THE JOURNAL OF BIOLOGICAL CHEMISTRY© 1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 272, No. 21, Issue of May 23, pp. 13810 –13815, 1997
Printed in U.S.A.

Uncoupling of Ligand-binding Affinity of the Bacterial Serine
Chemoreceptor from Methylation- and Temperature-modulated
Signaling States*(Received for publication, January 14, 1997)

Tomonori Iwama‡, Michio Homma§, and Ikuro Kawagishi§¶
From the ‡Department of Biotechnology, Division of Utilization of Biological Resource, Faculty of Agriculture,
Gifu University, Yanagido, Gifu 501-11, Japan and the §Division of Biological Science, Graduate School of Science,
Nagoya University, Chikusa-ku, Nagoya 464-01, JapanThe Escherichia coli chemoreceptor Tsr mediates tactic responses to serine, repellents, and changes in temperature. We have previously shown that the serinesensing ability of Tsr-T156C, which has a uniquecysteine in place of threonine at residue 156, is specifically inactivated by thiol-modifying reagents and that
L-serine protects the receptor from modification. In thisstudy, we demonstrated the correlation between protective effects of various attractants and their potencies toelicit attractant responses. This indirect binding assay
was used to monitor the affinity of the receptor for Lserine under various conditions. It has been demonstrated by in vitro assays that the ligand-binding affinities of Tsr and the related chemoreceptor Tar are
unaffected by changes in the methylation state of thereceptor. Using the serine protection assay, we re-examined this issue both in vitro and in vivo. The methylationlevels of Tsr-T156C did not affect its ligand-binding affinity. We also showed both in vitro and in vivo that theligand-binding affinity was unaffected by temperature.
These results suggest that the structure of the periplasmic domain of the receptor is uncoupled from the signaling states of the cytoplasmic domain. This ligandbinding assay system should be applicable to otherreceptors.

In many sensory systems, desensitization or adaptation to apersisting stimulus plays a crucial role in highly sensitive
detection of stimuli over a comprehensive range. Extracellularsignals (e.g. binding of ligands) are received and transduced
into intracellular signals (e.g. activation or inactivation of kinases) by cell surface receptors. Binding of a ligand to a receptor extracellular domain induces some changes in structure ofa receptor intracellular domain and, hence, in its activity. By
contrast, upon adaptation, the signaling activities of the intracellular domains are often down-regulated by intracellular
feedback regulatory systems (typically via covalent modification of the receptors). The effects of receptor down-regulation
may be limited to the intracellular domain or may induce a
global structural change in the receptor.
Four closely related chemoreceptors of Escherichia coli serve* This work was supported in part by a grant-in-aid for scientificresearch (to I. K.) from the Ministry of Education, Science and Cultureof Japan. The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
¶ To whom all correspondence should be addressed: Division of Biological Science, Graduate School of Science, Nagoya University,
Chikusa-ku, Nagoya 464-01, Japan. Tel.: 81 52 789 2993; Fax:
81 52 789 3001; E-mail: i45406a@nucc.cc.nagoya-u.ac.jp.

as model systems for investigating both transmembrane signaling and down-regulation (for reviews, see Refs. 1– 6), Tsr(for serine), Tar (for aspartate and maltose), Trg (for ribose andgalactose), and Tap (for dipeptide). These chemoreceptors function as homodimers regardless of ligand occupancy state (7),and the homodimer forms a ternary complex with a homodimerof a cytoplasmic autokinase CheA and two molecules of an
adaptor protein CheW (8). Each receptor monomer consists ofan N-terminal periplasmic ligand-binding domain, a C-terminal cytoplasmic signaling domain, and two membrane-spanning segments. Binding of a ligand to the interface between thetwo periplasmic domains triggers some structural change
within the receptor dimer. This structural change leads toactivation or inactivation of autophosphorylation of CheA andphosphotransfer from CheA to CheY. Phosphorylated CheY
binds to the flagellar motor and induce clockwise (CW)1 rotation of the motor (causing tumbling of the cell), which otherwise
rotates counterclockwise (CCW) (causing smooth swimming).
Adaptation is achieved via methylation and demethylation of
the chemoreceptors (9). Each chemoreceptor contains four to
six glutamic acid residues that are reversibly methylated.
Methyltransferase CheR catalyzes transfer of a methyl groupfrom S-adenosylmethionine to a glutamic acid side chain, andmethylesterase CheB hydrolyzes the methyl ester bond of amethylated glutamate residue. The latter enzyme also serves
as deamidase, converting specific glutamine residues of thenascent chemoreceptors to methylatable glutamic acid residues(10).
Recent in vitro studies (11–13) suggest that methylation of a
receptor modulates its signaling state but has little effect on its
ligand-binding affinity. This means that the structure of theperiplasmic ligand-binding domain is uncoupled from the signaling state of the cytoplasmic domain under some conditions.
However, contradictory results have been reported for receptorcontaining membranes (14). To explain the discrepancy, Lin etal. (13) suggested that association of chemoreceptors with cytoplasmic proteins, such as CheA and CheW, might influence
their ligand-binding behavior. Therefore, the effects of methylation and demethylation on ligand-binding affinity of receptors must be investigated under conditions closer to their native setting.
The four chemoreceptors are unique in that they function
also as thermosensors (15, 16). Tsr, Tar, and Trg are warm
1
The abbreviations used are: CW, clockwise; CCW, counterclockwise;Em, a methylated glutamic acid residue; NEM, N-ethylmaleimide;
IPTG, isopropyl-1-thio-b-D-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis; TM1, the first transmembrane segment; TM2, thesecond transmembrane segment; Cmr, resistance to chloramphenicol;
Tcr, resistance to tetracycline.

13810

This paper is available on line at http://www-jbc.stanford.edu/jbc/

Ligand-binding Affinity of the Serine Chemoreceptor
receptors that produce CCW signals upon a temperature increase and CW signals upon a temperature decrease, whereas
Tap is a cold receptor with opposite signaling behavior (16).
Temperature should affect the signaling states of the chemoreceptors, but it remains unclear how this can be achieved.
Does a change in temperature cause a global change in receptor
structure or just some local change within the cytoplasmic
signaling domain?
In this study, we investigated the effects of methylation/amidation and temperature on the ligand-binding affinity ofTsr. We have previously shown that a Tsr-specific attractant
L-serine protects Tsr-T156C, which has a unique cysteine residue at the ligand-binding site, from thiol-modifying reagents
such as N-ethylmaleimide (NEM) in a dose-dependent manner(17). This protective effect of L-serine should reflect the affinityof Tsr-T156C for L-serine. If so, this assay can be used tomonitor the ligand-binding affinity of the receptor under various conditions. In this report, we demonstrate a correlation
between the stimulus strength of various amino acid attractants detected by Tsr-T156C and their abilities to protect Cys156 from NEM modification. Using this assay system both invitro and in vivo, we found that the ligand-binding affinity of
Tsr is uncoupled from its signaling states modulated by covalent modification and temperature.

13811

FIG. 1. Sensing abilities of Tsr-T156C for various attractants.
HCB339 cells with Tsr-T156C were pretreated with 1 M glycerol andthen stimulated with various concentrations of L-serine (open circles),D-serine (closed circles), or L-alanine (open triangles). Changes insmooth swimming cells after 30 s were measured. All manipulations
were done at 25 °C. In this and the following figures, lines are drawn
just by eye.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids—All strains used in this work arederivatives of E. coli K-12. Strain HCB339 [Dtsr-7021 D(tar-tap)5201
trg::Tn10 thr leu his met rpsL136] (18) lacks all four chemoreceptors,
and strain CP553 [trg-100 Dtsr-7028 D(tar-cheB) leu his rpsL lac xyl ara
tonA tsx thi zab::Tn5] (19) lacks CheB and CheR, as well as all chemoreceptors. Plasmid pGAN1 (17) carries the promoterless mutant tsr
gene encoding the receptor protein with the single amino acid substitution, Thr-156 to Cys, placed downstream of the tac promoter. Plasmid
pRAB1 (20) carries the methylesterase gene cheB and the tetracyclineresistant gene tetA (Tcr). A cheR-overproducing plasmid pKB23 (11) anda cheB-overproducing plasmid pKB24 (11), both of which carry thechloramphenicol acetyltransferase gene cat (Cmr), were provided by M.
I. Simon of California Institute of Technology.
Chemicals—NEM, isopropyl-1-thio-b-D-galactopyranoside (IPTG),
phenylmethylsulfonyl fluoride (PMSF), and 1,10-phenanthroline werepurchased from Wako Pure Chemical Industries (Osaka, Japan). Octylb-D-glucopyranoside was purchased from Dojindo Laboratories (Kumamoto, Japan). N-[ethyl-1-14C]ethylmaleimide (40 mCi/mmol) and reagents for the bicinchoninic acid protein assay were products of Dupont
NEN and from Pierce, respectively.
Assays of Chemo- and Thermoresponses—Cells were grown at 30 °C
in tryptone-glycerol broth (1% Bacto-tryptone (Difco Laboratories, Detroit, MI), 0.5% NaCl, 0.5% glycerol) supplemented with 50 mg/ml
ampicillin (and 25 mg/ml chloramphenicol, when necessary). At late
exponential phase, cells were collected by centrifugation, washed twicewith motility medium (10 mM potassium phosphate buffer, pH 7.0, 0.1
mM EDTA, 10 mM sodium DL-lactate, 0.1 mM methionine), and resuspended in motility medium.
Temporal stimulation assays for chemoresponse were carried out asdescribed previously (21). The cells suspended in motility medium werepretreated with a repellent, 1 M glycerol, and then stimulated withvarious concentrations of a Tsr-mediated attractant, L-serine, D-serine,or L-alanine. The change in the smooth swimming fraction after 30 s
was measured. Addition of 1 M glycerol reduces the smooth swimming
fraction of the cells to nearly zero. Therefore, an increase in the smoothswimming fraction upon addition of an attractant represents the magnitude of an attractant response of the cells.
Temporal stimulation assays for thermoresponse were carried out asdescribed previously (20). Cells were cultured and washed as describedabove. A drop of cell suspension was placed on a glass slide mounted ona temperature control apparatus (22). When cells swam too smooth or
too tumble, glycerol or L-serine was added to the suspension. The
temperature was first increased from 20 to 30 °C and then decreased
from 30 to 20 °C.
Preparation of Membranes and Solubilization of Membrane Proteins—Membranes were prepared by the method of Foster et al. (23). A
fresh overnight culture of CP553 cells carrying pGAN1 was inoculated(1:100 dilution) into LB medium (1% Bacto-tryptone, 1% yeast extract,

0.5% NaCl) supplemented with 200 mg/ml ampicillin and grown at
30 °C. If the cells also carried pRAB1 (Tcr) or pKB23 (Cmr), 12.5 mg/ml
tetracycline or 25 mg/ml chloramphenicol was added. After 3 h, 1 mM
IPTG was added to the culture. After further incubation for 4 h, thecells were harvested by centrifugation, washed with motility medium,
and were treated with 100 mM NEM in the presence of 10 mM serine for
60 min at 0 °C. The cells were then collected again, resuspended in lysis
buffer (100 mM sodium phosphate, pH 7.2, 5 mM EDTA, 10% glycerol, 5
mM 1,10-phenanthroline, 2 mM PMSF) at 0 °C, and lysed by sonication(Heat Systems-Ultrasonics, Inc., model W-225). Unbroken cells and celldebris were removed by centrifugation for 20 min at 10,000 rpm in a
Sakuma 7B rotor at 4 °C. The supernatant was centrifuged for 60 min
at 40,000 rpm in an RP 50 –2 rotor (Hitachi Koki Co., Ltd.) at 4 °C. The
pellet was washed with wash buffer (50 mM sodium phosphate, pH 7.2,
2 M KCl, 10% glycerol, 5 mM EDTA, 5 mM 1,10-phenanthroline, 1 mM
PMSF) and then resuspended in a buffer containing 50 mM Tris-HCl,
pH 7.0, 10% glycerol, 5 mM 1,10-phenanthroline, and 1 mM PMSF at
0 °C. Protein concentration was determined by the bicinchoninic acidprotein assay system using bovine serum albumin as a standard. The
suspension was diluted with the same buffer to approximately 10 mg ofprotein/ml. Octylglucoside was added to 1.25%, and the mixture wasincubated for 20 min at 0 °C and centrifuged for 60 min at 40,000 rpm(Hitachi RP65 rotor) at 4 °C. The supernatant was stored at 4 °C.
Modification with Radioactive NEM—For modification of Tsr-T156C
in intact cells, N-[ethyl-1-14C]maleimide (10 mCi/mmol; 50 mM final
concentration) was added to the cells (A590 5 1.5), and the mixture wasincubated at 25 °C for 30 min. For membranes or octylglucoside extracts, the mixture was incubated at 25 °C for 6 min. When necessary,various concentrations of L-serine, D-serine, or L-alanine were addedprior to the addition of NEM. The reactions were terminated by theaddition of 5% trichloroacetic acid, and the samples were collected bycentrifugation. The precipitates were then subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21) and autoradiography. The labeled proteins was analyzed using Bio Imaging
Analyzer (Fuji Photo Film Co., Ltd., BAS-1000).
RESULTS

Protection of Cys-156 from NEM Modification Reflects the
Ligand-binding Affinity of Tsr-T156C—We first examined thesensing abilities of Tsr-T156C for various amino acid attractants. Plasmid pGAN1 carrying the tsr-T156C gene was introduced into strain HCB339, which is defective in all four chemoreceptor genes. As shown in Fig. 1, 40 mM L-serine or 1 mM
D-serine was required for attractant responses in 50% of thecells. Only a small response was induced by 50 mM or higher

13812

Ligand-binding Affinity of the Serine Chemoreceptor

FIG. 2. Protective effects of various attractants on modification of Tsr-T156C by radioactive NEM. Membranes (containing
approximately 4 mg of proteins) extracted from CheB2 CheR2 cells
overproducing Tsr-T156C were solubilized with 1.25% octylglucoside.
50 mM [14C]NEM was added to octylglucoside extracts in the presence ofvarious concentrations of an attractant. The mixtures were incubatedfor 6 min at 25 °C and were subjected to SDS-PAGE followed by autoradiography with an imaging plate. The relative intensities of labeled
Tsr-T156C were plotted. Open circles, L-serine; closed circles, D-serine;
open triangles, L-alanine.

concentrations of L-alanine. These results are essentially consistent with the published data for wild-type Tsr (24), that theaffinity of Tsr for L-alanine is lower than that for L-serine bymore than two orders of magnitude.
Next, we investigated the ability of various attractants toprotect against NEM modification. As shown in Fig. 2, theaddition of L-serine or D-serine to membrane proteins extractedwith octylglucoside significantly prevented NEM from reactingwith Tsr-T156C, but L-alanine appeared to have no protective
effect. Similarly, glycine and a-aminoisobutyric acid, a nonmetabolizable attractant, had no effect (data not shown). The
protective effect of D-serine was smaller than that of L-serine,
consistent with the fact that the concentration of D-serine for ahalf-maximal response of cells expressing Tsr-T156C is about
10-fold higher than that of L-serine. Similarly, the inability ofL-alanine to protect corresponds to the low apparent affinity of
Tsr-T156C for L-alanine. Therefore, we conclude that the relative extent of protection by an attractant against NEM modification of Tsr-T156C reflects the affinity of Tsr for theattractant.
Effects of Covalent Modification on Serine Protection in
Vitro—We next examined whether methylation of the chemoreceptor affects its affinity for attractants. Plasmid pGAN1
carrying the tsr-T156C gene was introduced into strain CP553,
which lacks CheB and CheR as well as all four chemoreceptors.
To modulate the methylation level of Tsr-T156C, the resulting
strain was further transformed with plasmids carrying either
the methylesterase/deamidase gene cheB or the methyltransferase gene cheR. Fig. 3A shows the effects of various concentrations of L-serine on NEM modification of Tsr-T156C in membrane preparations from CheB1 CheR2 or CheB2 CheR2 cells.
In these strains, the five major methyl-accepting residues ofTsr (collectively designated as QEQEE) should be modified asfollows. In CheB2 CheR2 cells, all five residues are unmodified(QEQEE); in CheB2 CheR1 cells, the Gln residues are notdeamidated, and the Glu residues are methylated (QEmQEmEm); and in CheB1 CheR2 cells, the Gln residues are deamidated, and the Glu residues are not methylated (EEEEE). In
fact, Tsr-T156C proteins in those cells showed distinct mobili-

FIG. 3. Effect of covalent modification on protection by L-serine. Membranes (approximately 6 mg of proteins) from CheB1 CheR2 or
CheB2 CheR1 cells were incubated with 50 mM [14C]NEM for 6 min at
25 °C in the presence of various concentrations of L-serine. A, samples
were subjected to SDS-PAGE and autoradiography. B, the relative
intensities of labeled Tsr-T156C were plotted as described in the legend
to Fig. 2. In the case of CheB2 CheR1 cells, the sum of all four bands
was plotted. Open circles, membranes of CheB1 CheR2 cells; closed
circles, membranes of CheB2 CheR1 cells.

ties on SDS-PAGE due to methylation or deamidation (25). In
each case, the extent of NEM labeling of Tsr-T156C decreased
considerably in the presence of higher concentrations of Lserine. The L-serine concentration required for 50% protection
is approximately 500 mM both for CheB2 CheR1 cells and for
CheB1 CheR2 cells (Fig. 3B). Similar results were obtainedusing octylglucoside-solubilized Tsr-T156C proteins (data notshown). These results suggest that covalent modification of
Tsr-T156C does not dramatically affect its affinity for L-serinein the absence of the cytoplasmic Che proteins, a conclusionconsistent with recent studies on Tar (11, 12) and Tsr (13).
Effects of Covalent Modification on Serine Protection in
Vivo—We then examined serine protection of Tsr-T156C from
NEM modification in intact cells. As shown in Fig. 4B, theL-serine concentration required for 50% protection of TsrT156C was approximately 1 mM for CheB2 CheR2 (QEQEE),CheB2 CheR1 (QEmQEmEm), and CheB1 CheR2 (EEEEE)
cells. This was not due to the ineffectiveness of the method
since about 100 mM D-serine was required for 50% protection of
Tsr-T156C expressed in CheB2 CheR2 cells (Fig. 4B). Fourbands of Tsr-T156C were detected for samples from CheB2
CheR1 cells due to differential methylation levels (Fig. 4A). So,
we further examined serine protection on each band. The slowest band on SDS-PAGE was considered to be the unmodified
form (QEQEE) of Tsr (Fig. 4C). All bands were protected from
NEM modification by L-serine to almost the same degree as theslowest band. Taken together, we conclude that covalent modification of Tsr-T156C does not dramatically change its affinity
to serine even in the presence of the cytoplasmic Che proteins.
However, since Tsr was overproduced, we cannot rule out thepossibility that many of the receptor proteins might not form
ternary complexes with CheW and CheA.

Ligand-binding Affinity of the Serine Chemoreceptor

13813

TABLE I
Thermosensing abilities of Tsr and Tsr-T156C in the
CheR2 backgrounds
Thermoresponses of cells expressing Tsr or Tsr-T156C were analyzed. Strains CP553 (CheB2 CheR2: abbreviated as B2R2) and CP553
transformed with the CheB-overproducing plasmid pKB24 (CheB11
CheR2: abbreviated as B11R2) were used as hosts for plasmids carrying the tsr or tsr-T156C gene. Cells were incubated at 20 °C prior to theassays for themoresponses. Temperature was increased to 30 °C andthen decreased to 20 °C as described under “Experimental Procedures.”S, smooth swimming; R, random swimming; T, tumbling; NT, not tested; 1, cells showed smooth swimming and tumbling upon an increase
and a decrease in temperature, respectively; 2, cells did not change
their swimming behavior upon temperature changes.
Thermoresponse

Chemoreceptor/host

Swimming patternat 25 °C

None

1 Serine

1 Glycerol

Tsr/B11R2
Tsr/B2R2
Tsr-T156C/B11R2
Tsr-T156C/B2R2

S/R
T
S/R
T

1
2
2
2

NT
1a
NT
1b

NT
NT
1c
NT

a
Thermoresponses were measured in the presence of 120 mM Lserine.
b
Thermoresponses were measured in the presence of 300 mM Lserine.
cThermoresponses were measured in the presence of 10% glycerol.

FIG. 4. Effect of covalent modification on serine protection in
intact cells. CheB1 CheR2, CheB2 CheR2 or CheB2 CheR1 cells
overproducing Tsr-T156C were incubated with 50 mM [14C]NEM for 30
min at 25 °C in the presence of various concentrations of L-serine orD-serine. A, samples were subjected to SDS-PAGE and autoradiography. Each Tsr protein with differentially modified form is indicated bya thin arrow on the right. Thick arrows on the left represent thepositions of bands, corresponding to the molecular weight of 66 kDa. B,
the relative intensities of labeled Tsr-T156C were plotted as describedin the legend to Fig. 2. Open circles, CheB2 CheR2 cells, L-serine; closed
circles, CheB2 CheR2 cells, D-serine; open triangles, CheB1 CheR2
cells, L-serine; closed triangles, CheB2 CheR1 cells, L-serine. C, therelative protection by L-serine of different methylated forms in the
CheB2 CheR1 cells. Open circles, total (bands 1– 4); closed circles, band
1; open triangles, band 2; closed triangles, band 3; open square, band 4.

Effects of Temperature on Serine Protection—We next examined whether temperature influences the ligand-binding affinity of the chemoreceptor. First of all, we had to examine
whether Tsr-T156C has thermosensing ability in the CheB2
CheR2 background since Tar does not have thermosensing

ability in the same background (20). The results are summarized in Table I. CP553 cells (Tsr2 Tar2 Trg2 Tap2 CheB2
CheR2) were transformed with plasmid pJUN11, which carries
the tsr-T156C gene. The resulting cells showed tumbling without any stimulus at 25 °C and did not change their swimming
behavior upon temperature changes. When tumbling was reduced by the addition of 0.3 mM L-serine, the cells showed
thermosensing abilities. The cells showed smooth swimming as
the temperature increased (from 20 to 30 °C) and then tumbling as the temperature decreased (from 30 to 20 °C). Similar
results were obtained for CP553 cells expressing wild-type Tsr
in the presence of 120 mM L-serine. An analysis by Western
blotting with anti-Tsr antiserum detected no change in mobility of either wild-type Tsr or Tsr-T156C during these behavioral responses, indicating that neither spontaneous deamidation nor degradation of the chemoreceptors had occurred (datanot shown). These results suggest that, unlike Tar, the primary, unmodified translational product of Tsr can mediate athermoresponse. That serine does not play an essential role inthermosensing by Tsr-T156C was verified as follows. To shift
the signaling bias toward smooth swimming (CCW flagellar
rotation) by deamidation, Tsr-T156C was expressed in CP553
cells carrying the CheB-overproducing plasmid pKB24(CheB11 CheR2). The resulting cells swam very smoothly evenwhen temperature was changed. However, in the presence of arepellent glycerol (10%), the deamidated form of Tsr-T156C
mediated a thermoresponse. We conclude that both the unmodified and the deamidated forms of Tsr-T156C have thermosensing abilities.
Next, we subjected Tsr-T156C expressed in the CheB2
CheR2 background to NEM modification in the presence ofvarious concentrations of L-serine. As shown in Fig. 5, both ina membrane preparation and in an octylglucoside extract, TsrT156C was protected by L-serine from NEM modification at 20,
25, and 30 °C to almost the same degree. By contrast, incubation at higher temperature (50 °C) almost completely abolishedthe protective effect of serine (data not shown), indicating (partial) denaturation of the protein. These results suggest that theligand-binding affinity of the chemoreceptor is not significantly
affected by a moderate change in temperature which causes
thermotaxis.

13814

Ligand-binding Affinity of the Serine Chemoreceptor
DISCUSSION

FIG. 5. Effect of temperature on serine protection. CheB2
CheR2 cells overproducing Tsr-T156C (A), and membranes (B) andsolubilized membrane proteins (C) from the cells were incubated with
50 mM [14C]NEM for 6 min at 20 °C (open circles), 25 °C (closed circles),or 30 °C (open triangles). The relative intensities of labeled Tsr-T156C
were plotted as described in the legend to Fig. 2.

We used sulfhydryl modification to investigate the ligandbinding affinity of Tsr-T156C under various conditions.
Whereas an attractant binds reversibly to Tsr, NEM covalently
attaches to the cysteine residue. Therefore, the amount of
NEM-labeled to Tsr-T156C increases even in the presence ofthe competitor during incubation. In this study, we could not
determine absolute values of the dissociation constants. Moredetailed analyses would be required for obtaining such values
as demonstrated for dissociation constants between an enzyme
and its substrate or inhibitors (26). However, when Tsr-T156C
was incubated with NEM for a fixed length of time in thepresence of L-serine, D-serine, or L-alanine, the magnitudes ofprotection were consistent with the apparent affinities of TsrT156C for those attractants. Therefore, we could compare relative affinities of Tsr for L-serine under various conditions
simply by monitoring the protective effect of L-serine on NEM
modification during a fixed length of time.
Using this assay system, we found that the serine-binding
affinity of Tsr is uncoupled from its methylation-modulated
signaling states both in vivo and in vitro. This is consistent
with recent in vitro studies on Tar (11, 12) and Tsr (13). These
results suggest that “reverse transmembrane signaling” fromthe inside to the outside of the cell membrane does not happen
in adaptation. However, a previous report (14) demonstrated
that methylation of Tsr and Tar substantially decreased theligand affinity of receptors in the membrane preparations. The
discrepancy might be explained by the fact that the latter study
did not monitor ligand binding to the receptors directly or bydifferences in the amount of the receptors in the membrane
preparation. Alternatively, the cytoplasmic Che proteins, especially CheA and CheW, which form a ternary complex with thechemoreceptor, might contribute to reduction of the ligandbinding affinity upon methylation, which cannot be observed
for the bare receptor, as has been discussed before (13). We
cannot rule out this possibility since Tsr-T156C was overproduced in our experiments, and some would not be in ternarycomplexes with CheA and CheW.
More surprisingly, we found that the serine-binding affinityof Tsr is also uncoupled from its temperature-modulated signaling states in vivo and in vitro. Thus, a moderate change intemperature in the range between 20 and 30 °C may not cause
a dramatic change in the receptor structure, such as monomerdimer transition or a profound conformational change, but may
cause some effects limited to the cytoplasmic domain. These
local structural changes should be the nature of thermosensing
by the chemoreceptor. The importance of the cytoplasmic domain in thermosensing by Tar has been suggested by a previous study (20), which showed that the primary translational
product of tar does not have thermosensing ability and theunmethylated and the heavily methylated forms of Tar function as warm and cold receptors, respectively. In this regard,
Tsr behaves differently. The unmodified form functions as awarm receptor (Table I), and it seems to lose thermosensing
ability upon methylation (27, 28). These differences between
Tsr and Tar might be related to differences in the number ofmethylation sites (25, 29). In any case, covalent modification in
the cytoplasmic domain dramatically affects the thermosensing
property of the receptor, a fact which is consistent with thenotion that the structure of the cytoplasmic domain is alteredby temperature changes.
Our results provide insight into the molecular architecture of
the receptor. That the ligand-binding domain may be fairly
stable in its overall organization is suggested by crystallographic analyses (30 –32). There is only a small difference
between the ligand-occupied and -free structures of a periplas-

Ligand-binding Affinity of the Serine Chemoreceptor
mic fragment of Tar. Recent studies suggest that the firsttransmembrane helices (TM1 and TM19) pair stably, and binding of the ligand causes some movement of the second transmembrane helix (TM2) relative to TM1-TM19 (33–36). However, it remains unclear what happens to the cytoplasmic
domain. Recent in vitro studies suggest that dimerization (oroligomerization) of the cytoplasmic domain of Tar plays a critical role (37, 38). On the other hand, the assays for genetic
complementation between two mutant receptors suggest that
only one intact cytoplasmic domain per receptor dimer is sufficient for signaling (39, 40). Therefore, the signaling mechanism may involve interactions between dimers, conformationalchanges within a single cytoplasmic domain, or both. In any
case, binding of an attractant followed by a relatively smallchange in the periplasmic domain triggers a dramatic inactivation of CheA kinase. Therefore, it is reasonable to assume
that the two different signaling states (CW and CCW; kinase
on and off) of the receptor are caused by a relatively large
structural change of the cytoplasmic domain. If so, covalentmodification and temperature changes should cause similar
structural changes in the cytoplasmic domain. However, undersuch conditions, the structure of the periplasmic domain remains unaffected, suggesting structural uncoupling between
the two domains across the membrane.
Acknowledgments—We thank Dr. M. I. Simon for generously providing plasmids, and S. Nishiyama and I. Tatsuno for collaborating in
preparation of antiserum. We especially thank Dr. J. S. Parkinson for
critically reading the manuscript.
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