From the Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina 27599-7290
Received for publication, December 19, 2000, and in revised form, February 27, 2001
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ABSTRACT |
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CheY is a response regulator in the well studied
two-component system that mediates bacterial chemotaxis.
Phosphorylation of CheY at Asp57 enhances its
interaction with the flagellar motor. Asn59 is located near
the phosphorylation site, and possible roles this residue may play in
CheY function were explored by mutagenesis. Cells containing CheY59NR
or CheY59NH exhibited hyperactive phenotypes (clockwise flagellar
rotation), and CheY59NR was characterized biochemically. A
continuous enzyme-linked spectroscopic assay that monitors
Pi concentration was the primary method for kinetic analysis of phosphorylation and dephosphorylation. CheY59NR
autodephosphorylated at the same rate as wild-type CheY and
phosphorylated similarly to wild type with acetyl phosphate and faster
(4-14×) with phosphoramidate and monophosphoimidazole. CheY59NR
was extremely resistant to CheZ, requiring at least 250 times
more CheZ than wild-type CheY to achieve the same dephosphorylation
rate enhancement, whereas CheY59NA was CheZ-sensitive. However, several
independent approaches demonstrated that CheY59NR bound tightly to
CheZ. A submicromolar Kd for CheZ binding to
CheY59NR-P or CheY·BeF Flagellated bacteria such as Escherichia coli and
Salmonella typhimurium move toward chemical attractants and
away from repellants by regulating the frequency with which their
flagella switch between counterclockwise
(CCW)1 rotation (which
results in smooth swimming) and clockwise (CW) rotation (which causes
the bacterium to tumble). Chemical information outside of the cell is
transmitted to the flagella by a network of chemotaxis proteins. Ligand
binding to the periplasmic portion of transmembrane receptors controls
the autophosphorylation of CheA, a kinase bound to the cytoplasmic
portion of the receptors. The phosphoryl group is then transferred from
a histidyl residue on CheA to an aspartyl residue on the freely
diffusible protein CheY. Phosphorylated CheY (CheY-P) binds to the FliM
protein in the flagellar switch, which increases the likelihood of CW
rotation (see Refs. 1-4 for recent reviews). CheY exhibits an
intrinsic autodephosphorylation activity, but this reaction occurs
slowly relative to the time scale of changes in in vivo
behavior (5), and the primary means of dephosphorylation of CheY in the
cell is via an auxiliary protein, CheZ. The proteins controlling
chemotaxis are an example of a two-component regulatory system, a large
family of systems employed in many organisms to mediate sensory
processes. An essential biochemical feature of these systems is
phosphotransfer from a histidyl residue on a sensor kinase
(e.g. CheA) to an aspartyl residue on a response regulator
(e.g. CheY) and subsequent hydrolysis of the phosphoryl
group from the response regulator (6).
A series of studies have combined to suggest basic mechanisms for the
phosphorylation and autodephosphorylation reactions of CheY, which are
likely applicable to other response regulators. CheY can receive a
phosphoryl group from small molecules such as acetyl phosphate (7) as
well as from CheA, evidence that the fundamental machinery for
phosphorylation resides on CheY. Both phosphorylation and
autodephosphorylation reactions are centered around a Mg2+
ion, and both structural (8) and mechanistic studies using small
molecules as models of the reaction chemistry (9, 10) are consistent
with direct substitution at the phosphoryl phosphorous via a
bipyramidal transition state for both reactions. In contrast, the
mechanism of CheZ-dependent CheY dephosphorylation is not known. CheZ activity is also Mg2+-dependent
(11), and CheZ does not catalyze the hydrolysis of acetyl phosphate
(7). These features are consistent with the possibility that CheZ may
act as a positive allosteric modulator of CheY autodephosphorylation.
Alternatively, CheZ may contribute its own residues to catalysis as
does a conventional phosphatase. Possible regulation of CheZ activity
by other chemotaxis proteins could help explain the ultrasensitivity of
chemotactic sensing in E. coli (12-14) or provide an
additional mechanism for chemotactic adaptation (15). The interaction
of CheZ and CheY-P has been reported to result in large oligomeric
complexes (16), which may be involved in CheZ regulation (15, 17).
The strict conservation among response regulators of essential active
site residues (18) as well as the relative orientation of these
residues in the folded proteins (19) reinforces the notion that the
basic phosphorylation reaction mechanisms for two-component systems are
conserved. Despite this consistency, the rates of the
autodephosphorylation reactions can differ over several orders of
magnitude for different systems (20). Therefore, it is plausible that
nonconserved residues in the vicinity of the active sites of response
regulators have evolved to regulate the rates of the phosphorylation
reactions in a way that meets the individual needs of a system.
Asn59, a nonconserved residue, has a central location in
the active site of E. coli CheY as does the analogous
residue in other CheY proteins and response regulators. The backbone
carbonyl of Asn59 directly chelates the active site
Mg2+ in both the inactive (8, 21) and active (22)
conformations, and the backbone amide directly interacts with a
fluorine atom in the activated
CheY·BeF Chemicals--
The calcium salt of monophosphoimidazole (MPI)
(23) and the potassium salt of phosphoramidate (PAM) (24) were
synthesized as described. Stock solutions of MPI were made fresh and
centrifuged before each use to remove small amounts of insoluble
calcium phosphate. Acetyl phosphate was from Aldrich.
Proteins--
The mutant genes cheY59NR, 59NA,
59NK, 59ND, and 59NE were made by
dut Chemotaxis Behavior--
All chemotaxis assays were performed on
K0641recA/pRBB40 strains. The plasmid pRBB40 carries both
cheY and cheZ so that any effect of possible CheY
overexpression is counteracted by the presence of a proportional amount
of CheZ (30). Rates of bacterial swarming on semi-solid agar plates
were carried out at 30 °C as described (31). The rotational behavior
of the bacterial flagella was determined by tethering analysis (13) and
the Hobson Tracker system was used to analyze rotating cells (33).
Kinetics of Phosphate Release--
The steady state rates of
release of inorganic phosphate from reactions containing CheY, MPI, and
CheZ were measured using an enzyme-linked spectroscopic assay (Enzchek
Pi Kit, Molecular Probes). In this assay, the reactions of
interest were carried out in the presence of purine nucleoside
phosphorylase and a guanine analog substrate,
2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG). Purine
nucleoside phosphorylase catalyzes the reaction of phosphate with MESG
to rapidly form a product that absorbs at 360 nm (34). In our
experiments, buffer (final concentration of 100 mM Hepes,
pH 7.0, 20 mM MgCl2), MESG (final concentration of 200 µM), and MPI were mixed in a cuvette and placed
into the spectrophotometer (Beckman DU7500 diode array), and the
absorbance at 360 nm was continuously monitored. Purine nucleoside
phosphorylase (5 µl; 0.5 units) was added, and an absorbance increase
(typically 0.2-0.4 units) resulted due to the presence of inorganic
phosphate in the MPI preparations. If appropriate, CheZ was then added. The addition of CheZ did not affect the absorbance of the sample. After
3 min in the cuvette chamber to ensure temperature equilibration, CheY
was added to initiate the reaction. After a short lag period (<10 s),
a linear increase in absorbance was observed due to release of
Pi from CheY-P. Linearity continued until an absorbance of about 1.6, whereupon the slope decreased gradually, as is expected at
high absorbance. The time course was monitored for 2-4 min, and the
slope of the absorbance change was determined by instrument software.
The total volume of the reaction was 450 µl, the temperature of the
cuvette was kept at 25 °C with electronic temperature control, and
mixing was done manually. The slopes were converted to µM Pi/s by using an empirically determined extinction
coefficient at 360 nm of 0.0091 µM Fluorescence--
All fluorescence measurements were carried out
using a PerkinElmer LS-50B spectrofluorimeter with a circulating water
bath for temperature control. Time courses for phosphorylation of CheY were monitored by tryptophan fluorescence using a stopped flow apparatus (Applied Photophysics RX2000) for rapid mixing of the reactants. Fluorescence was measured at an excitation wavelength of 292 nm and an emission wavelength of 346 nm, and all reactions were carried
out at 25 °C. Data were fitted to an exponential decay, which
yielded a first order rate constant (kobs),
which is a function of both the rates of phosphorylation and
dephosphorylation of CheY, as follows: kobs = kphosph
[phosphodonor]/Ks + kdephosph (33). The fluorescence
anisotropy of fluoresceinated CheZ was monitored as a function of CheY
concentration as described (16, 35).
Autodephosphorylation Rates--
Rates of autodephosphorylation
of CheY-P at room temperature were determined by electrophoretic
analysis of 32P]CheY-P as described (36). The buffer was
100 mM Tris, pH 7.5, containing 10 mM
MgCl2. Multiple (two to four) independent trials were
carried out for each CheY.
Analytical Gel Filtration--
Chromatography was carried out on
a high resolution Superose 12 column (1 × 30 cm) (Amersham
Pharmacia Biotech) using fast protein liquid chromatography with a flow
rate of 0.8 ml/min. CheY and CheZ were mixed in a 1:1 molar ratio
(final mix concentrations of 0.60 mg/ml CheY and 1.0 mg/ml CheZ) in 50 mM Tris, pH 7.5, 10 mM MgCl2. For
phosphorylating conditions, acetyl phosphate (final concentration of 20 mM) was added to the CheY/CheZ mixtures, allowed to
incubate for 3 min to allow for maximal phosphorylation, and then
chromatographed on the column that had been equilibrated with freshly
prepared 50 mM Tris, pH 7.5, 10 mM
MgCl2, 20 mM acetyl phosphate immediately
before application of the sample. Elution was monitored by ultraviolet
detection, and fractions (0.40 ml) were collected. The fractions that
corresponded to the observed peaks were pooled, concentrated by
Centricon 10 (Amicon/Millipore), and electrophoresed. Molecular weight
standards (Bio-Rad gel filtration standards kit and other individual
proteins from Sigma) were independently chromatographed on the same
column for estimation of the molecular weight of the species eluting
from the column.
Phenotypic Effects of Various Substitutions at Position 59 of
CheY--
Using site-directed mutagenesis, we made six substitutions
at position 59 (arginine, lysine, histidine, aspartate, glutamate, and
alanine). The effects of the mutations on chemotaxis were assessed by
measurement of the swarm rates and flagellar rotational biases of
strains containing the mutant genes (Table
I). All the mutant strains except that
containing CheY59NA swarmed at a significantly slower rate than the
wild-type strain. Decreased swarm rates can result from either an
increase or decrease in CCW bias (37). Strains containing CheY59NK,
CheY59ND, and CheY59NE displayed rotational biases more CCW than wild
type, with CheY59ND displaying the most extreme phenotype, with nearly
exclusively CCW behavior and inability to swarm. In contrast, cells
that contained CheY59NR or CheY59NH displayed extreme CW biases
relative to wild-type strains. Transformation of plasmids carrying
cheY59NR or cheY59NH into a CheA-deficient host
resulted in fully CCW behavior (data not shown), indicating that the CW
activity was dependent on CheY phosphorylation by CheA. Strong CW
behavior could be due to an increased rate of phosphorylation or
decreased rate of dephosphorylation, both of which would result in
increased CheY-P levels or enhanced binding of CheY-P to the flagellar
switch. Because we believed that any of these explanations would be
informative as to the role of position 59 in CheY function, we chose to
focus our biochemical studies on the basis for the CW behavior of the
CheY59NR protein.
Phosphorylation Properties of CheY Using Phosphate Release
Assay--
To assess the phosphorylation, autodephosphorylation, and
CheZ-dependent phosphorylation properties of CheY59NR (and
CheY59NA and wild-type CheY for comparison), we used a commercially
available enzyme-linked spectroscopic assay that continuously measures
phosphate concentration. The steady state rate of release of phosphate
for the reaction of CheY with MPI is a function of both the
phosphorylation and dephosphorylation rates. The data in Fig.
1A for wild-type CheY show
that as the concentration of MPI was increased, the rate of phosphate
release increased and eventually saturated. The sensitivity of the
Pi release rate to MPI concentrations indicates that the
observed rate is at least partially dependent on the phosphorylation
rate. Saturation occurs when autodephosphorylation becomes
rate-limiting. That saturation is due to limiting autodephosphorylation is evident because a similar titration carried out in the presence of
excess CheZ, which will greatly increase the dephosphorylation rate,
gave higher phosphate release rates, which continued to increase well
past 1 mM MPI (Fig. 1A). The
autodephosphorylation rate constant
(kdephosph) can be estimated from the
saturating rate by dividing the saturating rate by the concentration of
CheY. This gives a kdephosph value of 0.035 s
Similar titrations of CheY59NR and CheY59NA reveal several properties
of the phosphorylation reactions for these proteins (Fig.
1B). First, the phosphate release rates for the three CheY proteins all saturate at similar rates, implying that these proteins have similar autodephosphorylation rate constants
(kdephosph ~ 0.030-0.040
s CheZ Sensitivities--
Titrations of the phosphate release rate
with CheZ were carried out at 3 mM MPI, a concentration
where the phosphate release rates for all three proteins were
predominantly limited by autodephosphorylation (Fig. 1B).
For wild-type CheY, the rate of phosphate release increased with CheZ
and saturated by 150 nM, with 40-60 nM CheZ
required for half saturation (Fig. 2).
Saturation is expected when autophosphorylation becomes rate-limiting,
and the rate at which saturation occurs should correlate with the
phosphorylation rate. Like wild-type CheY, the phosphate release rate
for CheY59NA increased throughout the 0-100 nM CheZ range
but gave a lower saturation rate, implying a slower phosphorylation
rate (Fig. 2B), consistent with the results described above
(Fig. 1B, Table II). In contrast, CheY59NR demonstrated extreme CheZ resistance. There was no detectable change in phosphate release rate for CheZ concentrations up to 1 µM, showing
that CheZ had no effect, either negative or positive, on the rate of dephosphorylation of CheY59NR. A CheZ concentration of 12 µM (the concentration of CheY was 3.3 µM)
was required to increase the rate 68% relative to the rate in the
absence of CheZ (data not shown). Saturation of the rate was not
achieved and would be expected to occur at a rate much higher than that
of wild-type CheY (Table II). Given the rate increase by the highest
amount of CheZ, we estimate that CheY59NR requires a minimum of 250 times more CheZ to get the same rate enhancement as wild-type CheY and
CheY59NA. The exceptional CheZ resistance of CheY59NR would result in
high concentrations of CheY-P in the cell, thus accounting for the CW
phenotype of cells containing this protein. Finally, for comparison, the CheZ sensitivity of a previously characterized CheZ-resistant mutant, CheY23ND (27), was assessed using this assay. Asn23
is a surface residue located on Competition of CheY23ND and CheY59NR with Wild-type CheY for
CheZ--
The biochemical basis for the extreme CheZ resistance of
CheY59NR was explored further by assessment of the ability of the mutant protein to bind to CheZ. The abilities of the CheZ-resistant proteins CheY23ND and CheY59NR to affect CheZ phosphatase activity toward wild-type CheY was assessed by the phosphate release assay. The
catalytic effect of a small amount of CheZ (33 nM) for
dephosphorylation of wild-type CheY was determined by measuring the
difference in phosphate release rates in the presence and absence of
CheZ (Fig. 3; data point on ordinate).
This concentration of CheZ had no impact on the phosphate release rate
of CheY23ND or CheY59NR (Fig. 2A). The effect of this amount
of CheZ was then determined in the presence of increasing
concentrations of competing CheY23ND or CheY59NR. For CheY23ND the
difference in rates stayed the same despite the presence of an
extraneous CheZ-resistant CheY (Fig. 3). This result is consistent with
the previously established defect in CheZ binding of CheY23ND (27). In
contrast, the presence of CheY59NR inhibited CheZ activity toward
wild-type CheY (Fig. 3). When equimolar quantities of CheY59NR and
wild-type CheY were present, there was no detectable
CheZ-dependent increase in rate, implying that binding of
CheZ to CheY59NR was at least as tight as to wild-type CheY. Therefore
CheY59NR acts as a competitive inhibitor for CheZ activity toward
wild-type CheY.
Complex Formation between CheY59NR and CheZ--
The competition
experiment implied that CheY59NR, but not CheY23ND, was capable of
binding to CheZ with a sufficiently tight affinity to prevent
interaction with wild-type CheY. This suggested the possibility that
CheY59NR was capable of binding to CheZ but that the CheZ phosphatase
activity was disabled in the complex. To more directly assess binding,
mixtures of CheY and CheZ were analyzed by analytical gel filtration
under both nonphosphorylating and phosphorylating conditions. Gel
filtration chromatography of mixtures of wild-type CheY and CheZ (1 CheY chain:1 CheZ chain) resulted in clean separation of the two
proteins, both in the presence and absence of acetyl phosphate (Fig.
4, A, B, and
E), as has been previously observed (16). Based on the
calibration of the column by molecular mass standards, CheZ eluted at
an apparent molecular mass of 87 kDa (the molecular mass of
CheZ2 is 48 kDa), consistent with previous observations
(39) that have implicated an elongated structure for CheZ2.
CheY (molecular mass 14 kDa) eluted at an apparent molecular mass of 17 kDa. Similar mixtures of CheY59NR and CheZ showed the presence of two
peaks with the same mobilities as seen with wild-type CheY but a
reduction of the intensity of the CheY peak under phosphorylating
conditions (Fig. 4, C and D). Gel electrophoresis
showed that this was due to the co-elution of a portion of the CheY
(about one-third to one-half) with the CheZ (Fig. 4F). The
elution position of the peak that contained both CheY and CheZ was
identical to that of CheZ alone. Therefore, CheY59NR and CheZ formed a
phosphorylation-dependent complex that stays associated
through gel filtration chromatography. The complex has a size that was
indistinguishable, within the sensitivity of this method, from that of
the CheZ dimer. The inability to detect a wild-type CheY·CheZ complex
under the conditions used for gel filtration is likely because there is
very little phosphorylated CheY present due to the phosphatase activity
of CheZ.
CheY·CheZ complexes have been reported to form higher associative
states under phosphorylating conditions (15-17). One of the methods
used to detect the oligomers is fluorescence anisotropy measurements on
fluoresceinated CheZ (16). Because the gel filtration experiment
suggested that the CheY59NR·CheZ complex was similar in size to
CheZ2, it was of interest to assess the size of the CheY59NR·CheZ complex using fluorescence anisotropy. When CheY59NR was added to fluoresceinated CheZ under phosphorylating conditions, the
anisotropy increased and saturated at a similar anisotropy value as
observed for wild-type CheY (Fig. 5),
indicating that the anisotropy measurements were monitoring the same
event with both proteins and that CheZ forms a similarly sized complex
with CheY59NR-P as with wild-type CheY-P.
The anisotropy results also showed that the titration curve for
CheY59NR was shifted to lower CheY concentrations than for wild-type
CheY (Fig. 5). Notably, the titration curve for CheY59NR was
superimposable, within experimental error, with that for wild-type CheY
in the presence of BeF CheY-CheZ Interactions--
CheY59NR was extremely resistant to
CheZ (>250× wild-type, Fig. 2A) despite binding to CheZ at
least as well as wild-type CheY (Figs. 3-5). The CW behavior of cells
containing this mutant protein (Table I) was evidence that CheY59NR was
capable of interacting effectively with the switch and that the amino
acid substitution had no obvious deleterious effects on CheY
conformation. In keeping with this, CheY59NR had the same
autodephosphorylation rate as wild-type CheY and the same or better
(discussed below) rates of autophosphorylation (Table II), both
demonstrations that the active site geometry was intact. Therefore, the
behavior of CheY59NR appeared to be due to specific inhibition of CheZ
activity and not a conformational defect, which would be expected to
affect many activities. The fact that CheY59NA was sensitive to CheZ (Fig. 2B) implied that it is not the asparagine at position
59 that is necessary for CheZ sensitivity but rather that the arginine at this position inhibited CheZ activity. This combination of properties is unique; a previously characterized CheZ-resistant CheY
mutant, CheY23ND (Ref. 27 and this study) was defective in CheZ
binding. Likewise, several CheY mutants showed varying degrees of CheZ
resistance (5-50×) despite binding to CheZ but were incapable of
conferring CW behavior (42). The strong CheZ resistance of CheY59NR can
explain the CW phenotype of cells containing this protein as higher
than normal concentrations of intracellular CheY-P would result.
The result that an arginine substitution at position 59 allows binding
but not catalysis by CheZ implies that the presence of the arginine
prevents the critical residue(s) from achieving optimal positions for
catalysis. We used computer modeling (Insight II and Swiss
Protein Data Bank Viewer) using the coordinates for the
CheY·BeF
An alternative explanation for the behavior of CheY59NR is that the
arginine side chain at position 59 could adopt a conformation, perhaps
by interaction with the phosphoryl group, that sterically prohibits
access to the active site. This could prevent potential catalytic
residues, likely from CheZ, from physical access to the active site.
Current studies are directed at differentiation between these two
models by analysis of CheY proteins with single site substitutions in
the
Of the five other substitutions that were made at position 59 of CheY,
only CheY59NH gave the same strong CW rotational bias as CheY59NR
(Table I). Because the histidyl side chain can be positively charged
and is capable of participating in hydrogen bonding, it follows that
substitution at this position could result in similar interactions as
the arginyl substitution, which led to CheZ resistance. For example,
the plausibility of an interaction between the His59 and
Glu89 is also supported by computer modeling. However, the
correlation between CW rotational bias and positive charge did not
extend to the lysine substitution (Table I). Further studies are
necessary to determine if the CCW rotational bias of CheY59NK, in fact, reflects CheZ sensitivity or is instead due to another effect of the
substitution such as defective interaction with the flagellar switch.
The inability of the arginine or alanine substitutions at position 59 in CheY to affect the rate of autodephosphorylation contrasts with
results from the response regulator Spo0F, where changing the analogous
residue had a large impact on the autodephosphorylation rate (20).
Spo0F with the native lysine residue has a slow rate, whereas
substitution with an asparagine increases the rate. Because we did not
observe any change in autodephosphorylation rates for similar mutations
in CheY, we conclude that the trends observed with Spo0F are not
generally applicable to all response regulators.
Associative State of the CheY-P·CheZ
Complex---
Complexes of CheY-P and CheZ, like any
enzyme-substrate complex, are inherently difficult to study using
wild-type proteins because the rate of dephosphorylation of CheY is so
rapid that very little or no CheY-P is present at steady state. Thus,
gel filtration chromatography of mixtures of wild-type CheY and CheZ in
the presence of phosphodonor resulted in separate elution of the two
proteins (this study and Ref. 16). In contrast, we were able to isolate
a CheY59NR·CheZ complex by gel filtration (Fig. 4), presumably
because the rate of dephosphorylation is slow enough (approximately
equal to the autodephosphorylation rate) that there is appreciable
CheY-P in solution. However, it was surprising that this complex had an
elution position indistinguishable from that of CheZ2. In
our experiments, CheZ2 (molecular mass 48 kDa) eluted at a
position that corresponded to an apparent molecular mass of 87 kDa,
consistent with previous observations (39) that implicate an elongated
structure for CheZ2. The elution position of the
CheY59NR-P·CheZ complex is consistent with a CheZ2·CheY composition because such a complex would not be expected to show any
change in mobility by this method
(CheZ2·CheY2 is improbable due to the surplus
of CheZ in the eluted peak). Any higher aggregation of
CheZ2·CheY, which retained the elongated shape, would
result in earlier elution from the column, based on the mobility of
molecular weight standards, and this was not detected. However, we
cannot rule out the possibility that
CheZ4·CheY2 (molecular mass of 124 kDa) could
coelute with CheZ2 if the shape of the former complex was
near spherical. The indistinguishable elution positions of the
CheY59NR-P·CheZ complex and CheZ2 is in apparent
contradiction to the observation that CheY59NR-P caused an increase in
the anisotropy of fluoresceinated-CheZ, a change that has been ascribed
to an oligomerization process with the formation of species of about 200 kDa (16). It is conceivable that higher oligomers dissociated during the chromatography process. However, protein concentrations were
100-fold higher than in the fluorescence anisotropy experiment where
stable oligomers were observed, so this seems unlikely. Therefore,
despite isolation of a CheY-P·CheZ complex using gel filtration, we
were not able to confirm the higher aggregation of this complex by this method.
The titration curves showing the effect of CheY concentration on the
anisotropy of CheZ for CheY59NR-P and
CheY·BeF Mechanism of Autophosphorylation--
Whereas wild-type CheY and
CheY59NR had similar kobs values for
phosphorylation with acetyl phosphate, CheY59NR had significantly enhanced rates with PAM and MPI. Because the autodephosphorylation rates (kdephosph) were the same, this difference
reflected differences in the rate of phosphorylation
(kphosph/Ks). A
notable difference between the phosphodonors with an N-P linkage and
those with an O-P is the charge on the leaving group. Phosphoramidates
only react with CheY when they are positively charged (36) and will give a cationic leaving group, whereas the acetate leaving group is
negatively charged. Therefore, the positive charge at position 59, through charge/charge repulsion, may increase the probability that the
PAM or MPI substrate is oriented favorably for reaction.
The Pi Kinetic Assay Gives New Information about
Phosphorylation Kinetics--
In this study we demonstrate the
application of a commercially available enzyme-linked assay to
kinetically assess phosphotransfer reactions involving CheY and/or
CheZ. Favorable comparison of rate constants obtained by this assay to
parallel experiments using fluorescence to monitor the rate of
accumulation of CheY-P and 32P
measurement to directly measure loss of the phosphoryl group established the validity of the Pi release assay. The
Pi assay provides information unavailable from other
assays. For example tryptophan fluorescence, which monitors levels of
CheY-P, is not able to assess CheZ-dependent
dephosphorylation of CheY-P when either CheZ or CheY is defective in
the reaction. In this situation, the fluorescence of CheZ becomes large
compared with the increase in fluorescence due to dephosphorylation,
and large errors occur in subtraction of this background signal. In
addition, the assay can provide information about phosphorylation in
systems where fluorescence cannot be used due to lack of a fluorophore
near the phosphorylation site or where phosphorylated response
regulator does not accumulate. Inability to accumulate phosphorylated
response regulator could be due to either the absence of
phosphorylation or to slow phosphorylation relative to
dephosphorylation. These two scenarios can be distinguished using the
Pi assay, because Pi would be released due to
continuous turnover in the latter case.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ung
mutagenesis
(25) of the cheYZ plasmid pRBB40 (26), and cheY23ND was
found in a random mutant search as previously described (27). Wild-type
and mutant CheY proteins were purified from the
K0641recA/pRBB40 strain (26) according to published
procedures (28). CheZ was overexpressed from strain
K0642recA/pKCB1.134KE, which was made by site-directed
mutagenesis of pRBB40 to correct an inadvertent mutation at position
134 (29). CheZ was purified using a published protocol (28), except for
the following two changes. First, a 5-ml Hi-trap Q-Sepharose Column
(Amersham Pharmacia Biotech) was used in place of a MonoQ column
(Amersham Pharmacia Biotech) to allow for larger scale preparations.
Second, after gel filtration on Superose 12, a portion of the CheZ pool
(2-3 mg) was chromatographed on a 1.0-ml MonoQ column. This step
removed a contaminating enzymatic activity for MPI hydrolysis that was
evident with the phosphate assay (see below) as well as a small
amount of the CheZ proteolytic product CheZ1-181, which
sometimes forms during purification (29). The concentrations of CheY
and CheZ were determined by absorbance at 280 nm using extinction
coefficients of 10,200 M
1
cm
1 (CheY) and 16,700 M
1 cm
1
(CheZ). The extinction coefficients were determined empirically by
parallel measurements of absorbance and protein concentration by
quantitative amino acid analysis (The Protein Chemistry Laboratory of
University of North Carolina, Chapel Hill, NC).
1
cm
1 at pH 7.0. This is slightly less than a
published extinction of 0.011 µM
1
cm
1 measured at pH 7.6, as expected (34).
There was no increase in slope if larger amounts of purine nucleoside
phosphorylase were added, indicating that the observed rate was not
limited by the linked enzymatic reaction. PAM and acetyl phosphate
could not be used as phosphodonors in the enzyme-linked phosphate
assay because preparations of these compounds contained levels of
contaminating phosphate that gave off-scale absorbances at the
concentrations required for efficient phosphorylation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Phenotypes of K0641recA/pRBB40 strains containing CheY with
substitutions at position 59
1, which compares well with other
determinations (36, 38).
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Fig. 1.
The rate of release of inorganic phosphate as
a function of MPI concentration. Reactions were carried out as
described under "Experimental Procedures." A, wild-type
CheY (4.5 µM) in the presence ( ) and absence (
) of
1 µM CheZ. B, comparison of MPI titrations of
wild-type CheY (
), CheY59NR (
), and CheY59NA (
). The ionic
strength of the reactions were kept constant by the addition of sodium
chloride so that the final ionic strength was 40 mM.
1). This conclusion was confirmed by direct
determination of autodephosphorylation rates by following the
decomposition of [32P]CheY-P by gel electrophoresis and
phosphorimaging analysis. This analysis gave
kdephosph values of 0.036 ± .006 (1 S.D.)
s
1 (wild-type CheY), 0.035 ± 0.005 s
1 (CheY59NR), and 0.037 ± 0.008 s
1 (CheY59NA). Second, the shift of the
titration curve to a lower MPI concentration for CheY59NR indicates
that the phosphorylation rate at a given MPI concentration is faster
for this mutant than for wild-type CheY. Similarly, the shift of the
curve to the right for CheY59NA indicates slower autophosphorylation at
a given MPI concentration. These conclusions were supported by
fluorescence measurements of rates of CheY-P formation. With MPI as
phosphodonor, the observed rate constant for accumulation of CheY-P
(kobs) was more than 10-fold higher for CheY59NR
compared with wild-type CheY, whereas the rate for CheY59NA was about
2-fold lower than wild-type CheY (Table
II). Because the autodephosphorylation
rates are similar for the proteins (above), these differences in
kobs reflect differences in phosphorylation
rates
(kphosph/Ks).
The accelerated phosphorylation rate for CheY59NR was also observed with PAM as phosphodonor, as CheY59NR had a kobs
about four times higher than wild-type CheY. However, with acetyl
phosphate, CheY59NR had a slightly slower kobs
than wild-type CheY. Therefore the accelerated rate of phosphorylation
for CheY59NR occurred only with the nitrogen-phosphorous phosphodonors.
Possible mechanistic implications are discussed below.
Observed rate constants for CheY phosphorylation with various
phosphodonors measured by fluorescence
helix 1, about 20 Å from the active site Asp57. CheY23ND required 800-1000
nM CheZ to reach half of the possible rate acceleration
(Fig. 2A), about 15-20 times more than wild-type CheY. This
value is in reasonable agreement with a previous observation that
CheY23ND required about 50-fold more CheZ than wild-type CheY for a
similar rate acceleration (27).
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Fig. 2.
CheZ sensitivities of wild-type and mutant
CheY assessed by the rate of phosphate release. The rate of
release of phosphate was measured for wild-type CheY ( ), CheY59NR
(
), CheY59NA (
), and CheY23ND (
) in the presence of various
concentrations of CheZ. Wild-type CheY, CheY59NA, and CheY23ND were
present at 4.5 µM; CheY59NR concentration was 4.9 µM. Note the different CheZ concentration scales for
panels A and B.
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Fig. 3.
Competition of mutant CheZ-resistant
CheY with wild-type CheY for CheZ. The ordinate
represents the CheZ-dependent increase in the rate of
phosphate release for wild-type CheY in the absence (y
intercept) and presence of various concentrations of CheY 23ND
( ) or CheY 59NR (
), two CheZ-resistant CheY proteins. The
concentration of CheZ was 33 nM and that of wild-type CheY
was 3.3 µM.
View larger version (51K):
[in a new window]
Fig. 4.
Analytical gel filtration of CheY/CheZ
mixtures. Shown are absorbance elution profiles of mixtures
containing wild-type CheY (A and B) and CheY59NR
(C and D) after chromatography on Superose 12 under nonphosphorylating (A and C) and
phosphorylating (B and D) conditions. The
profiles are aligned with each other according to fraction number. The
arrows in A show the mobility of molecular mass
standards, with mass expressed in kDa. Each profile showed two peaks:
I, the early peak; II, the late peak. Fractions
composing each peak were pooled, concentrated, and run on SDS gels for
wild-type CheY (E) and CheY59NR (F). For each
gel, the lanes are peak I P (1) peak II
P
(2), peak I +P (3), peak II +P (4),
CheY standard (5), and CheZ standard (6).
View larger version (14K):
[in a new window]
Fig. 5.
Fluorescence anisotropy of fluoresceinated
CheZ in the presence of various CheY proteins. Aliquots of CheY
were added sequentially to a solution of fluoresceinated CheZ (0.20 µM), and the fluorescence anisotropy was recorded after
each addition. The conditions were wild-type CheY + 20 mM
acetyl phosphate ( ), wild-type CheY + 0.1 mM
BeF
), CheY59NR + 20 mM acetyl phosphate (
), and CheY 23ND + 20 mM acetyl phosphate (
). When indicated, acetyl phosphate
was added to CheZ before the addition of CheY.
BeF
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 and
4
of CheY. This interaction was not predicted to occur in inactive CheY
due to a different conformation of the
4/
4 loop. Precedent for
such an interaction comes from the structure of the response regulator FixJ, which has an arginine at position 56 (analogous to position 59 in
E. coli CheY) that interacts directly with Asp86
(analogous to CheY position 89) (43). There are also attractive interactions between Asn61 (analogous to position 59) and
two residues on the
4/
4 loop in the inactive structure of NarL
(44). Therefore, it is possible that CheZ binding to activated CheY
induces further movement of the flexible "nineties" loop, which
puts the necessary residue(s) (on CheY) into position to aid catalysis,
as proposed by Zhu et al. (42). It is conceivable that
Glu89 itself has a catalytic role in
CheZ-dependent dephosphorylation. An arginine at position
59 could prevent this event due to interactions with the loop. A search
for CheZ proteins using the Entrez search engine (NCBI) yielded CheZ
proteins from 7 bacterial species. It is noteworthy that the CheY
proteins of all 7 species contain an asparagine at the position
corresponding to position 59 of E. coli CheY as well as a
glutamate at position 89. In CheY proteins from other bacterial species
there are a variety of amino acids at these two positions.
4/
4 loop.
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ACKNOWLEDGEMENTS |
---|
We thank Ho Cho and David Wemmer for sharing
the coordinates of the
CheY·BeF
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM50860.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Psychiatry, University of North
Carolina, Chapel Hill, NC 27599-7160.
§ Present address: Grinnell College, Grinnell, IA, 50112.
¶ To whom correspondence should be addressed: Tel.: 919-966-2679; Fax: 919-962-8103; E-mail: bourret@med.unc.edu.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M011418200
2 D. Wemmer, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CCW, counterclockwise; CW, clockwise; MPI, monophosphoimidazole; PAM, phosphoramidate; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside; CheY-P, phosphorylated CheY.
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