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Journal of Bacteriology, May 2001, p. 3149-3159, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3149-3159.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Phosphoryl Transfer Domain of UhpB Interacts
with the Response Regulator UhpA
Jesse S.
Wright III and
Robert J.
Kadner*
Department of Microbiology, School of
Medicine, University of Virginia, Charlottesville, Virginia
22908-0734
Received 1 November 2000/Accepted 27 February 2001
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ABSTRACT |
Bacterial two-component regulatory systems control the expression
of target genes through regulated changes in protein phosphorylation. Signal reception alters the ability of a membrane-bound histidine kinase (HK) protein to transfer phosphate from ATP to a highly conserved histidine residue. The transfer of phosphate from the histidine to an aspartate residue on the cognate response regulator (RR) changes the ability of the latter protein to bind to target DNA
sequences and to alter gene transcription. UhpB is the HK protein which
controls production of the sugar phosphate transporter UhpT. Elevated
expression of full-length UhpB or of a soluble hybrid protein, GST-Bc,
which is glutathione S-transferase (GST) fused to the
cytoplasmic C-terminal portion of UhpB, results in complete blockage of
uhpT expression in a uhp+ strain.
This dominant-negative interference could result from the ability of
GST-Bc to bind and sequester the RR UhpA and to accelerate its
dephosphorylation. The portion of GST-Bc responsible for the
interference phenotype was localized using truncation, linker
insertion, and point mutations to the region between residues 293 and
366 flanking His-313, the putative site of autophosphorylation. Point
mutations which allow GST-Bc to activate uhpT expression or
which relieve the interference phenotype were obtained at numerous sites throughout this region. This region of UhpB is related to the
phosphoryl transfer domain of EnvZ, which forms half of an interdimer
four-helix bundle and is responsible for dimerization of its
cytoplasmic domain. The expression of GST fusion proteins carrying the
corresponding portions of EnvZ strongly interfered with the activation
of porin gene expression by OmpR. The GST-Bc protein accelerated
dephosphorylation of P-UhpA. Reverse transfer of phosphate from P-UhpA
to GST-Bc was observed in the presence of the metal chelator EDTA and
depended on the presence of His-313. Phosphate transfer from P-UhpA to
the liberated phosphoryl transfer domain also occurred. Taken together,
these results indicate that the phosphoryl transfer-dimerization domain
of UhpB participates in the specific binding of UhpA, in the control of
autokinase activity, and in the dephosphorylation of P-UhpA.
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INTRODUCTION |
In Escherichia coli, a
simple genetic circuit controls production of the
organophosphate-Pi antiporter UhpT, which is responsible for the uptake of many phosphorylated sugars. Induction of
uhpT gene expression in response to the presence of
extracellular glucose-6-phosphate (Glc6P) is mediated by an unusual
two-component regulatory system comprised of three proteins, the
histidine kinase (HK) UhpB, the response regulator (RR) UhpA, and the
membrane receptor UhpC (20). Unlike most HK proteins, the
N-terminal portion of UhpB is hydrophobic and contains 6 to 10 transmembrane segments (18). The transmembrane portion of
UhpB (residues 1 to 272) is thought to operate in complex with UhpC, so
that binding of periplasmic Glc6P to UhpC results in stimulation of the
autophosphorylation activity of UhpB (17). The cytoplasmic
C-terminal portion of UhpB (residues 273 to 500) contains the conserved
sequence motifs characteristic of the catalytic portion of HK proteins.
Phosphate transfer from UhpB to UhpA stimulates the ability of UhpA to
bind at the uhpT promoter and to activate its transcription.
Information on the domain organization, structure, regulation, and
phylogenetic relationships of HK and RR proteins is emerging (for
recent reviews, see references 10 and 41). HK proteins typically contain two distinct parts. The N-terminal signaling region
is variable in sequence and in the number of transmembrane segments.
Some HK proteins are cytoplasmic, many possess two transmembrane segments which divide the intracellular and extracellular portions of
the protein, and some have more complex transmembrane topology. The
well-studied membrane-inserted HK proteins function as homodimers in which the two transmembrane segments from each monomer combine to
form a four-helix signaling structure (reviewed in reference 7). The relative orientations of these transmembrane
helices are affected by the binding of specific effectors to the
periplasmic domain and transmit a conformational signal to the
cytoplasmic HK portion. The cytoplasmic C-terminal portion contains
several key elements and conserved homology motifs. The linker region is predicted to form a coiled coil, follows the last transmembrane segment, and participates in signal transduction (reviewed in reference
48). The linker region is typically followed by a domain
which forms two amphipathic 
-helices which can pair with each other
and with the corresponding region from the other monomer to form a
four-helix bundle which is responsible for dimerization of the
cytoplasmic portion of the HK (44). Except in CheA and related proteins, the dimerization domain contains the highly conserved
site of phosphorylation, typically a histidine, and the flanking
conserved H box sequences. This segment is called the phosphoryl
transfer domain. The other major functional domain of HK proteins is
the nucleotide-binding kinase domain, whose structures in EnvZ
(43) and CheA (2) resemble the ATP-binding domains of DNA gyrase and chaperone Hsp90 but not other protein kinases
(6). Examples of cross talk between noncognate HKs and RRs
have been documented (1, 8, 14, 32), but under physiological conditions there appears to be remarkable specificity in
allowing communication only between cognate pairs of HK and RR
proteins. Identification of the determinants which specify this
recognition should further our understanding of the action of
two-component regulatory systems.
The function of all three uhp regulatory genes is required
for uhpT expression in E. coli, indicating that
UhpC must allow activation of UhpB (18). In agreement, we
have found that expression of uhpB at a higher gene
copy number than that of uhpC prevents the induction of
uhpT (49), whereas co-overexpression
of uhpB and uhpC allows normal inducibility by
Glc6P. In addition, expression of the cytoplasmic C-terminal portion of
UhpB (residues 273 to 500) fused to glutathione
S-transferase (GST-Bc) conferred a strong dominant-negative
effect and completely blocked the expression of uhpT from
the wild-type chromosomal uhpABCT locus. The liberated HK
domain carried in GST-Bc was found to prevent both the binding of UhpA
at the uhpT promoter and transcription activation in vitro. This interference phenotype cannot be solely a consequence of the
ability of GST-Bc to accelerate dephosphorylation of P-UhpA. The
inhibitory effect of GST-Bc was seen even when UhpA could drive
uhpT transcription in its unphosphorylated state, as occurs with the phosphorylation-independent H170Y variant of UhpA carrying the
replacement of His-170 with Tyr or upon overexpression of UhpA. In the
latter case, GST-Bc even inhibited the activity of overexpressed UhpA
D54N, which is unable to be phosphorylated (4, 49). We
interpreted from these results that UhpB can sequester UhpA in an
inactive state.
Several variant forms of UhpB which were selected for their ability to
allow uhpT expression in the absence of UhpC function had a
reduced inhibitory effect in vivo when expressed as GST-Bc fusions
(49). Although GST-Bc does not exhibit autokinase
activity, a variant carrying the replacement of Arg-324 with Cys
(R324C), which confers UhpC independence in the context of full-length UhpB, exhibited both the ability to phosphorylate itself and to transfer that phosphate to UhpA. The GST-Bc R324C variant had not lost
the ability to sequester UhpA, which was revealed by the restoration of
the dominant-negative behavior when its autokinase activity was
eliminated by mutations affecting conserved residues in the catalytic N
or G boxes. The GST-Bc R324C variant also appeared to possess the
ability to dephosphorylate P-UhpA, since the phosphate transferred to
UhpA by the action of GST-Bc was much more labile than expected from
the half-life of P-UhpA formed in the absence of UhpB by phosphate
transfer from acetyl phosphate. The phosphatase activity of
GST-Bc was examined here. The region of GST-Bc required for the
interference or squelching phenotype was localized by analysis of
linker substitution, truncation, and point mutations to the phosphoryl
transfer domain. A similar specific squelching phenotype was seen when
the corresponding region of the HK EnvZ was expressed. This finding
suggests that the interference phenotype is a general manifestation of
the interaction of cognate HK-RR protein pairs.
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MATERIALS AND METHODS |
Strains and plasmids.
The E. coli strain RK1309
(uhp+) is derived from MC4100
[
(argF-lac)U169 araD139 rpsL 150 relA1 flbB5301 deoC1 ptsF25
rbsR22] but is recA and carries the
RZ5 uhpT-lacZ transcriptional fusion as a lysogen at the
att
site. Strain RK1307 is RK1309
uhp(B60-C437). All plasmids used in this study
were derived from pGEX3x-TEV and pJSW-B141 (49), which
encodes a hybrid protein in which residues 273 to 500 of UhpB are fused
to the C terminus of GST.
Purification of GST and His-tag fusions.
Procedures for the
expression and purification of UhpA and UhpB proteins fused to
His6 and GST tags were described previously (49).
-Galactosidase assays.
Regulation of the uhpT
promoter was determined from the level of -galactosidase expressed
from a uhpT-lacZ fusion carried as a single lysogen of phage
RZ5 (28). -Galactosidase assays were performed as
described previously (18). Overnight cultures were diluted
1:100 in minimal medium A supplemented with 1% glycerol (vol/vol),
0.5% Casamino Acids, and 1.5 mM MgSO4. When indicated, IPTG (isopropyl -D-thiogalactopyranoside) was added at
the time of subculture. Cells in exponential growth were induced with
0.25 mM Glc6P in 96-well microtiter plates containing 200 µl of
culture per well. After 40 min of induction at 37°C, cells were lysed with CHCl3-sodium dodecyl sulfate (SDS). The permeabilized
cells were mixed with Z buffer (29) and 2 mM
o-nitrophenyl--D-galactopyranoside, and the
time course of hydrolysis was measured at 415 nm over 5 min at 37°C
in a microplate reader (Molecular Devices). The rate of the enzyme
reaction was normalized for cell density. Reported values are the
averages of at least three experiments, with a variation of <10%.
Construction of linker insertions, truncations, and
chimeras.
The isolation of uhpB linker insertions
carrying a 12-bp oligonucleotide which introduced a PstI
recognition site and resulted in the insertion of a tetrapeptide after
UhpB residues 288, 345, 387, 411, 473, and 489 was previously described
(18). The numbering of all amino acid coordinates
corresponds to the wild-type protein. These insertions were transferred
into the coding region for GST-Bc. The PstI site in the
bla gene of plasmid pGEX3x-TEV was first removed by
restriction fragment exchange of the PstI-less
bla gene of plasmid pQE32 (Qiagen). As described previously
for the cloning of GST-Bc (49), linker insertions in the
uhpB coding region were cloned by PCR using appropriate
primers and VENT polymerase (New England Biolabs) from plasmid
templates which contain the PstI insertions in the
uhpABCT operon (17). The sequences of all PCR
primers are available upon request. Cloning of the PCR amplimers
yielded plasmids encoding variants of GST-Bc (UhpB residues 273 to 500)
carrying each insertion. The presence of the expected changes was
confirmed by PstI digestion and by DNA sequencing of the
coding region. Sequence determination was provided by the University of
Virginia Biomolecular Research Facility.
C-terminal truncations of GST-Bc removed sequences distal to the site
of the PstI linker and were constructed by digesting the
plasmids carrying GST-Bc linker insertion mutants with PstI and EcoRI, removing the overhangs by the addition of VENT
polymerase and nucleoside triphosphates at 70°C for 20 min, and
recircularizing the plasmid by ligation. The C-terminal truncations
removed the normal uhpB stop codon, but the pGEX3x vector
contains stop codons in all three reading frames directly after the
EcoRI site, resulting in the addition of a few nonspecific
residues to the C terminus of the truncated UhpB sequence. N-terminal
truncations were created by PCR with VENT polymerase using primers
which added a 5' BamHI site to allow the fusion of GST to
UhpB at residue 282, 288, or 293.
To facilitate the construction of hybrid proteins combining segments of
UhpB and EnvZ, an
EagI site was introduced into homologous
sequences of both genes. The nucleotide substitutions which introduced
the
EagI site after the coding region for residue 366 of
GST-Bc
required no change in amino acid sequence and created plasmid
pJSW142. The coding region for residues 296 to 450 of EnvZ was
amplified from the chromosome of strain MC4100 by PCR with VENT
DNA
polymerase using primers 1 (5'-CCGGGCAGGAG
CGGCCGATGGAAAT-3'
introducing an
EagI site) and 2 (5'-GATTTGAAGCTGGA
GAATTCCTATCCAGTATCTT-3'
introducing an
EcoRI site) (new restriction sites are
underlined).
The
EcoRI/
EagI-digested amplimer was
cloned into similarly digested
pJSW142 to create plasmid pJSW143, which
encodes the hybrid protein
designated GST-BZc. Similarly, the coding
region for residues
181 to 295 of EnvZ was amplified with primers 3 (5'-GGGGCGTGGCTGTTTATTC
GGATCCAGAACCGAC-3'
introducing a
BamHI site) and 4 (5'-CCGCCATTTCCAT
CGGCCGCTCCTGCCCGGT-3'
introducing an
EagI site), and the
EagI/
BamHI-digested amplimer
was cloned into
similarly digested pJSW142 to create plasmid pJSW144,
which encodes the
hybrid protein designated GST-ZBc. The introduced
EagI site
in
envZ resulted in a change of methionine-294 to arginine.
Plasmid pJSW145, which encodes GST-Zc, was made by appropriate
restriction fragment exchange. The correct nucleotide sequences
of all
plasmid inserts were
confirmed.
C-terminal truncations in which GST-Bc ends at residue 366 or GST-Zc
ends at residue 296 were created by digestion with
EagI
and
EcoRI, as described above for the construction of the other
C-terminal
truncations.
PCR mutagenesis.
Localized random mutagenesis of the portion
of the uhpB sequence encoding residues 293 to 366 was
performed by PCR with Taq polymerase (Life Technologies)
using modifications of a mutagenic reaction (3) as
described by Zhou and Blair (50). In brief, this method
takes advantage of the increased misincorporation exhibited by
Taq polymerase in the presence of Mn2+ and
suboptimal ratios of nucleoside triphosphate substrates. Each amplified
PCR fragment was digested with BamHI and EagI and ligated into plasmid pJSW142 digested with the same enzymes.
Western blot analysis.
Cell samples were adjusted to the
same optical density at 415 nm (OD415) and mixed with an
equal volume of 2× sample buffer, boiled for 5 min, and resolved by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were
transferred to nitrocellulose filters and blocked in Tris-buffered
saline with 5% milk solids and 0.05% Tween 20 overnight at 4°C. For
detection of GST fusion proteins the membranes were developed with
anti-GST monoclonal antibody 9D9 at 1:25,000 (gift of Amy Ma and
J. T. Parsons, University of Virginia) and horseradish
peroxidase-labeled rabbit anti-mouse immunoglobulin G antisera (Sigma
Immunochemicals) at 1:5,000. Peroxidase activity was localized using
the ECL bioluminescence kit (Kirkegaard and Perry) and exposed to film
(Kodak X-OMAT).
Assay of porin expression.
Overnight cultures of cells
expressing GST fusion constructs were diluted 1:100 into minimal medium
A supplemented with 1% glycerol (vol/vol), 0.5% Casamino Acids, and
1.5 mM MgSO4. Sucrose was added to a final concentration of
20% (vol/vol) for high-osmolarity conditions. After growth to
mid-exponential phase, cell volumes adjusted to the same
OD415 were harvested by centrifugation and disrupted by
sonication in 50 mM HEPES, pH 8.0. Following low-speed centrifugation
to remove unbroken cells, Triton X-100 was added to a final
concentration of 0.5% to solubilize the cytoplasmic membrane
components. The insoluble outer membrane fraction was pelleted by
ultracentrifugation at 110,000 × g for 60 min. The outer membrane proteins were resolved by SDS-PAGE with 4 M urea in 11%
polyacrylamide gels (24) and stained with CodeBlue (Pierce).
Protein phosphatase assay.
Acetyl
[32P]phosphate was prepared by the chemical method as
described previously (26). The concentration of acetyl
phosphate was determined as described by Skarstedt and Silverstein
(40) in comparison to a standard sample (Sigma).
His
6-UhpA with an extension containing six His residues at
the N terminus of UhpA (
49) was phosphorylated with 20 to
25
mM acetyl [
32P]phosphate by incubation at 37°C for 2 to 3 h in assay buffer
(50 mM HEPES [pH 8.0], 5 mM
MgCl
2, 1 mM dithiothreitol). Ni
2+-conjugated
agarose beads (100 µl of 50% slurry) were added and
incubated for 10 min at 25°C. Beads were washed three times with
100 ml of assay
buffer until the eluted radioactivity decreased
to background levels.
His
6-UhpA was eluted with 320 µl of assay
buffer
containing 250 mM imidazole. When indicated, EDTA or unlabeled
ATP was
added to the eluted protein at a final concentration of
15 or 1 mM,
respectively. The phosphatase assay was initiated
by the addition of
GST-Bc fusion proteins at 10% of the initial
molar concentration of
His
6-UhpA and incubation of the mixture
at 25°C. Samples
were removed at intervals and mixed with 2 µl
of 6× sample buffer
and 1 µl of 150 mM EDTA and placed on ice.
Proteins were resolved by
SDS-PAGE, and radioactive proteins on
dried gels were detected by a
PhosphorImager (Molecular
Dynamics).
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RESULTS |
Localization of the UhpA-docking region on UhpB.
To help
localize the portion of GST-Bc (containing UhpB residues 273 to 500)
which is necessary for the inhibition of UhpA action, we took advantage
of several Pstl linker insertions in this region
(18). When present in full-length uhpB, the
five mutations which introduce tetrapeptide insertions after residues 288 (designated UhpB-288::
4), 345, 411, 473, and 489 resulted in an uninducible Uhp
phenotype, whereas
insertion after residue 387 resulted in normal inducibility
(17). Each of these insertions was transferred into the
coding sequence for GST-Bc (Fig. 1A)
and examined for its effect on uhpT-lacZ expression in the
uhp+ strain RK1309 (Fig. 1B). Western blot
analysis using antibody to GST showed that most of the variant proteins
were produced at comparable levels, with the exceptions that a
substantial proportion of the GST-Bc-288::4 protein was
cleaved near the fusion junction and the level of
GST-Bc-411::4 was reduced. Three regulatory patterns were
observed. When expressed in the absence of IPTG induction, the GST-Bc
derivatives with insertions at residues 387, 473, and 489 exhibited the
same strong dominant-negative interference as did GST-Bc, suggesting
that these insertions did not affect the region involved in the docking
of UhpA. In contrast, GST-Bc variants carrying insertions at residues
288, 345, and 411 showed no interference effect. The
GST-Bc-288::4 variant even conferred a low level of
expression in the absence of Glc6P.
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FIG. 1.
Effect of tetrapeptide insertions and C-terminal
truncations on the interference phenotype exerted by GST-Bc. (A)
Schematic representation of the UhpB transmitter region indicating the
location of the tetrapeptide insertions and truncation endpoints
relative to conserved motifs in HK proteins (H, N, and G boxes). (B)
Basal expression levels of GST-Bc tetrapeptide insertion variants
detected by Western immunoblot with detection of the GST moiety by
monoclonal antibody 9D9 and their effect on uhpT-lacZ
expression in RK1309 (uhp+) cells in the absence
or presence of IPTG (25 µM). (C) The data shown are as in panel B but
are for the GST-Bc C-terminal truncations. Cells were grown in the
absence or presence of Glc6P (G6P).
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Expression of the fusion proteins was increased by the addition of 25 µM IPTG to derepress the
tac promoter driving their
transcription. The
uhpT-lacZ activity was then measured
(Fig.
1B). Under these conditions, the GST-Bc-288::
4
variant conferred
high basal expression comparable to the Glc6P-induced
level. This
variant was able to restore constitutive Uhp expression in
strain
RK1307 (
uhpBC) (data not shown), which suggests
that this insertion
in the putative linker region might activate the
autokinase activity
of GST-Bc. A similar constitutive phenotype had
been observed
for the E295G/E302K double substitution in the same
vicinity (
49).
The insertion at residue 345 lacked the
interference phenotype
even when amplified, and the insertion at
residue 411, which confers
some lability, showed a modest increase in
inhibition of
uhpT-lacZ expression following induction with
IPTG. The same patterns of
dominant-negative behavior were observed
when the GST-Bc::
4 variants
were expressed in a strain
carrying the phosphorylation-independent
and constitutively active
uhpA H170Y allele (data not shown).
These results showed
that the insertion at position 288 altered
regulation and that at
position 345 abrogated the interference
with UhpA
function.
Interference by truncated UhpB regions.
C-terminal truncations
of GST-Bc were made that deleted UhpB sequences beyond the site of each
linker insertion (Fig. 1C). Western immunoblot analysis which detected
the GST moiety showed that all truncated GST-Bc variants produced
polypeptides which were of the expected size but were present in
reduced amounts relative to GST-Bc. In comparison to the full-length
derivatives with linker insertions, most of the C-terminal truncations
showed a reduced ability to inhibit uhpT-lacZ expression in
the presence of Glc6P but without IPTG, except GST-Bc(273-411), which
showed strong inhibition. Upon induction with 25 µM IPTG, all
truncations except the shortest one, GST-Bc(273-345), showed strong
inhibition of uhpT expression. As was found with the linker
insertion derivatives, amplified expression of all truncated
derivatives except GST-Bc(273-345) also strongly interfered with
transcription driven by the phosphorylation-independent UhpA H170Y
variant (data not shown). Thus, the portion of UhpB from residues 273 to 387, containing the phosphoryl transfer domain, is sufficient for
the interference phenotype.
The UhpB region from residues 273 to 305 is predicted to form a
coiled-coil motif (
38) and shows some similarity to linker
regions of other HK proteins. Some GST-Bc truncation derivatives
in
which the UhpB sequences started at residue 282, 288, or 293
were made
(Table
1). Each of the N-terminal
truncation variants
blocked
uhpT expression as completely as
did the intact GST-Bc
fusion, indicating that residues 273 to 293 were
not required
for the interference phenotype. Interestingly, when these
N-terminal
truncations were combined with the R324C substitution, which
results
in constitutive, UhpC-independent activation of autokinase
activity,
the resulting
uhpT-lacZ expression was increased
more than 10-fold
relative to the GST-Bc R324C protein. The elevated
activity of
all three truncation derivatives was independent of Glc6P
induction
or the presence of the chromosome-encoded UhpB or UhpC
protein.
Thus, truncations into the linker region, which may extend
from
residues 273 to 305, resulted in enhanced autokinase or
phosphotransfer
activity when the autokinase activity was triggered by
the R324C
substitution, but the truncations did not allow autokinase
activity
by themselves. Other changes in this linker region could
activate
UhpB function by themselves, namely the insertion at residue
288,
the double substitution at residues 295 and 302, and the L293P
substitution described below.
Hybrids of UhpB and EnvZ.
Several chimeric proteins which
joined portions of UhpB and EnvZ were made to test whether replacement
of the nucleotide-binding domain of UhpB with that from EnvZ would
allow Uhp expression and to test for consequences of overexpression of
the homologous domains from another two-component regulated system. The
fusion junction was chosen on the basis of the domain boundaries seen in the structure of the EnvZ transmitter (34, 43). A
proline residue in the linker region between the dimerization domain
and the nucleotide-binding domain of EnvZ was also found to occur in a
region of sequence similarity in UhpB. The coding regions around both
proline residues were altered by the introduction of an EagI
restriction site at comparable portions of both genes, thereby
facilitating construction of the desired hybrid proteins by restriction
fragment exchange. The hybrids carried the four possible combinations
of the phosphoryl transfer domains of UhpB (residues 273 to
366) and EnvZ (residues 181 to 296) with the C-terminal
nucleotide-binding HK domains of UhpB (residues 367 to 500) and EnvZ
(residues 297 to 450) (Fig. 2, first
panel). These regions were coupled to GST as in GST-Bc.
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FIG. 2.
Expression of chimeric proteins containing segments of
the UhpB and EnvZ transmitter domains and their effect on UhpT and
OmpF-OmpC expression. The left panel shows a schematic portraying the
domain organization of the GST fusion chimeras between UhpB (dark
region) and EnvZ (light region). The second panel shows the effect of
fusion protein expression on uhpT-lacZ expression in RK1309
(uhp+) cells measured by -galactosidase activity in the
presence of Glc6P. The third panel displays the effect of chimera
fusion expression on outer membrane porin expression levels (A, OmpA;
F, OmpF; C, OmpC) in RK1309 cells under moderate (0% sucrose) and high
(20% sucrose) osmolarity conditions. The results of these experiments
are summarized in the fourth panel and show whether expression of the
reporter protein occurred (+) or was inhibited ( ).
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Western immunoblot analysis showed that all four fusions were expressed
at levels comparable to that of GST-Bc (data not shown).
Both GST-Bc
(UhpB sequence from 273 to 500) and GST-BZc (UhpB
sequence from 273 to
366 and EnvZ sequence from 297 to 450) completely
blocked Glc6P-induced
uhpT-lacZ expression in strain RK1309
(
uhp+) (Fig.
2, second panel). In contrast,
GST-ZBc (EnvZ sequence
from 181 to 296 and UhpB sequence from 367 to
500) and GST-Zc
(EnvZ sequence from 181 to 450) had no apparent effect
on
uhpT expression. This result confirmed that the region
required for
the inhibitory effect of GST-Bc lies between UhpB residues
273
and 366. In addition, the EnvZ ATP-binding domain, which is active
for phosphate transfer (
5,
51), showed no phosphate
transfer
to the H box of UhpB, as deduced from the lack of
uhpT-lacZ expression
in the presence of the GST-BZc
variant.
Cells expressing these hybrid proteins were assayed for their effect on
the levels of the porins OmpF and OmpC, whose transcription
depends on
the phosphorylation of OmpR by EnvZ. Changes in medium
osmolarity alter
the kinase and phosphatase activities of EnvZ,
resulting in the
expression of OmpF at a low osmolarity but of
OmpC at a high osmolarity
(
35). Here, cells were grown in minimal
medium A in the
absence or presence of 20% sucrose as an osmolyte.
In this minimal
medium, roughly equal amounts of OmpF and OmpC
were expressed by cells
grown in the absence of sucrose, and OmpC
was the major species after
growth with sucrose. Strikingly, the
presence of either GST-ZBc or
GST-Zc almost completely abolished
expression of both OmpF and OmpC
under both growth conditions
(Fig.
2, third panel). In contrast,
GST-Bc and GST-BZc had no
apparent effect on porin levels or on
their osmoregulation. As
expected from their decreased porin
expression, cells expressing
GST-ZBc and GST-Zc grew slowly in liquid
media and formed small
colonies on Luria-Bertani agar plates. The lack
of cross-inhibition
of Uhp signaling by GST-Zc or GST-ZBc and of porin
regulation
by GST-Bc or GST-BZc indicates that the specific recognition
of
the cognate RR is determined by the phosphoryl transfer
domain.
The GST-BZc hybrid protein was modified to carry the R324C
substitution, which activates the autokinase activity of GST-Bc,
to
test whether this substitution would allow the EnvZ nucleotide-binding
domain to phosphorylate the UhpB-derived H box. This GST-BZc R324C
variant showed no
uhpT-lacZ expression in RK1307
(
uhpBC) and
exerted strong interference in RK1309
(
uhp+), just like the GST-BZc hybrid did (data
not shown). This absence
of phosphate transfer between noncognate
phosphoryl transfer and
nucleotide-binding domains in the same molecule
points to a high
specificity for their interaction as well as for
interaction with
the RR
protein.
Activity of the liberated H box domains.
Derivatives of GST-Bc
and GST-Zc with truncations of the nucleotide-binding domain
terminating at residues 366 and 296, respectively, were examined. They
were assayed for their effect on uhpT-lacZ expression and
porin synthesis (Fig. 2). The truncated form of GST-Bc expressing UhpB
residues 273 to 366, called GST-Bc366, exhibited a complete
dominant-negative effect on Uhp expression even without IPTG induction.
This interference effect was stronger than that exerted by the other
C-terminal truncations shown in Fig. 1, perhaps owing to the greater
stability of this domain without the appended portions of the kinase
domain. As was seen with the chimeric GST-Bc and GST-BZc proteins, the
GST-Bc366 variant had no effect on porin expression. An
even shorter GST-Bc variant containing residues 293 to 366 of UhpB was
also able to block Uhp signaling completely (data not shown).
The corresponding domain liberated from EnvZ, called
GST-Zc
296, had the same strong interference on OmpR
signaling for porin
expression as did the GST-Zc protein. It had no
effect on
uhpT-lacZ expression. These results show that the
expression of the liberated
phosphoryl transfer domain is sufficient
for the interference
behavior of the full-length chimeric
proteins.
Variants of GST-Bc with altered signaling.
Genetic analysis of
the functional role of amino acid residues in the phosphoryl transfer
domain used two selection procedures for the isolation of mutants with
altered regulatory behavior. The coding region for UhpB residues 293 to
366 was mutagenized by PCR and resected into the unmutagenized plasmid
to restore the sequence of GST-Bc (residues 293 to 500). In the first
selection, variants of GST-Bc which activate uhpT expression
were selected as Uhp+ transformants able to grow on
fructose-6-phosphate as a carbon source following introduction of the
mutagenized plasmid into the uhpBC strain RK1307.
Plasmids isolated from these colonies were transformed into
naïve RK1307 cells to confirm that the mutant phenotype was
associated with the plasmid. Sequencing of the coding region for the
phosphoryl transfer domain from the variant plasmids showed that 12 of
24 variants carried the previously described R324C substitution
(49). The remaining 12 variants carried 10 unique single
and 1 double substitution between residues 293 and 358. The nature of
the amino acid changes in these gain-of-function mutations, which may
also stimulate autokinase activity like the R324C substitution does,
are presented in Table 2. All of the single amino acid substitutions affected residues in the two predicted -helical regions or the joining loop of the phosphoryl transfer domain, except for the L293P mutation in the proposed linker region (see below).
The second genetic selection procedure involved the isolation of GST-Bc
variants which had lost the interference phenotype.
The pool of
mutagenized plasmids described above was introduced
into the
uhp+ strain RK1309. Ampicillin-resistant
variants able to grow on
fructose-6-phosphate as a carbon source were
selected. This loss
of interference is the same null phenotype which
would be given
by the empty vector or one encoding an inactive GST-Bc
protein.
As expected, this selection yielded many more colonies than
did
the selection for gain of autokinase function. Hence, 50 colonies
were randomly chosen for analysis by Western immunoblot, which
showed
that only 14 of the 50 isolates produced full-length GST-Bc
at levels
comparable to that of the transformant carrying the
unmutagenized
plasmid. Plasmids obtained from these 14 isolates
were transformed into
naïve RK1309 for phenotypic analysis. The
growth properties and
levels of
uhpT-lacZ expression indicated
that all 14 variants had lost their dominant-negative character,
even when their
expression was increased by the addition of 25
µM IPTG (Table
3).
As expected, some of the noninterfering mutants were able to confer
constitutive, Glc6P-independent activation of Uhp expression.
These
included one isolate carrying the double substitution L293P
plus
D356A, one isolate carrying the Y355C substitution, and three
isolates
carrying the R324C substitution. Note that substitutions
in the same
positions, namely L293P, R324C, Y355H, and D356A,
had been identified
in the previous selection for constitutive
uhpT expression.
The other nine noninterfering mutants were defective
in both activation
and interference and resulted from five unique
single substitutions and
three double substitutions (Table
3).
These positions are scattered
among the same positions as the
up-regulated mutations. Some
substitutions for the same amino
acid had different phenotypes, namely
I316F and L352Q conferred
constitutive activation, whereas I316T and
L352P resulted in loss
of interference without activation of
expression. Thus, numerous
positions in the putative helical regions of
the phosphoryl transfer
domain appear to affect both the regulation of
autokinase activity
and the ability to interact with
UhpA.
Dephosphorylation of P-UhpA by GST-Bc.
Many HK proteins can
accelerate the dephosphorylation of their cognate RR protein. The
cophosphatase activity of GST-Bc was expected to exist based on the
finding that the turnover of P-UhpA was much more rapid when it was
formed by phosphate transfer from GST-Bc R324C than when it was
produced in the absence of GST-Bc by phosphate transfer from acetyl
phosphate (4, 49). To demonstrate directly this
cophosphatase activity, GST-Bc was added to 32P-labeled
His6-UhpA prepared by incubation with acetyl
[32P]phosphate and then purified by elution from an
Ni2+-chelate matrix. The addition of the wild-type form of
GST-Bc greatly increased the rate of dephosphorylation of P-UhpA
compared to the rate in the presence of GST (Fig.
3A). Dephosphorylation of P-UhpA appeared to be increased somewhat in the presence of 1 mM
ATP. The phosphatase activities of the NarX and NarQ proteins are
independent of ATP (36), whereas those of NtrB
(22) and EnvZ (15) depend on ATP as a
cofactor.
View larger version (48K):
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|
FIG. 3.
Dephosphorylation of P-UhpA by GST-Bc. (A) UhpA (15 µM) was phosphorylated by incubation with 20 mM acetyl
[32P]phosphate as described in Materials and Methods.
P-UhpA was isolated by adsorption to Ni2+-conjugated
agarose beads, washed, and eluted to remove excess acetyl
[32P]phosphate. P-UhpA dephosphorylation was measured
following further incubation in the presence of 1.5 µM GST or GST-Bc
in the absence of ATP (first panel), in the presence of 1 mM ATP
(second panel), and in the presence of 15 mM EDTA (third panel).
Samples were removed at the indicated times and separated by SDS-PAGE.
The location of phosphorylated proteins was determined by
PhosphorImager analysis. (B) Reverse phosphotransfer was detected by
incubation of P-UhpA with GST-Bc variants carrying substitutions in the
H box (H313E and H313Q) or the catalytic domain (H428Q) (49) or with
the nucleotide-binding domain removed (GST-Bc366). Following incubation with P-UhpA,
portions were removed at 0, 5, 10, 20, and 30 min and analyzed as
described for panel A. Hydrolysis of P-UhpA was determined in the
absence of 15 mM EDTA (panels 1 and 3) and in its presence (panels 2 and 4).
|
|
Reverse phosphotransfer from UhpA to GST-Bc.
Transfer of
phosphate from P-UhpA to GST-Bc was observed to occur under certain
conditions. The removal of Mg2+ typically prevents
phosphorylation of the receiver domain of RR proteins, and chelation of
Mg2+ once phosphotransfer has occurred can stabilize the
phosphorylated form of the protein (25). When the
phosphatase assay with GST-Bc was carried out in the presence of Mg
ions, all of the label released from P-UhpA was of low molecular weight
and was presumably Pi. When the phosphatase assay was
carried out in the presence of the metal chelator EDTA, the half-life
of P-UhpA in the sample incubated with GST was greatly extended, from
10 to 20 min to around 60 min (Fig. 3A). In these experiments, the
amount of label on UhpA was fairly low and extended exposure on the
PhosphorImager screen was required for detection. The resultant
reduction in the signal-to-background ratio prevented convincing
quantification of the rates of phosphate release, although the general
differences in lifetimes are clear. The rate of dephosphorylation of
P-UhpA by GST-Bc was substantially slowed in the presence of EDTA but still showed a half-life of <10 min. Strikingly, label was seen to be
transferred from P-UhpA to GST-Bc when EDTA was present. This labeling
of GST-Bc was transient and the label was released from both proteins
by 60 min.
The effect of several mutations affecting conserved residues in GST-Bc
on the dephosphorylation of P-UhpA and on reverse phosphate
transfer to
GST-Bc was studied (Fig.
3B) using shorter time intervals
than in the
previous experiment. In this experiment, GST-Bc accelerated
dephosphorylation of P-UhpA, but at a much lower rate than was
seen for
Fig.
3A. As expected, phosphate transfer from P-UhpA
to GST-Bc was seen
only when EDTA was present. Two variant forms
of GST-Bc in which the
presumed site of phosphorylation, His-313,
was changed to Glu or Gln
(H313E or H313Q) did not show formation
of phosphorylated GST-Bc. These
two variants seemed to retain
some ability to accelerate
dephosphorylation of P-UhpA. In contrast,
the GST-Bc variant with the
H428Q substitution in the N box, which
is also completely defective for
autokinase activity (
49), showed
dephosphorylation of
P-UhpA comparable to that of GST-Bc. Phosphate
transfer to the H428Q
variant of GST-Bc occurred in the presence
of
EDTA.
The GST-Bc fusion carrying UhpB residues 273 to 366 (GST-Bc
366) and lacking the HK domain appeared to be able
to dephosphorylate
P-UhpA, but at a lower rate than the other GST-Bc
proteins. This
truncated protein was able to accept phosphate from
P-UhpA in
the presence of EDTA and displayed a low level of phosphate
transfer
in the absence of EDTA. Taken together, these results show
that
GST-Bc can accelerate dephosphorylation of P-UhpA and that reverse
phosphotransfer to GST-Bc can be trapped in the presence of EDTA
or the
absence of the HK nucleotide-binding domain. Reverse phosphotransfer
requires the presence of His-313, but the data do not allow us
to
conclude whether His-313 is the site of phosphorylation or
whether
reverse transfer is an obligate step in P-UhpA
dephosphorylation.
| |
DISCUSSION |
UhpA-docking domain.
The expression of UhpB or its cytoplasmic
C-terminal segment as a GST fusion protein results in specific blockage
of the Uhp signaling process by inactivation of the transcription
activator UhpA. Dephosphorylation of P-UhpA is shown to be catalyzed by GST-Bc and presumably also occurs with the full-length protein, but the
squelching phenotype occurs even under conditions where phosphorylation
of UhpA is not involved. The most likely explanation for the squelching
phenotype involves the binding or sequestration of UhpA rather
than dephosphorylation of P-UhpA or the formation of inactive
mixed multimers of GST-Bc with the wild-type UhpB. Localization of the inhibitory portion of UhpB, whose
liberated expression prevents UhpA action, might identify the
UhpA-docking surface on UhpB by a method analogous to the domain
liberation approach of Morrison and Parkinson (30).
Probable recognition surfaces on several RR proteins for their cognate
HK proteins have been identified (e.g., see references
11,
42,
and
45), but there is less detailed knowledge about
the
recognition determinants on HK proteins for the cognate RR
proteins.
The CheY-docking domain on the HK CheA was identified
as the P2 domain
by the domain liberation technique (
30) and
was
subsequently cocrystallized with CheY (
27,
47). Most other
HK proteins lack a P2 domain, as shown in the structure of the
EnvZ
protein (
43,
44). Our studies here analyzing the
regulatory
consequences of expressing various segments of the
C-terminal
half of UhpB localized the region responsible for the
interference
phenotype to between residues 293 and 366. The
insertion at residue
288 affected the interference phenotype by
activating UhpB function,
hence affecting regulation rather than UhpA
docking. Several other
substitutions in the proposed linker region
upstream of the phosphoryl
transfer domain also changed UhpB
regulation from its default
state of kinase-off. The phosphoryl
transfer domain of UhpB contains
the conserved His-313 residue, which
is required for all known
phosphotransfer processes, and the flanking
residues in the H
box. The corresponding region of EnvZ conferred the
same phenotype
by strongly inhibiting the action of its cognate RR,
OmpR. This
interference by docking and sequestration of the RR may be a
general
feature of HK proteins but may not be detected in wild-type
cells
owing to the considerable excess of OmpR molecules relative to
EnvZ molecules (
35).
The lack of activation of porin expression by GST-Zc was unexpected
because some soluble forms of EnvZ exhibit high and unregulated
autokinase activity in vitro (
5,
33,
39). These forms of
EnvZ retain substantial amounts of the input domain (
16).
To
our knowledge, the activity of the cytoplasmic portion of EnvZ
(residues 181 to 450) as used here has not been previously studied.
The
lack of activation could result from the presence of the appended
GST
moiety or of the M294R mutation formed by the
EagI site
substitution.
It remains to be shown whether the dominant-negative
effect of
GST-Zc or GST-ZBc on porin expression results from
sequestration
of OmpR (
34), as proposed for the effect by
GST-Bc on UhpA,
or from its action as P-OmpR phosphatase (
15,
51). Nonetheless,
the portion of UhpB which appears to be
involved in the binding
of UhpA overlaps the homologous region of a
VanS-derived peptide
which binds VanR (
46), and portions
of NtrB and EnvZ which are
necessary for phosphatase activity on NtrC
and OmpR, respectively
(
19,
23).
The interference by the phosphoryl transfer domains present in GST-Bc
or GST-Zc with UhpA or OmpR action was highly specific:
expression of
the UhpB domain had no effect on OmpR function,
or conversely,
expression of the EnvZ domain had no effect on
UhpA function. Thus, the
interference phenotype is not a general
disruption of two-component
signaling but must reflect a specific
interaction between the HK and
its cognate RR. Whether the interference
reflects a process that occurs
in wild-type cells expressing full-length
proteins at normal levels
needs further
study.
Phosphate transfer.
The ability of GST-Bc to accelerate
dephosphorylation of P-UhpA was demonstrated using P-UhpA
prepared by incubation with acetyl phosphate. This activity is seen in
both GST-Bc and its constitutively active R324C variant
(49). The activation of autokinase activity in the R324C
variants thus appears not to result from the loss of its cophosphatase
activity. Reverse transfer of phosphate from P-UhpA to GST-Bc was found
to occur in the presence of EDTA and to be dependent on His-313. There
are many possible explanations for this behavior. Metal chelation
strongly retards the rate of spontaneous or GST-Bc-catalyzed
dephosphorylation of UhpA as well as the rate of phosphate transfer to
UhpA (4, 49). It is possible that Mg chelation by EDTA
changes the reaction mechanism so that reverse transfer can occur only
under these conditions. This seems unlikely, because reverse
phosphotransfer has been observed for other HK-RR pairs in the absence
of EDTA. Chelation may trap phosphate transferred to GST-Bc by
preventing its transfer back to UhpA. Perhaps P-GST-Bc is subject to
Mg-dependent hydrolysis by its nucleotide-binding portion. Unlike the
situation with EnvZ and NtrB (14, 22, 31),
dephosphorylation of P-UhpA by GST-Bc did not require the presence
of ATP, although ATP may stimulate it somewhat (but see reference
51). Interestingly, the truncated species
GST-Bc366, which lacks the nucleotide-binding domain, exhibited a low level of reverse phosphotransfer from P-UhpA in
the absence of EDTA. Our conclusion at this time is that the phosphoryl
transfer domain of UhpB can recognize and bind UhpA and can accept
phosphate from P-UhpA.
A role for reverse phosphotransfer in RR dephosphorylation has been
proposed for EnvZ, PhoR, and ArcA (
5,
9,
37).
Some HK
proteins lose phosphatase activity when the histidine
at the site of
phosphorylation is changed to certain residues
but not to others
(
13,
21,
39,
51). This variable dependence
on the identity
of the substitute for the histidine residue questions
the role of the
His in the phosphatase reaction and suggests that
reverse
phosphotransfer is not the only route by which HK proteins
can
accelerate dephosphorylation of their cognate RR protein.
Some
substitutions for the histidine might disrupt the surrounding
active
site and alter the protein conformation, which is important
for
phosphatase action or RR binding. In the case of GST-Bc, replacement
of
His-313 with Glu or Gln prevented both autokinase activity
and reverse
phosphotransfer from P-UhpA but did not prevent the
interference
phenotype, which probably requires only the binding
of UhpA. It remains
to be determined how these substitutions affect
UhpB activity in the
context of the full-length
protein.
Model for dimerization domain.
In many HK proteins, the H box
is located downstream of the linker region and is part of the
dimerization domain which, in the cases of EnvZ and Spo0B, was shown to
form part of a four-helix bundle. The linker region plays an
important role in coupling transmembrane signal reception to regulation
of autokinase and/or phosphatase activity (48). This
region is predicted to form a coiled-coil structure based on its heptad
repeat character. Interestingly, EnvZ does not require the integrity of
this coiled-coil region for autophosphorylation, phosphotransfer, or
phosphatase activity in vitro, suggesting that the points of contact
with OmpR do not lie in this region (33). For UhpB,
various structure prediction models suggest that the likely linker
region of residues 273 to 305 following the last transmembrane segment
can form a coiled-coil structure. GST-Bc variants with truncations of
this region with fusion junctions at UhpB residues 282, 288, and 293 did not confer activation of GST-Bc kinase activity and still showed
the strong interference phenotype, as did the GST-Bc(273-500) species,
indicating that the putative linker region is not involved in contact
with UhpA or in keeping the kinase activity in a silent state. However,
these truncations in combination with the constitutively active R324C
substitution showed about 10 times more activity than the R324C variant
with the intact linker region. This finding, together with the
constitutive phenotypes of the 288-4, E295G/E302K, and L293P
variants, supports the importance of the linker region in regulating
the phosphate transfer process.
Although the greatest sequence similarity in this region of HK proteins
is in the H box, there is substantial relatedness
throughout the
phosphoryl transfer and dimerization domain, as
shown in Fig.
4 (
12). Several secondary
structure prediction
programs suggested that the regions of UhpB from
residues 305
to 327 and 342 to 363 could form two
-helices. The ends
of these
putative helices were not convincingly assigned, and the
position
of the loop separating the homologous helices in EnvZ was not
correctly identified by these programs. Nevertheless, display
of the
residues forming the predicted four-helix bundle in UhpB
as helical
wheels revealed some interesting features (Fig.
4).
The helix
containing residues 305 to 327 (helix 1) displays helical
faces of
three distinct properties, one hydrophobic, one basic,
and one acidic.
The second helix, containing residues 342 to 363
(helix 2), also shows
strong amphipathic character, with a hydrophobic
face and a polar face
but without the prominent charge segregation
seen in helix 1. It is
likely that the interfaces between helices
in the four-helix bundles
are through their hydrophobic surfaces,
as in EnvZ.
View larger version (62K):
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|
FIG. 4.
Sequence alignment and secondary structure prediction of
the UhpB four-helix bundle domain. (A) Alignment of the four-helix
bundle regions of representative transmitters. Helix 1 and helix 2 correspond to the helices defined in the structure of this region of
EnvZ (44). Based on the EnvZ structure, the residues in helix 1 and
helix 2 that contribute to the interhelical and dimer packing interface
are denoted with an asterisk and shaded gray. The phosphorylated
histidine is shaded black. (B) Helical wheel diagram of proposed
interhelical interface of the UhpB monomer. Helix 1 is viewed from its
N terminus (N) and helix 2 is viewed from its C terminus (C).
Hydrophobic residues are shaded gray and His-313 is shaded black. The
heavy arc along both helical wheel diagrams corresponds to the putative
interhelical hydrophobic packing interface with the other UhpB monomer.
Substitutions in the UhpB H box domain from Tables 2 and 3 that result
in gain of function (upward arrow) or loss of function (downward arrow)
or different substitutions that result in both phenotypes (two-headed
arrow) are shown on the helical wheels and on the primary sequence in
panel A. (C) Helical wheel diagram of EnvZ helix 1. The dashed oval
corresponds to the residues involved in the interhelical and dimer
interface of EnvZ helix 1 as determined by nuclear magnetic resonance.
The relative positions of the active site His-243 in EnvZ and His-313
in UhpB helix 1 can be compared with the location of the hydrophobic
interfaces denoted by the dashed oval.
|
|
Strikingly, His-313 in UhpB is exposed at the boundary between the
hydrophobic face and the basic face of helix 1. Similar
exposure
of this highly conserved histidine at the boundary between
the
hydrophobic and polar faces was also predicted for all other
HK
proteins of the same homology family as UhpB, which we examined.
On the
other hand, in EnvZ and related HK proteins, this histidine
is exposed
in the middle of the polar face. The suggested structure
of the
four-helix bundle shows that most of the mutations which
affected
GST-Bc function fall on or near the hydrophobic face
of each helix. In
EnvZ several amino acid substitutions which
affected autokinase
regulation were located in the X region (
12)
at the
C-terminal end of helix 2. It is likely that disruption
of helical
packing might increase or decrease access of the histidine
to the
nucleotide-binding domain, thereby altering its activity
as an
autokinase. We suggest that the His in UhpB is normally
not accessible
to the nucleotide-binding domain, thereby accounting
for the lack of
autokinase activity in the wild-type protein.
The UhpA-binding residues
of UhpB have not been identified yet.
However, the UhpA-binding surface
is likely to include the poorly
conserved residues in the loop and
adjoining helices in the four-helix
bundle. It is intriguing that a few
gain-of-activity mutations
affected residues on the polar faces of both
helices. Future studies
will address the effect of these changes on
UhpA binding and test
whether the access of the histidine to
phosphorylation can be
affected by the binding of inducer and the
presence of
UhpC.
| |
ACKNOWLEDGMENTS |
We appreciate helpful discussions with Qing Chen, Igor
Olekhnovich, Bob Nakamoto, and Phil Matsumura. We thank Amy Ma and J. T. Parsons for the monoclonal antibody to GST.
This work was supported by research grant GM38681 from the National
Institute of General Medical Sciences. J.S.W. was a predoctoral trainee
supported in part by training grant CA09091 from the National Cancer Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Virginia School of Medicine, P.O. Box
800734, Charlottesville, VA 22908-0734. Phone: (804) 924 2532. Fax:
(804) 982 1071. E-mail: rjk{at}virginia.edu.
| |
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Journal of Bacteriology, May 2001, p. 3149-3159, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3149-3159.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
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