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Originally published In Press as doi:10.1074/jbc.M206245200 on August 23, 2002
J. Biol. Chem., Vol. 277, Issue 44, 42151-42156, November 1, 2002
Allosteric Enhancement of Adaptational Demethylation by a
Carboxyl-terminal Sequence on Chemoreceptors*
Alexander N.
Barnakov ,
Ludmila A.
Barnakova, and
Gerald L.
Hazelbauer§¶
From the Washington State University, Pullman, Washington
99164-4660 and the § Department of Biochemistry,
University of Missouri, Columbia, Missouri 65211
Received for publication, June 23, 2002, and in revised form, August 21, 2002
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ABSTRACT |
Sensory adaptation in bacterial chemotaxis is
mediated by covalent modification of chemoreceptors. Specific glutamyl
residues are methylated and demethylated in reactions catalyzed by
methyltransferase CheR and methylesterase CheB. In Escherichia
coli and Salmonella enterica serovar typhimurium,
efficient adaptational modification by either enzyme is dependent on a
conserved pentapeptide sequence at the chemoreceptor carboxyl terminus,
a position distant from the sites of modification. For CheR-catalyzed
methylation, previous work demonstrated that this sequence acts as a
high affinity docking site, enhancing methylation by increasing enzyme
concentration near methyl-accepting glutamates. We investigated
pentapeptide-mediated enhancement of CheB-catalyzed demethylation and
found it occurred by a distinctly different mechanism. Assays of
binding between CheB and the pentapeptide sequence showed that it was
too weak to have a significant effect on local enzyme concentration.
Kinetic analyses revealed that interaction of the sequence and the
methylesterase enhanced the rate constant of demethylation not the
Michaelis constant. This allosteric activation occurred if the sequence was attached to chemoreceptor, but hardly at all if it was present as
an isolated peptide. In addition, free peptide inhibited demethylation of the native receptor carrying the pentapeptide sequence at its carboxyl terminus. These observations imply that the allosteric change
is transmitted through the protein substrate, not the enzyme.
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INTRODUCTION |
Sensory systems are designed to adapt to persistent stimulation.
In bacterial chemotaxis, adaptation is accomplished through covalent
modification of transmembrane chemoreceptors (1). Specific glutamyl
residues in the chemoreceptor cytoplasmic domain (2) are methylated by
methyltransferase CheR (3) to form carboxyl methyl esters.
These esters can be hydrolyzed by methylesterase CheB (4). In the well
studied chemosensory systems of Escherichia coli and
Salmonella enterica serovar typhimurium (see Refs. 5-7 for
reviews), chemoreceptors have four to six methyl-accepting glutamyl
residues, four of which are at conserved positions (2, 8-11) (Fig.
1). Control of receptor methylation and
demethylation determines steady state cellular behavior, the ability of
the chemosensory system to provide exact adaptation over a wide dynamic range, and the process of molecular memory. Thus the means by which
receptor modification is modulated are of great interest.
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Fig. 1.
The chemoreceptor dimer and CheB.
At the left and middle are ribbon diagram models
of a chemoreceptor (20) and the methylesterase CheB (50) drawn to the
same scale. The two subunits of the receptor homodimer are shown in
red and blue, respectively, sites of adaptational
modification on the receptor are marked by red balls and
positions of the carboxyl-terminal pentapeptide sequence (NWETF) are
indicated. The boundaries of the cytoplasmic membrane are indicated by
the two horizontal lines. On the right is an
enlargement of the model of CheB, with labels for the catalytic and
regulatory domains and indications of the sites of catalysis,
phosphorylation, and binding to the NWETF sequence.
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Chemoreceptors form complexes with the chemotaxis-specific
histidine kinase CheA, enhancing an otherwise low rate of
autophosphorylation (12) and providing phosphoryl groups for transfer
to two response regulators, CheY and CheB (13). Phospho-CheY binds the
flagellar rotary motor and determines the direction of rotation (14). CheB is phosphorylated on its regulatory domain, activating its catalytic domain (15) (Fig. 1). Binding of chemoattractant to receptor
reduces kinase activity (12) and thus cellular levels of phospho-CheY,
resulting in a change in rotational bias and thus an altered pattern of
swimming (16). However, these changes are short-lived because an
increase in ligand occupancy also initiates the feedback loop of
adaptation. Stimulated receptors accumulate methyl groups through a
combination of a reduced cellular level of phospho-CheB and occupancy
specific activation of methyl-accepting sites (17, 18). Increased
methylation creates a compensatory change in the receptor-kinase
complex (19) that restores CheA activity to its null,
receptor-activated state even though the increased level of attractant persists.
What determines the efficiency of adaptational methylation and
demethylation? Sites of modification are spaced seven apart in the
receptor sequence (2), are on solvent-exposed surfaces of the
chemoreceptor cytoplasmic domain (20), and are bracketed by sequences
that share common features and influence kinetic preferences among
sites (8-10, 21). However, a crucial determinant is at the
chemoreceptor carboxyl terminus, separated from the sites
of modification. In E. coli and Salmonella, a
conserved sequence,
asparagine-tryptophan-glutamate-threonine-phenylalanine (NWETF), at the
carboxyl terminus of high abundance chemoreceptors (Fig. 1) interacts
with the methyltransferase (22) and the methylesterase (23, 24).
Chemoreceptors lacking the pentapeptide naturally (the low-abundance
receptors) or as the result of engineered truncations or mutations are
inefficiently methylated, demethylated, and deamidated (23,
25-28) and are ineffective on their own at mediating tactic response
and directed movement (28-32). Such receptors mediate effective taxis
only with the assistance of NWETF-containing receptors (28, 29, 31,
32).
How does the NWETF pentapeptide at the carboxyl terminus of a receptor
enhance adaptational modification? For methyltransferase CheR, it
provides a high affinity docking site that increases enzyme
concentration near the methyl-accepting glutamates (22). In the current
work we investigated enhancement of CheB-catalyzed demethylation by the
pentapeptide sequence. We found that it occurs through a distinctly
different mechanism of allosteric activation.
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EXPERIMENTAL PROCEDURES |
Proteins and Peptides--
Procedures described previously were
used to produce, purify, and quantify CheA, CheW, and membrane-embedded
chemoreceptors (23, 33), produce and purify CheB and CheBc (24), and
synthesize and purify peptides (23). CheB was quantified using
 280 = 13,700 M 1
cm1. This value was determined by quantitative amino acid
analysis of >99% pure E. coli CheB obtained using a NWETF
affinity column, with a S.E. ± 1200. It was approximately midway
between two rather different values for CheB published previously, one
determined experimentally (34), the other calculated from amino acid
composition (35). Because of these differences, we did five independent determinations by quantitative amino acid analysis, each using at least
10 amino acids, and obtained consistent values with a standard error
<9%. As a control, we determined 280 for bovine serum
albumin (Pierce Chemical) and found a value within 10% of the accepted
one. Concentrations of NWETF and related peptides were determined using
the tryptophan 279.8 = 5,600 (36).
Receptor Demethylation--
Kinetic analyses of demethylation
were performed on forms of Tar with the gene-designated two glutamates
and two glutamines ("2E-2Q") at the sites of adaptational
modification, produced in cells lacking other chemotaxis proteins,
isolated in native membrane, and methylated in vitro to 0.4 to 0.6 3H-methyl groups per receptor monomer (23). In assay
mixtures, phospho-CheB was generated by the presence of previously
autophosphorylated CheA or by providing activated CheA in complex with
chemoreceptor and CheW (23). Phosphorylation of CheB by
receptor-activated CheA was quite effective (see "Results");
autophosphorylated CheA was somewhat less effective, as previously
illustrated (23). To form activating complexes, 10 µM
membrane-embedded Tar pp (2E-2Q), 5 µM CheA, and 5 µM CheW in 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM dithiothreitol, and 10% glycerol
(TEDG) containing 50 mM KCl and 10 mM
MgCl2 (TEDGKM) were incubated at room temperature. After
1 h membrane vesicles containing 3H-methyl-labeled
receptors (Tar or Tarpp) were added to final receptor concentrations
from 1 to 10 µM. 10 s later ATP and CheB were added
to 1 mM and 0.05 to 0.5 µM, respectively. At
least three samples were taken over periods during which the rate of demethylation was essentially linear (60 to 180 s, depending on the particular experimental conditions). Initial rates were determined by linear least squares fits through the points. In experiments testing
activation of Tarpp demethylation by free peptides, receptor-CheA complexes were formed as above except that 10 µM Tarpp
used to form complexes with CheA and CheW carried 3H-methyl
groups that provided the substrate for phospho-CheB, no additional
methylated receptors were added to the mixture after the 1-h
incubation, and a peptide was present from the beginning of the
incubation at concentrations ranging from 0.4 to 1.6 mM.
Binding of Isolated NWETF to CheB--
Isothermal titration
calorimetry was performed with a MicroCal Omega instrument (MicroCal
Inc., Northampton, MA). The data were analyzed using a
single-set-of-sites model from which were obtained values for binding
stoichiometry and association constants. Equilibrium dialysis was
performed using radiolabeled pentapeptide NWETF and pure CheB.
Additions of amino acids to the pentapeptide amino terminus do not have
a detectable effect on interaction with CheB (23), so we introduced
radiolabel by modification of the amino-terminal amino group with
N-succinimidyl [2,3-3H]propionate (Amersham
Biosciences). Radiolabeled reagent at ~1 mCi/ml was added to
pure NWETF peptide at 10 mg/ml in 50 mM sodium phosphate
buffer, pH 7.2. After incubation overnight, the mixture was dialyzed
extensively against TEDGKM. Radiolabeled pentapeptide was mixed with
nonradioactive pentapeptide to create specific activity ~3 cpm/pmol.
For binding assays, 100-µl samples of an ~50 µM
solution of CheB in TEDGKM were placed in dialysis tubing (Spectrapore,
molecular weight cutoff = 14,000), sealed at one end by a
Spectrapore closure and the other end stretched over a piece of plastic
tubing. The bag was immersed in 5 ml of a stirred solution of
radiolabeled peptide in TEDGKM at 4 °C. Dialysis was continued until
equilibrium was reached as judged by samples taken in the course of the
dialysis from the open end of the tube (~2 days). At equilibrium, the
amount of pentapeptide bound to CheB was determined from duplicate
determinations of the difference between radioactivity inside the
dialysis bag and in the dialysis buffer.
Binding of Receptor-borne NWETF to CheB--
Studies were
performed using radiolabeled CheB-S164C produced in strain RP3098
harboring a form of pCW/cheB (24) in which codon 164 was
altered to code for cysteine, thus eliminating catalytic activity (37).
Cells were grown in a minimal salts medium to ~3 × 108 cells/ml and
isopropyl-1-thio- -D-galactopyranoside was added to 0.25 mM. One hour later, a
L-[U-14C]amino acid mixture (Amersham
Biosciences) was added. After 10 min the culture was rapidly cooled in
an ice-water bath, the cells were processed and CheB was purified as
described (23, 33). Membrane vesicles containing Tsr or Tar were
produced as described (33). For experiments using vesicles with a high
content of Tsr, membranes were purified on a sucrose gradient (35).
Binding assays were based on those of Gegner et al. (35).
50-µl mixtures of receptor-containing membranes, ~25
µM radiolabeled CheB-S164C, and 0.5 mg/ml bovine serum
albumin in TEDG were incubated 10 min at room temperature and
centrifuged at 50,000 rpm for 15 min in a TLA100.4 rotor. Two 20-µl
samples of the resulting supernatant were analyzed by liquid
scintillation counting. In these experiments, receptor was not
methylated and thus there were no methyl ester substrate sites to which
CheB might bind.
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RESULTS |
Kinetic Measurements--
Previously, we compared demethylation of
Tar and Tarpp, its truncated derivative lacking the NWETF sequence,
at a single receptor concentration and found that rates of
CheB-catalyzed demethylation for intact receptor were at least 20-fold
greater than rates for receptor lacking the pentapeptide sequence (23). This was the case whether CheB was in the absence or presence of a
means of generating its activated form, phospho-CheB (23). We were
interested in performing more extensive kinetic analyses comparing
demethylation of Tar and Tarpp by determining initial velocities of
demethylation over a range of substrate concentration. A quantitative
comparison was not possible for demethylation catalyzed by
unphosphorylated CheB because demethylation of Tarpp was so ineffective that reliable rates could not be obtained to compare with
the substantially faster rates of Tar demethylation (see Ref. 23 for an example).
However, quantitative comparison could be made for
demethylation reactions catalyzed by phospho-CheB. Plots of
initial velocity as a function of substrate (methyl group)
concentration (Fig. 2) document the
impressive enhancement of receptor demethylation provided by the
carboxyl-terminal sequence. A double reciprocal plot (inset
Fig. 2) illustrates that this enhancement is the result of a difference
in Vmax (the y axis intercept) and
not in Km (x axis intercept). These
differences were seen whether phospho-CheB was produced using
phosphorylated CheA or by providing CheA activated in complex with
chemoreceptor (see "Experimental Procedures"). The latter protocol
was more efficient in activation (23), but in both protocols,
phospho-CheB was created independently of the methylated
receptor substrates that were being compared. We repeated experiments like those in Fig. 2, testing several concentrations of
CheB. From three such experiments, we calculated the mean values (± S.E.) for Km and kcat, which
is Vmax normalized to the amount of enzyme. For
Tar, Km = 2.75 ± 0.9 µM and kcat = 187 ± 49 mmol of methanol
s1 mol CheB1; for Tarpp,
Km = 2.3 ± 0.5 µM and
kcat = 7.9 ± 1.7 mmol methanol
s1 mol CheB1. Thus the presence of
receptor-borne NWETF increased catalytic efficiency, as measured by
kcat, over 20-fold but did not alter the
apparent affinity of enzyme for substrate, as reflected in Km. Because phospho-CheB is short lived, only a
fraction of the CheB in our experiments would have been in the
phosphorylated form (38), and thus the kcat
values we determined are underestimates of the true values (39).
However, because the level of phospho-CheB would have been the same
whether the methylated substrate was Tar or Tarpp, the difference
between the kcat values we determined for the
two forms of the receptor provided a valid indication of the effect of
the receptor carboxyl-terminal pentapeptide. In addition, the
kcat value we determined for demethylation
of Tar (~190 mmol of methanol s1 mol of
CheB1) is comparable with the value for Tar determined by
Anand and Stock (~150 mmol of methanol s1 mol
CheB1) using phosphoramidate to create a steady state of
~65% phosphorylated CheB (39). This implies that our conditions
generated a similar fraction of phospho-CheB.
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Fig. 2.
Kinetics of demethylation of Tar and
Tarpp catalyzed by phospho-CheB. Initial
rates of demethylation were determined for methylated,
membrane-embedded Tar or Tarpp in the presence of phospho-CheB
generated by activated CheA in complex with a different chemoreceptor
and CheW (see "Experimental Procedures") and plotted as a function
of concentration of methyl groups. Fits to the Michaelis-Menten
equation (the lines shown) provided Km
and Vmax values of 1.9 µM and 7.0 pmol/min for Tar, and 1.3 µM and 0.23 pmol/min for
Tarpp. The inset shows double reciprocal plots of the
same data with lines drawn using the kinetic parameters from the
Michaelis-Menten fits.
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If interaction of CheB and the receptor-borne sequence enhanced
demethylation by changing kcat but not
Km, then the enzyme missing the pentapeptide-binding
site should catalyze demethylation of Tar and Tarpp at essentially
the same velocities and with a Km essentially
unchanged from the intact enzyme. We tested this using CheBc, the
carboxyl-terminal catalytic domain of the enzyme (Fig. 1) that is
missing the regulatory domain and linker and thus lacks the
pentapeptide-binding site at the juncture of those domains (24). As
shown in Fig. 3, CheBc catalyzed
demethylation of Tar and Tarpp with almost the same efficiency. This
was a striking contrast to the vast difference in velocities for the two substrates when demethylated by intact enzyme (Fig. 2). In addition, the Km values for CheBc catalysis as
determined in multiple experiments (2.1 ± 0.7 and 2.5 ± 0.5, for Tar and Tarpp, respectively) were essentially unchanged
from the values for intact phospho-CheB (2.75 ± 0.9 and 2.3 ± 0.5 µM, respectively) or for the value for intact but
unphosphorylated CheB acting on Tar (2.9 ± 1.4 µM).
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Fig. 3.
Kinetics of demethylation of Tar and
Tarpp catalyzed by CheBc. Initial rates
of CheBc-catalyzed demethylation were determined for methylated,
membrane-embedded Tar and Tarpp, and plotted as a function of
concentration of methyl groups. Fits to the Michaelis-Menten equation
(the lines shown) provided Km and
Vmax values of 5.0 µM and 0.36 pmol/min for Tar, and 6.3 µM and 0.37 pmol/min for
Tarpp.
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Binding of CheB to Pentapeptide NWETF--
Wu et al.
(22) used isothermal titration calorimetry to determine that CheR bound
to the isolated NWETF pentapeptide in a 1:1 complex with a
Kd ~ 2 µM. We used the same
procedure to characterize binding of CheB to the identical peptide and
found a stoichiometry close to 1:1 (1:0.85) but a Kd
of ~160 µM, indicating much weaker binding (Fig.
4). This weak interaction was unexpected
so we investigated binding by a second technique, equilibrium dialysis.
We labeled NWETF by amino-terminal modification with
[3H]propionate and used the radiolabeled pentapeptide in
equilibrium dialysis experiments with CheB (Fig.
5). Analysis of the binding revealed a
stoichiometry of 1:1 and a Kd of 130 µM, confirming that CheB bound the receptor pentapeptide
much less strongly than did CheR.
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Fig. 4.
Isothermal titration calorimetry of binding
of pentapeptide NWETF to CheB. A solution of 0.7 mM
NWETF pentapeptide in TEDG was injected (17 injections, 15 µl each)
into a 110 µM solution of pure CheB at 26 °C. The plot
shows background-subtracted, integrated, and normalized data and a fit
derived from the single-set-of-sites model. The parameters of that fit
were Kd = 157 µM and stoichiometry of
1 CheB to 0.85 pentapeptide.
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Fig. 5.
Scatchard analysis of binding of radiolabeled
NWETF to CheB. The data are from three equilibrium dialysis
experiments for [3H]propionate-labeled NWETF binding to
pure CheB in the dialysis bag at 54 µM.
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We considered the possibility that pentapeptide sequence at its natural
location, the carboxyl terminus of a chemoreceptor, might be bound more
strongly by CheB than free peptide. Thus we tested binding of
radiolabeled CheB to the NWETF-bearing chemoreceptor Tar, assaying
depletion of radiolabel from a binding mixture after centrifugation of
receptor-containing vesicles (35). If CheB bound to receptor-borne
pentapeptide with an affinity comparable with the 2 µM
Kd of the CheR-pentapeptide complex, then assays
with receptor-borne pentapeptide at 1-10 µM should have revealed substantial binding. This was not the case, so we investigated higher concentrations using Tsr-containing membranes, which can be
isolated with higher receptor content (35). Binding was detected for
Tsr-borne pentapeptide at concentrations above 10 µM, but there were difficulties in testing pentapeptide concentrations >50
µM because the volume of membrane vesicles became
technically limiting. At 50 µM receptor-borne
pentapeptide, binding was not close to saturation. Extrapolation from
the limited data provided estimates of Kd ~ 150 µM and stoichiometry ~1:1, consistent with values for
free pentapeptide.
Allostery--
Studies of both binding and kinetics indicated that
interaction of CheB and receptor-borne pentapeptide sequence
enhanced demethylation by affecting catalysis, not by increasing local enzyme concentration. The pentapeptide-enzyme interaction occurs at
a site separated from the catalytic interaction, so this is an
allosteric effect. The substrate as well as the enzyme is a protein,
thus allostery could alter CheB, the protein substrate or both.
If the principal effect of pentapeptide-CheB interaction were
allosteric activation of the enzyme, then it should not be crucial whether the interacting sequence were attached to the receptor or
present as a free peptide. In such a case, CheB would be activated for
enhanced demethylation of Tarpp by the presence of sufficient free
pentapeptide to occupy its binding site on CheB. To test this
possibility, we compared demethylation of Tarpp in the absence and
presence of NWETF at concentrations 5-10-fold above its
Kd, but observed little difference (data not shown).
It was possible that NWETF binding did not induce an allosteric
activation because the amino end of the sequence had a charged amino
group not present in the receptor-borne sequence. Thus we tested
DPNWETF, the NWETF sequence extended at its amino terminus by the two
amino acids that occur naturally at those positions in native Tar and
is effective in binding CheB (23). The extended peptide caused only
marginal activation of Tarpp demethylation. We then considered the
possibility that introduction into an assay mixture of a charged
peptide at concentrations up to 2 mM could inhibit
demethylation nonspecifically, masking specific activation. For this
reason, we utilized control peptides with inverted sequences (FTEWN and
FTEWNPD) that should have the same nonspecific effects as the natural
sequences but do not bind to CheB (23). At concentrations from 0.1 to 2 mM, these peptides reduced the rates of Tarpp
demethylation only slightly (~20% at the highest concentrations).
Thus even after adjusting for the possible nonspecific effect, the
natural peptide sequences caused little activation. For example, as
illustrated in Fig. 6 demethylation of
Tarpp by phospho-CheB was hardly affected by the presence of a
potentially activating peptide (the extended pentapeptide DPNWETF) in
comparison to activity in the presence of the control, inverted
peptide. The peptide caused a ~0.3-fold enhancement (0.28 ± 0.12 in six measurement using four different preparations of
membrane-embedded receptor), less than 2% of the ~25-fold
enhancement provided by receptor-borne pentapeptide (Fig. 6,
right-hand bar).
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Fig. 6.
Effects of free and receptor-borne peptide on
demethylation catalyzed by phospho-CheB. The bars show
normalized initial rates of demethylation for methylated receptor in
the absence of the conserved peptide sequence (control), in the
presence of free peptide at concentrations sufficient for substantial
binding to the enzyme (free), and with the peptide at its natural
location at the carboxyl terminus (attached). In six experiments using
four preparations of 3H-methyl-labeled Tarpp,
demethylation rates were determined in the presence of peptide DPNWETF,
the extended form of pentapeptide NWETF (free), or FTEWNPD,
which has an inverted sequence (control), and were expressed
relative to the "control" rate. In three experiments the peptides
were at 0.8 mM and in three they were at 0.95 mM. The first two bars show the control rate
(normalized to 1 and thus without error bars) and the mean, normalized
rate and standard deviation (1.28 ± 0.12) in the presence of free
peptide. The third bar displays the rate of
demethylation catalyzed by phospho-CheB for Tar, a receptor
carrying the NWETF sequence at its carboxyl terminus, relative to the
rate for Tarpp, a form of that same receptor lacking the sequence.
This relative rate (23.7 ± 6.2) was calculated from values in the
text.
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The minimal effect of free peptide implied that allosteric enhancement
of demethylation required attachment of the NWETF sequence to the
receptor. If this were the case, then free pentapeptide should act as
an inhibitor for demethylation of an NWETF-bearing receptor. We
investigated inhibition using the NWETF pentapeptide, its
seven-residue, extended form, and the corresponding inverted-sequence peptides to control for nonspecific effects. As seen in a
representative experiment (Fig. 7), the
native sequence inhibited demethylation of intact Tar catalyzed by
phospho-CheB. Inhibition was half-maximal at a peptide concentration
~4-fold above the 150 µM kd of the
peptide-enzyme complex, as might be expected for competition with
pentapeptide tethered to receptor and thus present at a high effective
concentration (40). In any case, the high concentrations of free
peptide necessary to inhibit demethylation by phospho-CheB (Fig.
7), indicated that phosphorylated enzyme did not have a substantially
higher affinity for pentapeptide than the ~150 µM kd we measured for unphosphorylated CheB (Figs. 2
and 3).
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Fig. 7.
Inhibition of CheB-catalyzed demethylation by
free peptide. In experiments similar to those for Fig. 2, 4 µM Tar carrying 3H-methyl groups was
incubated with 5 µM CheA and 5 µM CheW for
1 h to allow formation of complexes of activated CheA in the
presence of the extended pentapeptide DPNWETF or the inverted, control
peptide FTEWNPD at the indicated concentrations. Reactions were
initiated by addition of CheB and ATP to 0.08 µM and 1 mM, respectively. For each concentration of extended
peptide, initial rates of demethylation were expressed as a percentage
of the rate in the presence of the control peptide and plotted as a
function of peptide concentration. The curve is a fit of the data to a
model that assumes that the initial rate is directly proportional to
the fraction of CheB interacting with receptor-borne pentapeptide and
that the extended peptide acts as a competitive inhibitor of that
interaction.
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DISCUSSION |
We found that enhancement of CheB-catalyzed demethylation by the
NWETF pentapeptide sequence at the carboxyl terminus of a chemoreceptor
occurred in a way distinctly different from its enhancement of
CheR-catalyzed methylation. For the methylation reaction, the sequence
provides a high affinity docking site for the CheR methyltransferase
and thus increases the effective concentration of the enzyme in the
neighborhood of substrate methyl-accepting sites (22). In contrast,
interaction of CheB with receptor-borne pentapeptide enhanced
demethylation allosterically. Consistent with an allosteric action, the
sequence bound too weakly to the CheB methylesterase to cause a
significant increase in local concentration of the enzyme.
Different Mechanisms of Enhancement--
Of the two kinds of
CheR-interaction sites on a chemoreceptor, the strongest binding is not
at the sites of catalysis (the methyl-accepting glutamyl residues) but
instead at the carboxyl-terminal NWETF sequence. CheR binds with the
same dissociation constant of ~2 µM to pentapeptide
alone, to the isolated cytoplasmic domain of chemoreceptor Tsr carrying
the pentapeptide sequence and three methyl-accepting sites, and to
intact receptor carrying the sequence and from one to five
methyl-accepting sites, but exhibits no detectable binding to isolated
cytoplasmic domains containing methyl-accepting sites but missing the
carboxyl-terminal sequence (22). Differences in rates of methylation
for Tar and Tarpp imply that binding of CheR to its substrate sites
is ~20-fold weaker than binding to the pentapeptide sequence (26).
Taken together, these observations show that the sequence acts as a
high affinity docking site for the methyltransferase, enhancing the
methylation reaction by recruiting the enzyme to the vicinity of its
substrate sites and thus increasing the probability of binding at the
methyl-accepting glutamyl residues.
Our data indicate that this is not the mechanism by which
receptor-borne pentapeptide enhances CheB-catalyzed
demethylation. Kinetic analysis of demethylation revealed that the
NWETF sequence at the carboxyl terminus of a receptor substrate
enhanced kcat not Km, and
thus indicated that pentapeptide-enzyme interaction affected catalysis
not binding. It is interesting to note that phosphorylation, which
activates CheB independent of interaction with the pentapeptide (23),
also enhances kcat rather than
Km (39). Enhancement of kcat
not Km was consistent with the weak binding we
observed for the enzyme and pentapeptide sequence, a binding
insufficient to enhance enzyme action by increasing concentrations of
CheB near substrate sites. As documented in Figs. 4 and 5, CheB bound
to the free pentapeptide NWETF with a Kd ~ 150 µM, and the concentrations of free peptide necessary to
inhibit phospho-CheB activity (Fig. 7) indicated that phosphorylation
of the enzyme did not make the affinity substantially stronger.
Investigation of CheB binding to the sequence at its natural location
at the carboxyl terminus of a chemoreceptor indicated a similarly weak
interaction. The short lifetime of phospho-CheB kept us from performing
direct measurements of binding of phosphorylated enzyme to
receptor-borne pentapeptide, but a strong binding for only this
combination could not explain enhancement by the receptor-borne pentapeptide of demethylation catalyzed by CheB that is not
phosphorylated (23), nor the effect of the receptor-borne pentapeptide
on kcat rather than Km.
Estimates of the cellular content of chemoreceptors range from 2,500 to
10,000 (41-43) and the content of CheB has been estimated at 2,000 (44). These values translate to cellular concentrations between 4 and
16 µM for chemoreceptors and ~3 µM CheB.
At such concentrations, a CheB-pentapeptide complex with a
Kd of ~150 µM, would involve only a
small proportion of total cellular CheB and this complex would not
create a significant increase in concentration of CheB in the
neighborhood of its sites of enzymatic action.
There is a disparity between the ~150 µM
Kd of pentapeptide and CheB or phospho-CheB and the
Km of ~3 µM for demethylation by
either CheB or phospho-CheB. We do not fully understand this disparity,
but it can be understood if we assume that Km
approximates Kd for enzyme and substrate methyl
ester (see below for the conditions under which this assumption would
be valid). In this case, the interaction of the enzyme and substrate
site would be significantly stronger that the interaction of the enzyme
and receptor-borne pentapeptide. This stronger binding, as reflected in
Km, would not be a function of the presence or
absence of the pentapeptide sequence on a receptor (this is what we
observe), and allosteric enhancement of CheB-mediated catalysis would
be the result of binding of pentapeptide to enzyme bound to or near the
substrate site. Using Km to approximate Kd is valid if the rate constant of dissociation for unreacted substrate from the enzyme-substrate complex is substantially higher than kcat and the catalytic mechanism
does not involve a covalent intermediate. These issues are currently
under investigation. No matter what the affinity of enzyme for its
substrate sites, the weak affinity of enzyme for pentapeptide indicates
that this interaction could not assist CheB in passing through a
receptor array by the "molecular brachiation" process postulated
for CheR (40).
Kinetic parameters for phospho-CheB acting on NWETF-containing
receptor, Km ~ 2.8 µM and
kcat ~ 190 mmol of methanol s1
mol of CheB1, are quite similar to the parameters for
CheR, which are Km = 2.1 µM and
kcat = 170 mmol s1 mol of
CheR1 (44). This is consistent with the dynamic
equilibrium of adaptational modification, in which the population of
unstimulated or adapted receptor is maintained at a constant level of
methylation by a balance between rates of methylation and demethylation
(45).
Allosteric Activation of Demethylation--
The
pentapeptide-binding site on CheB is separated from the active site
(24) and thus enhancement of catalysis through an effect on the enzyme
would be an effect of binding at a site distance from the site of
catalysis, i.e. an allosteric effect. However, the substrate
itself is a protein, so it was possible that interaction of its
carboxyl-terminal sequence with the methylesterase could allosterically
alter the receptor to enhance demethylation. The data are consistent
with this second alternative, that allosteric activation is principally
through the receptor, not the enzyme. If binding of the NWETF sequence
induced an allosteric activation in the methylesterase, then it should
not be crucial whether this sequence were present in its natural
location at the carboxyl terminus of a chemoreceptor or as a free
pentapeptide. Fig. 6 documents that free pentapeptide provided only a
few percent of the activation created by the receptor-borne sequence.
If allosteric activation required that the pentapeptide sequence be
coupled to the chemoreceptor, then free pentapeptide should inhibit
demethylation of receptors carrying the carboxyl-terminal sequence.
Fig. 7 documents that this is the case. Taken together, these two
results imply that the vast majority of the enhancement of
demethylation generated by interaction of the receptor-borne sequence
and CheB is conveyed through a change in the receptor, not the enzyme.
Thus the primary allosteric effect appears to be through the protein
substrate. However, the issues require additional investigation,
because we have not yet demonstrated directly induction of a
conformation change in the receptor by interaction of CheB and
receptor-borne pentapeptide. Such investigations are underway.
Substrate-transmitted Allostery and Signal
Amplification--
Chemotactic response in E. coli exhibits
striking signal amplification (46-49). The mechanism of this
amplification is not yet understood, but recent observations have
implicated methylesterase CheB as a crucial participant (48, 49). If
CheB binding to receptor-borne, carboxyl-terminal pentapeptide induces
substrate-transmitted allostery, this conformational change could be
related to the role of CheB in signal amplification.
| |
ACKNOWLEDGEMENTS |
We thank Gerhard Munske for synthesis of
peptides, Linda L. Randall for assistance and consultation in
calorimetry, and Wing-Cheung Lai for help in preparation of the text
and figures.
| |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Research Grant GM29963 (to G. L. H.) from the NIGMS.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: 3-Dimensional Pharmaceuticals, Inc., Eagleview
Corporate Center, 665 Stockton Dr., Exton, PA 19341.
¶
To whom correspondence should be addressed. Tel.:
573-882-4845; Fax: 573-882-5635; E-mail:
hazelbauerg@missouri.edu.
Published, JBC Papers in Press, August 23, 2002, DOI 10.1074/jbc.M206245200
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ABSTRACT
INTRODUCTION