determined Kd represents the
apparent dissociation constant for the formation of the
ternary complex; because the CD signal is only sensitive to the
formation of the ternary complex, it is not possible to measure the
true dissociation constants directly for each of the possible binary
complexes.
The measured affinity of CcpA binding to DNA is in a range that allowed
us to determine the stoichiometry for the interaction between Ser(P)
HPr and the CcpA dimer from the change in the CD signal. The
oligonucleotide used contains only one cre sequence and is
therefore sufficient for only one CcpA dimer. The change in CD signal
during a titration of 40 µM cre DNA and 40 µM CcpA dimer was essentially linear up to 80 µM Ser(P) HPr (data not shown); further additions had no
further effect on the CD spectrum, indicating that saturation has been
reached. These results indicate that the ternary complex contains two
molecules of Ser(P) HPr.
The CD results demonstrate the existence of a ternary complex formed
between CcpA, Ser(P) HPr, and cre DNA. This supports the
hypothesis that Ser(P) HPr is necessary for CcpA to bind specifically and with high affinity to cre sequences. Although these
results do not directly rule out the possibility that the function of Ser(P) HPr is to increase the affinity of CcpA for DNA, specific or
otherwise, the differences in the CD spectra for CcpA + DNA and CcpA + DNA + Ser(P) HPr suggest that Ser(P) HPr does alter the mode of DNA
binding by CcpA. Other evidence also indicates that the interaction is
specific to cre sequences; DNase I protection studies of
CcpA in the presence of Ser(P) HPr show protection only within
cre sequences (8, 9, 13), suggesting that a ternary complex
is specifically formed with the cre sequence only.
In an effort
to characterize CcpA and its interaction with either DNA or Ser(P) HPr
further, we performed limited proteolysis experiments. Full-length CcpA
(monomeric mass of ~39 kDa) was subjected to proteolysis using
trypsin, chymotrypsin, or clostripain, and the time points were
analyzed by SDS-polyacrylamide gel electrophoresis. In each case, a
fragment of ~31 kDa was generated within the first minute and
remained relatively resistant to further proteolysis (Fig.
3). Samples from each digest were
analyzed by MALDI-TOF mass spectroscopy to determine the prominent
cleavage sites. Some fragments whose identity could be unambiguously
assigned based upon their molecular mass and cleavage sites for each
enzyme are indicated in representative mass spectra for proteolysis
samples (Fig. 4). The ~31-kDa fragments
observed as protease-resistant bands on the SDS gels all correspond to
a large C-terminal fragment that begins between residues 54 and 59 and
extends to the C terminus of the protein, Lys332.
The domain structure of CcpA has been predicted based on its strong
sequence homology to other helix-turn-helix regulators such as LacI,
GalR, and PurR (3, 25) and is shown schematically in Fig. 4. The
initial proteolytic fragments are consistent with this proposal; for
each protease, cleavage occurs in the region between the DNA binding
helix-turn-helix domain and the C-terminal domain, producing a stable
C-terminal fragment. The observation that all three proteases cleave
between residues 54 and 59 suggests that this region is most likely
the separation point between the DNA binding domain and the C-terminal
domain.
Limited proteolysis experiments were also performed in the presence of
Ser(P) HPr, cre DNA, or both to determine whether the formation of a binary or ternary complex would lead to protection of
CcpA from cleavage. Under the conditions used for these experiments (typically 20-30 µM CcpA monomer, 100 µM
Ser(P) HPr, and 60 µM DNA), no change in resistance to
proteolysis was observed; this is in contrast to results of similar
experiments with PurR, its corepressor, and its cognate DNA sequence
(26, 27). These results indicate that the susceptibility of the linker
between the DNA binding helix-turn-helix domain and the C-terminal
domain is not greatly altered by the formation of any of the possible complexes or that the dissociation of any complex with CcpA occurs sufficiently fast to allow proteolysis to occur.
The CD results
demonstrate the formation of a ternary complex between the cre-DNA,
CcpA, and Ser(P) HPr. Previous elution-retardation experiments
indicated that Ser(P) HPr interacts directly with CcpA (14); however,
the question remains as to which regions on CcpA and Ser(P) HPr are
important for this interaction. Interactions between HPr and its PTS
protein partners have been characterized by NMR spectroscopy (28-31).
We employed a similar approach, in which
[1H-15N]HSQC spectra of
15N-labeled HPr (or Ser(P) HPr) are monitored as a function
of added CcpA. Because CcpA was not isotopically
15N-labeled, its effect on HPr can be observed directly in
these spectra.
Based upon sequence similarities to proteins with known
three-dimensional structures such as LacI (32) and PurR (33), we
reasoned that the C-terminal effector domain is likely responsible for
binding Ser(P) HPr; the N-terminal helix-turn-helix domain presumably
binds DNA, as has been observed for a proteolytic fragment of LacI
corresponding to the helix-turn-helix DNA binding domain (34). To test
this hypothesis, the C-terminal domain was generated by limited
proteolysis and purified. Quantities of the C-terminal domain (CcpA-C)
sufficient for NMR studies were isolated by treatment of CcpA with
clostripain, followed by purification by gel filtration. Similar to
intact CcpA, which is a dimer (3, 15), CcpA-C elutes on a gel
filtration column with an apparent molecular mass of ~60 kDa,
indicating that this domain, isolated from the DNA binding domain, also
exists as a dimer in solution (data not shown).
Aliquots of purified CcpA-C were titrated into a sample of 0.4 mM 15N-labeled Ser(P) HPr, and NMR spectra were
collected following each addition. Fig. 5
shows [15N-1H]HSQC spectra at various points
in the titration. Upon the addition of 0.25 mol equivalents CcpA-C, a
subset of resonances (indicated in Fig. 5A) either broaden
dramatically or disappear completely. As additional CcpA-C is added,
most peaks in the spectrum completely disappear. This behavior is
indicative of line broadening of resonances whose environment is
altered by the interaction between Ser(P) HPr and CcpA-C.
Unfortunately, due to the large size of the complex (~80 kDa; the
CcpA-C dimer at ~62 kDa and two Ser(P) HPr molecules at 9 kDa each),
the resonances for Ser(P) HPr in the complex are not observed. The
gradual disappearance of nonperturbed resonances, in contrast to the
very rapid disappearance of a subset of resonances, suggests that
exchange between free and bound is occurring in the fast to
intermediate time regime (relative to the chemical shift differences of
resonances in the free and bound
states).2 Therefore,
resonances that are perturbed most and consequently have the largest
chemical shift changes due to the formation of the complex should
disappear early in the titration, allowing for the determination of
which portions of Ser(P) HPr are most affected in its interaction with
CcpA-C.
Unfortunately, similar experiments with intact CcpA were impossible due
to insufficient solubility of the intact protein. A similar titration
of unphosphorylated HPr showed neither changes in intensity nor line
width changes, indicating that CcpA-C does not bind unphosphorylated
HPr, even at millimolar concentrations (Fig. 5D). Thus, the
NMR results confirm that the C-terminal domain is responsible for the
formation of a binary complex with Ser(P) HPr and that this interaction
absolutely requires that Ser46 be in its phosphorylated
state.
The residues that are selectively broadened at low concentrations of
CcpA form a contiguous surface on Ser(P) HPr (Fig.
6). The binding surface comprises two
DISCUSSION The results presented here shed light on the general mechanism for
CcpA-mediated CR and suggest a particular order of events from the
initial phosphorylation of HPr at Ser46 to the repression
of transcription by CcpA. The observation that a specific complex is
formed between Ser(P) HPr and CcpA in the absence of DNA suggests that
this interaction must precede the recognition of cre
sequences. The apparent increase in affinity for cre DNA by
CcpA in the presence of Ser(P) HPr and the observation of the ternary
complex also support this sequence of events.
It is remarkable to consider the effect of Ser46
phosphorylation on the binding of CcpA. Considering the apparent
Kd for the formation of the ternary complex (4.5 µM), phosphorylation of Ser46 on HPr leads to
an increase in affinity for CcpA by at least 3 orders of magnitude over
unphosphorylated HPr (no binding is observed at 0.5 mM HPr
and CcpA, Fig. 5D). NMR studies have revealed that
phosphorylation of Ser46 does not induce a conformational
change in HPr (36). Thus, CcpA must specifically recognize the
phosphoserine moiety, suggesting that the interaction has a large
electrostatic component.
It is important to note that the two proteins are from different
species; the CcpA used is from B. megaterium, and the HPr is
from B. subtilis. However, the HPrs from these two species exhibit 81% sequence identity, and the CcpAs exhibit 76% sequence identity. More importantly, however, there are only three amino acid
differences between the two species of HPr in the binding site for CcpA
as identified by the present results (Fig. 6); residues Thr21, Ala25, and Gln56 in B. megaterium are replaced with Val21, Thr25,
and Ala56 in B. subtilis. Therefore, the
apparent Kd measured in this heterologous system is
probably similar to either homologous system.
The crystal structure of another family member, the
PurR-DNA-hypoxanthine ternary complex (33) provides insight into the mode of DNA binding by CcpA. Upon binding to DNA, a leucine
(Leu56) from each monomer of PurR is inserted between bases
through the minor groove of the DNA, resulting in a bend in the DNA of ~45 °. A sequence comparison between CcpA and PurR indicates that this leucine is conserved, as are a number of residues preceding this
side chain that make contacts to the DNA phosphate backbone in the
PurR-DNA complex structure. The large changes in the DNA CD spectrum we
observe are consistent with a significant structural change in the DNA,
suggesting that CcpA, in the presence of Ser(P) HPr, may interact with
DNA in a manner similar to PurR. This conclusion is supported by nearly
identical CD spectral changes observed with PurR and its cognate DNA
sequence.3
The apparent structural similarity of CcpA to other helix-turn-helix
proteins is interesting in light of its mode of action. Other
helix-turn-helix proteins require small molecules for regulation, which
upon binding cause the protein to bind to specific DNA sequences and
affect transcription in some manner. Although the end result is the
same, CcpA must bind another protein, namely Ser(P) HPr, to manifest a
change in its DNA binding ability. A structural insight as to how this
might occur can be gained from the three-dimensional structures of LacI
and its complex with the coeffector
isopropyl-1-thio- FOOTNOTES ACKNOWLEDGEMENTS We thank David Baker for use of the CD
spectrometer, Peter Brzovic for assistance with MALDI-TOF mass
spectroscopy, and Richard Brennan for initial proteolysis suggestions,
for providing us with purified PurR and DNA, and for helpful
discussions.
REFERENCES
Volume 272, Number 42,
Issue of October 17, 1997
pp. 26530-26535
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding of the Catabolite Repressor Protein CcpA to Its DNA
Target Is Regulated by Phosphorylation of its Corepressor HPr*
,
University of Washington, Department of
Biochemistry and Biomolecular Structure Center, Seattle, Washington
98195-7742, the ¶ Lehrstuhl für Mikrobiologie, Institut
für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander
Universität Erlangen-Nürnberg, Staudstrasse 5, 91058 Erlangen, Germany, and the § Institut de Biologie et Chime
des Protéines, CNRS, 7 passage du Vercors, F-69367
Lyon Cedex 07, France
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
region of 29 B. subtilis genes (5).
-GGGCTGATTTGAAAGCGCTAACAGGG-3 and its complement, where
the underlining denotes the 14-base pair consensus cre
sequence (5, 24). The presence of an equimolar amount of CcpA dimer (30 µM DNA duplex and 30 µM CcpA dimer) results in a moderate change in the DNA CD spectrum compared with free DNA;
there is a slight decrease in ellipticity near 265 nm and a slight
increase in ellipticity at ~285 nm. However, the presence of Ser(P)
HPr (60 µM) results in much more pronounced changes, with
a maximal decrease in ellipticity occurring at ~267 nm. The observation that the CD spectrum of DNA in the presence of CcpA is
different from the free DNA spectrum suggests that there is some
nonspecific interaction between the protein and DNA, even at 150 mM NaCl. However, it is clearly different from what is observed in the presence of Ser(P) HPr. Identical results are obtained
under low ionic strength conditions; although the spectrum of the free
DNA is slightly different, the spectra of DNA + CcpA and DNA + CcpA + Ser(P) HPr are superimposable with the spectra shown in Fig. 1 (data
not shown).
Fig. 1.
CD spectra of cre DNA. DNA
(30 µM) in the presence of CcpA dimer (30 µM) or CcpA and Ser(P) HPr (60 µM) are
shown. Experiments were performed in 10 mM Tris-HCl (pH
7.5) containing 150 mM NaCl.
[View Larger Version of this Image (26K GIF file)]
Fig. 2.
CD titration of DNA with CcpA. A,
CD spectra of cre DNA (5 µM) in the presence of excess
HPr (40 µM) at the various concentrations of CcpA (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, and 15.0 µM CcpA). B, plot of the ellipticity at 267 nm
as function of [CcpA]. The fit is for a model of a two-state binding
reaction in which the complex is assumed to be formed from two
components and assumes a single binding site on the DNA for CcpA.
Experiments were performed in 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl.
[View Larger Version of this Image (25K GIF file)]
Fig. 3.
SDS-polyacrylamide gel electrophoresis
analysis of the limited proteolysis of CcpA. Coomassie-stained
SDS-polyacrylamide gels (14%) of proteolysis time courses of CcpA by
trypsin (A), chymotrypsin (B), and clostripain
(C). The time points in A are: 0, 1, 2, 5, 10, 20, 30, and 60 min after the addition of protease. The time points in
B are: 0, 1, 2, 5, 10, 20, 30, 45, and 60 min after the
addition of protease. The time points in C are: 0, 2, 5, 10, 20, 30, and 60 min after the addition of protease. The numbers to the left of each gel indicate the
molecular mass of the standards (in kDa).
[View Larger Version of this Image (29K GIF file)]
Fig. 4.
MALDI-TOF mass spectra of proteolytic digests
of CcpA. Mass spectra shown are for the 2-min time points taken
during limited proteolysis by trypsin (A), chymotrypsin
(B), and clostripain (C). A schematic of the
proposed domain structure of CcpA based on sequence homology to PurR is
shown above A.
[View Larger Version of this Image (33K GIF file)]
Fig. 5.
NMR titration of Ser(P) HPr with CcpA-C.
[1H-15N]HSQC spectra of a titration of
15N-labeled Ser(P) HPr (0.4 mM) with CcpA-C.
Residues that disappear completely upon addition of 0.25 equivalents of
CcpA-C (monomer) are indicated in A. The spectrum shown in
D is of unphosphorylated HPr (0.5 mM) with 1.2 equivalents of CcpA (monomer). (Note: the three intense peaks that
persist in the spectrum of Ser(P) HPr are due to a contaminant.)
[View Larger Version of this Image (27K GIF file)]
-helices in HPr; Helix A, which includes residues within the active
site of HPr (residues near His15), and Helix B, which
includes Ser46. Interestingly, this surface is very similar
to the surface recognized by both enzyme I (30) and enzyme IIA (29, 31)
of the PTS. The observation that the binding site for CcpA encompasses
His15 suggests that CcpA can also discriminate whether HPr
is phosphorylated at His15, consistent with previous
results suggesting that CcpA does not interact with HPr phosphorylated
at His15 (14) or mutated at His15 (35).
Interestingly, the interaction of HPr with enzyme IIA and probably
enzyme I is diminished upon phosphorylation at Ser46,
allowing the PTS to sense the phosphorylation state of
Ser46 in HPr. Thus, the regulatory phosphorylation of
Ser46 in HPr effectively coordinates the functions of the
import of sugars and the synthesis of catabolic enzymes.
Fig. 6.
Location of the binding site for CcpA-C on
Ser(P) HPr. Stereoview of HPr showing the residues that are
selectively broadened by CcpA-C as a dot surface; these residues are:
14-17, 21-27, 43, 44, 46-56, 78, 81, and 82. The location of
Ser46 and the active site residue His15 are
also indicated. The residues not shaded blue in Helix A are Pro18 (which there is no amide proton to detect in the NMR
spectra), Ala19, and Thr20; the latter two
residues exhibit some broadening but not to the extent that is observed
with the other shaded residues. This figure was made using MidasPlus
(37) and the coordinates for the refined solution structure of B. subtilis HPr (Protein Data Bank entry 2HID) (38).
[View Larger Version of this Image (68K GIF file)]
-D-galactopyranoside (32). In this
case, binding of isopropyl-1-thio--D-galactopyranoside to LacI results in a conformational change within the C-terminal effector domain that is proposed to reposition the DNA binding domains
of each monomer relative to one another. Whether Ser(P) HPr performs
its regulatory role in an analogous manner must await future
characterization of this system.
To whom correspondence should be addressed: University of
Washington, Dept. of Biochemistry and Biomolecular Structure Center, Box 357742, Seattle, WA 98195-7742. Fax: 206-543-8394; E-mail: klevit{at}u.washington.edu.
1
The abbreviations used are: CR, catabolite
repression; CcpA-C, C-terminal proteolytic domain of CcpA; HSQC,
heteronuclear single quantum coherence spectroscopy; MALDI-TOF,
matrix-assisted laser desorption time-of-flight mass spectrometry;
Ser(P) HPr, HPr phosphorylated at Ser46; PTS,
phosphoenolpyruvate sugar phosphotransferase system.
2
In the fast to intermediate exchange regime, the
observed line width for a peak will be the sum of the
population-weighted average of the line widths of the free and bound
species and an additional chemical exchange contribution that scales as
fffb
fb2,
where ff and fb are the
mole fractions of the free and bound species and fb
is the difference in chemical shift (in Hz) between the free and bound
species. This predicts that all resonances that do not undergo a change
in chemical shift upon complex formation will disappear (broaden) from
the spectrum as a function of mole fraction of bound species, whereas
resonances that undergo a change in chemical shift will broaden and
disappear early in the titration due to the squared dependence on the
chemical shift perturbation. This is precisely the behavior observed in
the titration of CcpA-C into Ser(P) HPr.
3
B. Jones, R. Klevit, and R. Brennan, unpublished
results.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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  B. Joseph, S. Mertins, R. Stoll, J. Schar, K. R. Umesha, Q. Luo, S. Muller-Altrock, and W. Goebel Glycerol Metabolism and PrfA Activity in Listeria monocytogenes J. Bacteriol., August 1, 2008; 190(15): 5412 - 5430. [Abstract] [Full Text] [PDF]
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 V. Chaptal, F. Vincent, V. Gueguen-Chaignon, V. Monedero, S. Poncet, J. Deutscher, S. Nessler, and S. Morera Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme J. Biol. Chem., November 30, 2007; 282(48): 34952 - 34957. [Abstract] [Full Text] [PDF]
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D. M. Daigle, L. Cao, S. Fraud, M. S. Wilke, A. Pacey, R. Klinoski, N. C. Strynadka, C. R. Dean, and K. Poole Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa J. Bacteriol., August 1, 2007; 189(15): 5441 - 5451. [Abstract] [Full Text] [PDF]
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 J. Deutscher, C. Francke, and P. W. Postma How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 939 - 1031. [Abstract] [Full Text] [PDF]
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M. A. Schumacher, G. Seidel, W. Hillen, and R. G. Brennan Phosphoprotein Crh-Ser46-P Displays Altered Binding to CcpA to Effect Carbon Catabolite Regulation J. Biol. Chem., March 10, 2006; 281(10): 6793 - 6800. [Abstract] [Full Text] [PDF]
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A. J. Wolfe The Acetate Switch Microbiol. Mol. Biol. Rev., March 1, 2005; 69(1): 12 - 50. [Abstract] [Full Text] [PDF]
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C. I. Caescu, O. Vidal, F. Krzewinski, V. Artenie, and S. Bouquelet Bifidobacterium longum Requires a Fructokinase (Frk; ATP:D-Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism J. Bacteriol., October 1, 2004; 186(19): 6515 - 6525. [Abstract] [Full Text] [PDF]
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T. Maurer, S. Meier, N. Kachel, C. E. Munte, S. Hasenbein, B. Koch, W. Hengstenberg, and H. R. Kalbitzer High-Resolution Structure of the Histidine-Containing Phosphocarrier Protein (HPr) from Staphylococcus aureus and Characterization of Its Interaction with the Bifunctional HPr Kinase/Phosphorylase J. Bacteriol., September 1, 2004; 186(17): 5906 - 5918. [Abstract] [Full Text] [PDF]
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A. Maze, G. Boel, S. Poncet, I. Mijakovic, Y. Le Breton, A. Benachour, V. Monedero, J. Deutscher, and A. Hartke The Lactobacillus casei ptsHI47T Mutation Causes Overexpression of a LevR-Regulated but RpoN-Independent Operon Encoding a Mannose Class Phosphotransferase System J. Bacteriol., July 15, 2004; 186(14): 4543 - 4555. [Abstract] [Full Text] [PDF]
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 M. G. Silveira, M. Baumgartner, F. M. Rombouts, and T. Abee Effect of Adaptation to Ethanol on Cytoplasmic and Membrane Protein Profiles of Oenococcus oeni Appl. Envir. Microbiol., May 1, 2004; 70(5): 2748 - 2755. [Abstract] [Full Text] [PDF]
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 C. D. Esteban, K. Mahr, V. Monedero, W. Hillen, G. Perez-Martinez, and F. Titgemeyer Complementation of a {Delta}ccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains Microbiology, March 1, 2004; 150(3): 613 - 620. [Abstract] [Full Text] [PDF]
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J. M. Inacio, C. Costa, and I. de Sa-Nogueira Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis Microbiology, September 1, 2003; 149(9): 2345 - 2355. [Abstract] [Full Text] [PDF]
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J. Abranches, Y.-Y. M. Chen, and R. A. Burne Characterization of Streptococcus mutans Strains Deficient in EIIABMan of the Sugar Phosphotransferase System Appl. Envir. Microbiol., August 1, 2003; 69(8): 4760 - 4769. [Abstract] [Full Text] [PDF]
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S. Nessler, S. Fieulaine, S. Poncet, A. Galinier, J. Deutscher, and J. Janin HPr Kinase/Phosphorylase, the Sensor Enzyme of Catabolite Repression in Gram-Positive Bacteria: Structural Aspects of the Enzyme and the Complex with Its Protein Substrate J. Bacteriol., July 15, 2003; 185(14): 4003 - 4010. [Full Text] [PDF]
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 S. Fieulaine, S. Morera, S. Poncet, I. Mijakovic, A. Galinier, J. Janin, J. Deutscher, and S. Nessler X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr PNAS, October 15, 2002; 99(21): 13437 - 13441. [Abstract] [Full Text] [PDF]
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I. Mijakovic, S. Poncet, A. Galinier, V. Monedero, S. Fieulaine, J. Janin, S. Nessler, J. A. Marquez, K. Scheffzek, S. Hasenbein, et al. Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: A relic of early life? PNAS, October 15, 2002; 99(21): 13442 - 13447. [Abstract] [Full Text] [PDF]
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S. R. Khan and N. Banerjee-Bhatnagar Loss of Catabolite Repression Function of HPr, the Phosphocarrier Protein of the Bacterial Phosphotransferase System, Affects Expression of the cry4A Toxin Gene in Bacillus thuringiensis subsp. israelensis J. Bacteriol., October 1, 2002; 184(19): 5410 - 5417. [Abstract] [Full Text] [PDF]
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K. G. Hanson, K. Steinhauer, J. Reizer, W. Hillen, and J. Stulke HPr kinase/phosphatase of Bacillus subtilis: expression of the gene and effects of mutations on enzyme activity, growth and carbon catabolite repression Microbiology, June 1, 2002; 148(6): 1805 - 1811. [Abstract] [Full Text] [PDF]
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J. A. Marquez, S. Hasenbein, B. Koch, S. Fieulaine, S. Nessler, R. B. Russell, W. Hengstenberg, and K. Scheffzek Structure of the full-length HPr kinase/phosphatase from Staphylococcus xylosus at 1.95 A resolution: Mimicking the product/substrate of the phospho transfer reactions PNAS, March 19, 2002; 99(6): 3458 - 3463. [Abstract] [Full Text] [PDF]
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S. Thomas, D. Brochu, and C. Vadeboncoeur Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System J. Bacteriol., September 1, 2001; 183(17): 5145 - 5154. [Abstract] [Full Text] [PDF]
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V. Monedero, O. P. Kuipers, E. Jamet, and J. Deutscher Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis J. Bacteriol., June 1, 2001; 183(11): 3391 - 3398. [Abstract] [Full Text]
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M. W. Fields and J. B. Russell The glucomannokinase of Prevotella bryantii B14 and its potential role in regulating {beta}-glucanase expression Microbiology, April 1, 2001; 147(4): 1035 - 1043. [Abstract] [Full Text]
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S. Chaillou, P. W. Postma, and P. H. Pouwels Contribution of the phosphoenolpyruvate:mannose phosphotransferase system to carbon catabolite repression in Lactobacillus pentosus Microbiology, March 1, 2001; 147(3): 671 - 679. [Abstract] [Full Text]
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I. Jankovic, O. Egeter, and R. Brückner Analysis of Catabolite Control Protein A-Dependent Repression in Staphylococcus xylosus by a Genomic Reporter Gene System J. Bacteriol., January 15, 2001; 183(2): 580 - 586. [Abstract] [Full Text]
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P. T. C. van den Bogaard, M. Kleerebezem, O. P. Kuipers, and W. M. de Vos Control of Lactose Transport, beta -Galactosidase Activity, and Glycolysis by CcpA in Streptococcus thermophilus: Evidence for Carbon Catabolite Repression by a Non-Phosphoenolpyruvate-Dependent Phosphotransferase System Sugar J. Bacteriol., November 1, 2000; 182(21): 5982 - 5989. [Abstract] [Full Text]
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J. B. Warner, B. P. Krom, C. Magni, W. N. Konings, and J. S. Lolkema Catabolite Repression and Induction of the Mg2+-Citrate Transporter CitM of Bacillus subtilis J. Bacteriol., November 1, 2000; 182(21): 6099 - 6105. [Abstract] [Full Text]
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A. J. Turinsky, T. R. Moir-Blais, F. J. Grundy, and T. M. Henkin Bacillus subtilis ccpA Gene Mutants Specifically Defective in Activation of Acetoin Biosynthesis J. Bacteriol., October 1, 2000; 182(19): 5611 - 5614. [Abstract] [Full Text]
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V. Dossonnet, V. Monedero, M. Zagorec, A. Galinier, G. Pérez-Martínez, and J. Deutscher Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion J. Bacteriol., May 1, 2000; 182(9): 2582 - 2590. [Abstract] [Full Text]
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J.-M. Jault, S. Fieulaine, S. Nessler, P. Gonzalo, A. Di Pietro, J. Deutscher, and A. Galinier The HPr Kinase from Bacillus subtilis Is a Homo-oligomeric Enzyme Which Exhibits Strong Positive Cooperativity for Nucleotide and Fructose 1,6-Bisphosphate Binding J. Biol. Chem., January 21, 2000; 275(3): 1773 - 1780. [Abstract] [Full Text] [PDF]
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E. Küster-Schöck, A. Wagner, U. Völker, and W. Hillen Mutations in Catabolite Control Protein CcpA Showing Glucose-Independent Regulation in Bacillus megaterium J. Bacteriol., December 15, 1999; 181(24): 7634 - 7638. [Abstract] [Full Text]
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P. Plamondon, D. Brochu, S. Thomas, J. Fradette, L. Gauthier, K. Vaillancourt, N. Buckley, M. Frenette, and C. Vadeboncoeur Phenotypic Consequences Resulting from a Methionine-to-Valine Substitution at Position 48 in the HPr Protein of Streptococcus salivarius J. Bacteriol., November 15, 1999; 181(22): 6914 - 6921. [Abstract] [Full Text]
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E. Küster, T. Hilbich, M. K. Dahl, and W. Hillen Mutations in Catabolite Control Protein CcpA Separating Growth Effects from Catabolite Repression J. Bacteriol., July 1, 1999; 181(13): 4125 - 4128. [Abstract] [Full Text]
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R. A. Burne, Z. T. Wen, Y.-Y. M. Chen, and J. E. C. Penders Regulation of Expression of the Fructan Hydrolase Gene of Streptococcus mutans GS-5 by Induction and Carbon Catabolite Repression J. Bacteriol., May 1, 1999; 181(9): 2863 - 2871. [Abstract] [Full Text]
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I. Martin-Verstraete, J. Deutscher, and A. Galinier Phosphorylation of HPr and Crh by HprK, Early Steps in the Catabolite Repression Signalling Pathway for the Bacillus subtilis Levanase Operon J. Bacteriol., May 1, 1999; 181(9): 2966 - 2969. [Abstract] [Full Text]
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D. Brochu and C. Vadeboncoeur The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene J. Bacteriol., February 1, 1999; 181(3): 709 - 717. [Abstract] [Full Text]
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E. J. Luesink, C. M. A. Beumer, O. P. Kuipers, and W. M. De Vos Molecular Characterization of the Lactococcus lactis ptsHI Operon and Analysis of the Regulatory Role of HPr J. Bacteriol., February 1, 1999; 181(3): 764 - 771. [Abstract] [Full Text]
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J. Behari and P. Youngman A Homolog of CcpA Mediates Catabolite Control in Listeria monocytogenes but Not Carbon Source Regulation of Virulence Genes J. Bacteriol., December 1, 1998; 180(23): 6316 - 6324. [Abstract] [Full Text]
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A. J. Turinsky, F. J. Grundy, J.-H. Kim, G. H. Chambliss, and T. M. Henkin Transcriptional Activation of the Bacillus subtilis ackA Gene Requires Sequences Upstream of the Promoter J. Bacteriol., November 15, 1998; 180(22): 5961 - 5967. [Abstract] [Full Text]
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 C. M. Fraser, S. J. Norris, G. M. Weinstock, O. White, G. G. Sutton, R. Dodson, M. Gwinn, E. K. Hickey, R. Clayton, K. A. Ketchum, et al. Complete Genome Sequence of Treponema pallidum, the Syphilis Spirochete Science, July 17, 1998; 281(5375): 375 - 388. [Abstract] [Full Text]
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Y. Takeda, K. Takase, I. Yamato, and K. Abe Sequencing and Characterization of the xyl Operon of a Gram-Positive Bacterium, Tetragenococcus halophila Appl. Envir. Microbiol., July 1, 1998; 64(7): 2513 - 2519. [Abstract] [Full Text]
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A. Galinier, M. Kravanja, R. Engelmann, W. Hengstenberg, M.-C. Kilhoffer, J. Deutscher, and J. Haiech New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression PNAS, February 17, 1998; 95(4): 1823 - 1828. [Abstract] [Full Text] [PDF]
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G. Wang, M. Sondej, D. S. Garrett, A. Peterkofsky, and G. M. Clore A Common Interface on Histidine-containing Phosphocarrier Protein for Interaction with Its Partner Proteins J. Biol. Chem., May 26, 2000; 275(22): 16401 - 16403. [Abstract] [Full Text] [PDF]
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J. A. Encinar, G. V. Mallo, C. Mizyrycki, L. Giono, J. M. Gonzalez-Ros, M. Rico, E. Canepa, S. Moreno, J. L. Neira, and J. L. Iovanna Human p8 Is a HMG-I/Y-like Protein with DNA Binding Activity Enhanced by Phosphorylation J. Biol. Chem., January 19, 2001; 276(4): 2742 - 2751. [Abstract] [Full Text] [PDF]
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