THE JOURNAL OF BIOLOGICAL CHEMISTRY© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 273, No. 46, Issue of November 13, pp. 30110 –30115, 1998
Printed in U.S.A.

Intersubunit Interaction between Transmembrane Helices of theBacterial Aspartate Chemoreceptor Homodimer*(Received for publication, March 30, 1998, and in revised form, June 1, 1998)

Tohru Umemura, Ichiro Tatsuno‡, Manabu Shibasaki§, Michio Homma, and Ikuro Kawagishi¶
From the Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, JapanThe transmembrane domain that connects the extracellular and intracellular domains of cell-surface receptors must play a critical role in signal transduction.
Here, we report studies of the interaction between thetransmembrane helices (TM1 and TM2) of the Escherichia coli aspartate chemoreceptor (Tar). Tar exists asa homodimer regardless of its state of ligand occupancy.
A particular residue substitution in TM1 (A19K) abolishes the signaling ability of Tar. This signaling defectcan be suppressed by single residue substitutions in
TM2 (W192R, A198E, V201E, and V202L). We have foundthat these suppressors can be divided into two groups.
A198E and V201E (class 1) almost completely suppress
the defects caused by A19K, and this suppression occurs
between two subunits of the Tar dimer. In contrast,
W192R and V202L (class 2) fail to suppress some signaling defects, and their suppression does not occur between subunits. Because disulfide-crosslinking studies
predict that residues 198 and 201 point toward residue
19 of the partner subunit, we propose that the class 1
suppressors form an intersubunit salt bridge with Lys19. Indeed, A19K was suppressed by several additional
aspartate or glutamate substitutions on the same face of
TM2 occupied by residues 198 and 201. None of theseintersubunit salt bridges perturb signaling function,suggesting that the mechanism of transmembrane signal propagation does not involve large displacements(such as extensive rotation) of the TM1 and TM2 helices
relative to each other.

Cell-surface receptors detect extracellular signals and convert them into intracellular signals. Their extracellular andintracellular domains are connected by transmembrane (TM)1
domains, which typically consist of a-helices. In addition to
supporting the molecular architecture of the proteins, these
TM domains must also play a critical role in signal transduction across the cytoplasmic membrane.
The aspartate chemoreceptor (Tar) of enteric bacteria is well
suited for studying the function of TM domains (for reviews, see* This work was supported in part by a grant-in-aid for scientificresearch from the Ministry of Education, Science and Culture of Japan(to I. K.). 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.
‡ Present address: Dept. of Bacteriology, Institute of Medical Science,
University of Tokyo, Minato-ku, Tokyo 108-0071, Japan.
§ Present address: Kowa Research Institute, Kowa Co., Ltd., 1-25-5
Kannon-dai, Tsukuba, Ibaraki 305-0856, Japan.
¶ To whom correspondence should be addressed. Tel.: 81-52-7892993; Fax: 81-52-789-3001; E-mail: i45406a@nucc.cc.nagoya-u.ac.jp.
1
The abbreviations used are: TM, transmembrane; MSA, minimal
semisolid agar; TM1, transmembrane region 1; TM2, transmembraneregion 2; TSA, tryptone semisolid agar.

Refs. 1– 4). Escherichia coli Tar also mediates responses tomaltose by interacting with liganded maltose-binding protein.
Tar is a homodimeric protein (5) with a subunit molecular massof about 60 kDa. It has two TM helices, TM1 and TM2. Unlike
some homodimeric eukaryotic receptors with tyrosine-kinase
activity, Tar apparently does not undergo monomer-dimertransitions during signaling, because some disulfide-crosslinked dimers are fully active (5, 6). Therefore, binding ofligands to Tar is thought to trigger a conformational change
within the receptor dimer, which must include some displacement of the TM domains with respect to each other.
The Tar dimer forms a stable ternary complex with a homodimer of the autophosphorylating histidine kinase CheA
and two molecules of the coupling protein CheW (7, 8). When
aspartate or another attractant ligand binds to Tar, CheA
activity is inhibited. Otherwise, phosphorylated CheA transfers the phosphoryl group of the response regulator CheY, andphospho-CheY promotes clockwise rotation of the flagellar motor and thereby causes the cell to tumble. When phospho-CheY
is not bound to it, the motor rotates counterclockwise, and thecell swims smoothly.
The periplasmic, ligand-binding domain of Tar has been
crystallized both in the presence and absence of aspartate (9).
Each monomer contains four a-helices (a1, a2, a3, and a4),
which form a four-helix bundle. The longer helices, a1 and a4,
are contiguous with TM1 and TM2, respectively, and they form
a quasi four-helix bundle with the a19 and a49 helices of theirpartner subunit. Recent studies suggest that the a-helical pair
TM1/a1–TM19/a19, which constitutes an interface between thetwo subunits of the receptor dimer, is rather static (10 –15). In
contrast, binding of ligand to the receptor dimer is thought tocause a displacement (a tilt, rotation or vertical slide) of a4/
TM2 relative to the TM1/a1–TM19/a19 pair.
In this context, it is relevant to note that a single amino acidsubstitution (A19K) in TM1 abolishes the signaling ability of
Tar without impairing the aspartate-binding ability of the receptor (16). Many intragenic suppressors of A19K were isolated, and four of them caused residue substitutions in TM2(W192R, A198E, V201E, and V202L). None of these suppressors abolish receptor function in the absence of the original
A19K mutation. Consistent with this observation, TM2 (17–19)
is relatively tolerant for substitutions. TM2 of Tar can be replaced by that of the related serine chemoreceptor (Tsr), or viceversa, without destroying receptor function (17). On the other
hand, certain substitutions at position 204 in TM2 (I204F,
I204Y, and I204W) impair the signaling ability of Tar (18), andseveral other mutations causing defects in signaling were identified in TM2 of the related ribose-galactose chemoreceptor(Trg) (19).
In this study, we examined how mutations in TM2 suppress
the TM1 mutation A19K. The original TM2 suppressors could
be divided into two groups: A198E and V201E almost com-

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Transmembrane Helices of the Aspartate Receptor
pletely suppress the defects caused by A19K, whereas W192R
and V202L fail to suppress some of the defects. Moreover, theformer two mutations, but not the latter two, can suppress
A19K even if they are present in the partner subunit of the
dimer. A19K can also be suppressed by the introduction ofnegatively charged residues (Asp or Glu) at position 205 or Asp
at position 201, suggesting that intersubunit suppression by
A198E or A201E results from formation of a salt bridge between TM1 and TM29. This finding places some clear constraints on the possible mechanisms of TM signaling.
EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids—All strains used in this study arederivatives of Escherichia coli K-12. Strains RP4372recA (F2 thi thr leu
met eda rpsL D(tar-tap)5201 tsr-1 recA) (20) and KO607 (Dtsr-7021
D(tar-tap)5201 Dtrg-100 recA) (21), both of which were provided by K.
Oosawa of Nagoya University, were used as the plasmid hosts in chemotaxis assays. Strain DH5a (F2 l- recA1 hsdR17 endA1 gyrA96
supE44 relA1 thi-1 D(argF-lacZYA)U169 f80dlacZDM15) (22) was used
for plasmid construction. A pBR322-based plasmid, pAK101, carries thewild-type tar gene (20). Its derivatives carrying tar genes encoding
Tar-A19K or Tar-A19K with suppressors (W192R, A198E, V201E,
V202L) were provided by K. Oosawa. Plasmid pIT6 was constructed bysubcloning a tar-containing DNA fragment into the plasmid vector
pSU18 (23), which contains the P15A replicon and the chloramphenicolacetyltransferase gene.
Mutagenesis of Tar—DNA manipulations were carried out using
standard methods. Site-directed mutagenesis was performed by a twostep polymerase chain reaction (24) using plasmid pAK101 as the
template and primers synthesized by Sawadi Technology (Tokyo). Polymerase chain reaction was carried out using ExTaq polymerase(Takara Shuzo, Kyoto) with 25 cycles of denaturing at 96 °C for 5 min,annealing at 58 °C for 30 s, and extension at 72 °C for 1 min. The
polymerase chain reaction products were cloned between the XbaI and
KpnI sites of pAK101. EcoRV fragments (1.2 kilobases) containing thedesired mutations were introduced into pAK101 or pIT6. The DNA
sequences were verified by the dideoxy chain termination method.
Swarm Assay of Chemotaxis—Chemotaxis was examined in tryptone
semisolid agar (TSA) (1% tryptone, 0.5% NaCl, 0.25% agar) or minimal
semisolid agar (MSA) (0.05 M potassium phosphate buffer (pH 7.0), 1
mM MgSO4, 1 mM glycerol, 0.1 mM each of threonine, leucine, histidine,
methionine, 1 mg/ml thiamine, 1 mM (NH4)2SO4, and 0.25% agar),
supplemented with 0.1 mM aspartic acid or maltose as needed. Ampicillin and/or chloramphenicol were added as required. Semisolid agar
was inoculated with aliquots of exponential-phase cultures (about 4 3
106 cells) and incubated at 30 °C.
Temporal Assay of Chemotactic Response—Temporal-stimulation assays were carried out essentially as described previously (25). Cells
were grown at 30 °C in TG broth (1% tryptone, 0.5% NaCl, 0.5% (w/v)
glycerol) with ampicillin and/or chloramphenicol. When necessary, 0.2%
maltose was added. Cells were harvested in late exponential phase,
washed twice with motility medium (10 mM potassium phosphate buffer(pH7.0), 0.1 mM EDTA, 0.1 mM methionine, 10 mM sodium DL-lactate),and resuspended in motility medium at room temperature. The swimming pattern of the cells was observed with a dark-field microscope andrecorded on videotape. For time-course assays, the fraction of smoothswimming cells was determined every 30 s after the addition of an
attractant. Video images were analyzed using an Argus-10 image processor (Hamamatsu Photonics K. K., Shizuoka). In an image integrated
for 1 s, smooth-swimming and tumbling cells gave linear and blurreddot traces, respectively. The smooth-swimming fraction was defined as
the percent fraction of smooth-swimming cells per total swimming cells.
Analysis of Methylation Patterns—Receptor methylation was assayed as described previously (26). Cells expressing wild-type or mutant Tar proteins were grown, harvested, and washed as describedabove. A chemoeffector was added to cells suspended in motility medium, and the suspension was then incubated at room temperature for
30 min. The cells were collected by centrifugation and suspended in
SDS-loading buffer (67 mM Tris-HCl (pH 6.8), 8% glycerol, 1% SDS,
0.003% bromphenol blue) supplemented with 7.7% 2-mercaptoethanol.
Samples were boiled for 3 min and subjected to SDS-polyacrylamide gelelectrophoresis. Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore Japan, Tokyo) using a semidry blotting
apparatus (Biocraft, Tokyo). Anti-Tsr-T156C serum (27), which crossreacts with Tar, and alkaline phosphatase-conjugated goat anti-rabbit
IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) were used

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TABLE I
Properties of the Tar-A19K z Sup homodimers
Wild-type and mutant Tar receptors were expressed in KO607, whichlacks Tar and the related chemoreceptors Tsr, Trg, and Tap. Swarming
ability was examined in MSA containing 0.1 mM aspartate (Asp) or 0.1
mM maltose (Mal). In temporal-stimulation assays, cells expressing any
receptor other than Tar-A19K responded to aspartate (Asp), maltose(Mal), and glycerol (Glyc). After prolonged incubation with these chemoeffectors, the cells were examined to see if they had adapted to thestimuli. Significant (1), weak (6), or no (2) adaptation was observed.
Swarming ability

Adaptation ability

Tar

Wild type
A19K
A19K z W192R
A19K z A198E
A19K z V201E
A19K z V202L
a

Asp

Mal

Asp

Mal

Glyc

1
2
1
1
1
1

1
2
6
1
1
6

1
NAa
1
1
1
1

1
NA
6
1
1
6

2
NA
1
6
1
1

NA, not applicable due to lack of an initial response.

as the first and the second antibodies, respectively. Protein-antibody
complexes were visualized in 5 ml of AP buffer (0.1 M Tris-HCl (pH 9.5),
1 M NaCl, 5 mM MgCl2) supplemented with 33 ml of nitroblue tetrazolium solution (50 mg/ml in 70% (v/v) dimethylformamide) and 16.5 ml of
5-bromo-4-chloro-3-indolylphosphate solution (50 mg/ml).
RESULTS

Identification of Two Classes of TM2 Suppressors ofA19K—We characterized the Tar proteins having the A19K
substitution and a TM2 suppressor on the same subunit (collectively referred to as Tar-A19KzSup) (Table I). These proteins
were expressed in strain KO607, which lacks all of the methylaccepting chemoreceptors except Aer, which mediates aerotactic responses (28, 29). The resulting transformants were tested
for their swarming ability. In TSA and MSA-aspartate (MSAAsp), cells expressing any Tar-A19KzSup protein formed
swarms like those of cells expressing wild-type Tar. In MSAmaltose (MSA-Mal), however, cells expressing TarA19KzW192R or V202L did not swarm well, whereas theswarms made by cells expressing Tar-A19KzA198E or V201E
were comparable with those of cells expressing wild-type Tar.
In temporal-stimulation assays, responses to aspartate or
maltose of cells expressing any Tar-A19KzSup were similar to
those of cells expressing wild-type Tar, and the threshold attractant concentrations (apparent sensitivities to the attractants) were similar. However, adaptation to maltose was impaired in cells expressing Tar-A19KzW192R or V202L, whereas
cells expressing Tar-A19KzA198E or V201E adapted normallyto maltose. In contrast, cells expressing any Tar-A19KzSup
adapted to aspartate indistinguishably from cells expressing
wild-type Tar.
These results suggest that the TM2 suppressors can be classified into two groups. Class 1 suppressors (A198E and V201E)
almost completely suppressed the signaling and adaptation
defects caused by A19K. Class 2 suppressors (W192R and
V202L) did not suppress some of the defects, notably those inadaptation to maltose.
Trans Suppression of A19K by the Class 1 Suppressors—To
address how the TM2 substitutions suppress defects caused by
A19K, we tested their ability to suppress an A19K substitution
in the partner subunit. We reported previously that A198E
suppresses A19K in trans and used this system to ask whether
a receptor dimer with only one intact signaling domain canmediate an attractant response to aspartate (30). Subsequently, we constructed a series of mutant Tar proteins having
one of the TM2 suppressors and the aspartate-binding site
alteration T154P. These mutant proteins (collectively referred
to as Tar-T154PzSup) were co-expressed with Tar-A19K instrain RP4372recA, which lacks Tar and the related chemore-30112

Transmembrane Helices of the Aspartate Receptor

FIG. 1. The strategy for intersubunit suppression. A, a functional (1) homodimer of Tar with A19K (star) and its suppressor (Sup)(circle); B, co-expression of Tar-A19K and Tar-T154PzSup. Tar-Sup isfunctional, and so the mutation T154P (triangle), which causes a severe
defect in aspartate sensing, was introduced into Tar-Sup. The functionof the heterodimer of Tar-A19K and Tar-T154PzSup (right) can bemonitored, because neither of the homodimers (left and center) can
mediate an aspartate response (2).

ceptors Tsr and Tap (Fig. 1). Immunoblots verified that all of
the mutant proteins were expressed (data not shown). Essentially similar results were obtained when the mutant proteins
were expressed in strain KO607.
These cells were then tested for their swarming ability in
MSA-Asp (Fig. 2) and TSA (not shown). In TSA or MSA-Asp,neither of these Tar derivatives alone supported formation ofswarm rings, indicating that the homodimers of these Tarproteins do not mediate an attractant response to aspartate, as
expected. However, cells expressing both Tar-A19K and TarT154PzA198E or V201E swarmed well in both TSA and MSAAsp. In contrast, neither W192R nor V202L in trans could
suppress the defect in swarming caused by A19K.
We then examined the attractant responses of these cells to
aspartate directly, using the temporal-stimulation assay (Fig.
3). Again, cells expressing Tar-A19K or any Tar-T154PzSup
protein alone did not respond to aspartate (data not shown).
However, cells expressing Tar-T154PzA198E or V201E with
Tar-A19K, did respond to aspartate. The concentration of aspartate required for a half-maximal response was similar tothat of cells expressing wild-type Tar. However, cells co-expressing Tar-A19K and Tar-T154PzW192R or V202L did not
respond, even to 0.01 M aspartate. These results indicate thatthe class 1 suppressors, but not the class 2 suppressors, cansuppress the defect in signaling caused by the A19K substitution on the partner subunit (intersubunit suppression).
Identification of Suppressors of A19K among Asp or Glu
Substitutions at Residues Near A198 and V201—The class 1(intersubunit) suppressors A198E and V201E introduce a negatively charged residue into TM2, whereas the other suppressors introduce a positively charged or uncharged residue. Furthermore, disulfide-crosslinking studies of TM1 and TM2 (10,11, 31, 32) predict that residues 198 and 201 of one subunit faceresidue 19 of the partner subunit (Fig. 4). Therefore, we propose that intersubunit suppression results from formation of asalt bridge between the e-amino group of Lys-19 and the g-carboxyl group of the suppressing Glu residue (Glu-198 or
Glu-201).
To test this hypothesis, we introduced Asp or Glu into positions on the face of TM2 helix predicted to be facing the partner
subunit (Fig. 4). Immunoblots demonstrated that the mutant
proteins were expressed (data not shown). Cells expressingthese proteins were tested for their swarming ability (Fig. 5). In
MSA-Asp, they produced three types of swarms (Fig. 5). (i)Cells expressing Tar-A19KzA198E, V201D, V201E, L205D, or
L205E produced swarm rings that were as sharp as althoughsmaller than that of cells expressing wild-type Tar. (ii) Cells
expressing Tar-A19KzI204D or I204E produced small and dif-

FIG. 2. Swarming abilities of RP4372recA cells co-expressing
Tar-A19K and Tar-T154PzSup from compatible plasmids. Overnight cultures were spotted onto MSA containing ampicillin, chloramphenicol, and 0.1 mM aspartate. The plate was incubated at 30 °C. The
Tar proteins present are: a, wild-type Tar; b, no Tar; c, Tar-A19K; d,
Tar-T154P; e, Tar-A19K 1 Tar-T154P; f, Tar-A19K 1 TarT154PzW192R; g, Tar-A19K 1 Tar-T154PzA198E; h, Tar-A19K 1 TarT154PzV201E; i, Tar-A19K 1 Tar-T154PzV202L; j, Tar-T154PzW192R;
k, Tar-T154PzA198E; l, Tar-T154PzV201E; m, Tar-T154PzV202L.

FIG. 3. Aspartate-sensing abilities of heterodimers containing
Tar-A19K and Tar-T154PzSup. Various concentrations of aspartate
were added to a suspension of cells expressing wild-type Tar (●) or
co-expressing Tar-A19K with Tar-T154P (E), Tar-T154PzW192R (L),
Tar-T154PzA198E (‚), Tar-T154PzV201E (M), or Tar-T154PzV202L (ƒ).
After 20 s, the smooth-swimming fractions of the cells were measured.

fuse swarms. (iii) Cells expressing Tar-A19KzA198D, A208D, or
A208E produced no swarm ring. Essentially, similar swarming
patterns were observed in TSA (data not shown).
In temporal-stimulation assays, the first group of cells
showed almost the same threshold for aspartate as cells expressing wild-type Tar (Fig. 6A), despite the varied diametersof the swarms formed by the mutants. The second group of cells
did not give a significant response immediately after the addition of aspartate (Fig. 6B). However, their smooth-swimming
fractions increased up to 30% within 30 s (data not shown). In
contrast, the third group of cells did not respond to aspartate atall (Fig. 6C). These results demonstrate that A19K can besuppressed by Asp or Glu substitutions for Ala-198, Val-201,
and Leu-205 (except A198D), suggesting that the basis of suppression really is the formation of a salt bridge between Lys-19
and an introduced negatively charged residue.

Transmembrane Helices of the Aspartate Receptor

30113

FIG. 4. Schematic representation of the TM domains of the Tar
homodimer. The relative orientation of the putative a-helices (TM1,
TM19, TM2, and TM29) is based on disulfide-crosslinking studies (10,11, 31, 32). The view is from the periplasmic surface (after Ref. 10).
Closed circles, positions at which substitutions created class 1 suppressors; gray circles, positions at which substitutions generated class 2
suppressors; hatched circles, positions at which Asp or Glu substitutions did not fully suppress A19K. The circle with a black center at
position 198 indicates that A198E suppressed, whereas A198D did not.

FIG. 5. Suppression of A19K by Asp or Glu substitutions inTM2. RP4372recA cells expressing each mutant Tar were examined fortheir swarming ability in MSA containing ampicillin and 0.1 mM aspartate as described in the legend to Fig. 2. The Tar proteins present are:a, wild-type Tar; b, no Tar; c, Tar-A19K; d, Tar-A19KzA198D; e, TarA19KzV201D; f, Tar-A19KzI204D; g, Tar-A19KzL205D; h, TarA19KzA208D; i, Tar-A19KzA198E; j, Tar-A19KzV201E; k, TarA19KzI204E; l, Tar-A19KzL205E; m, Tar-A19KzA208E.

We also examined the expression levels and methylation
patterns of these mutant Tar proteins by immunoblotting (Fig.
7). Multiple methylation of a chemoreceptor by CheR causes
stepwise increases in its mobility in SDS-polyacrylamide gelelectrophoresis (33–36). All of the mutant receptors were detected in whole cell lysates, although their amounts and levelsof methylation varied substantially. Stimulation of methylation by the addition of aspartate was observed with receptors
that mediated responses to aspartate (Tar-A19KzA198E,
V201D, V201E, L205D, and L205E) but not with those thatmediated little or no responses to aspartate (Tar-A19KzA198D,
I204D, I204E, A208D, and A208E).
Trans Suppression of A19K by Asp or Glu Substitutions in
TM2—We also tested for trans suppression of A19K by the Asp
or Glu substitutions. In MSA-Asp, RP4372recA cells co-expressing Tar-A19K with Tar-T154PzV201D, L205D, or L205E
produced swarm rings comparable with those of cells expressing wild-type Tar (Fig. 8). In contrast, cells co-expressing TarA19K with Tar-T154PzA198D, I204D, I204E, A208D, or A208E
produced little or no swarm ring. Essentially similar swarming

FIG. 6. Aspartate-sensing ability of homodimers of Asp- or Glusubstituted Tar-A19K. Responses to aspartate mediated by wild-type
Tar (●), Tar-A19K (E), or Tar-A19K with A198D (L), A198E (l),V201D (‚), V201E (Œ), I204D (ƒ), I204E (), L205D (M), L205E (f),
A208D (”), or A208D (–) were determined as described in the legend to
Fig. 3. A, cells that showed aspartate responses comparable with thatmediated by wild-type Tar; B, cells that showed marginal responses toaspartate; C, cells that did not respond to aspartate.

patterns were observed in TSA (data not shown). These results
demonstrate that V201D, L205D, and L205E can suppress
A19K in trans. Thus, V201D, L205D, and L205E are also class
1 suppressors.
DISCUSSION

In this study, we examined how single amino acid substitutions in TM2 suppress the detrimental substitution A19K in
TM1. A19K does not affect the ligand binding, but it does
abolish signaling ability (16). Our results divide the four suppressors in TM2 into two groups. The A198E and V201E substitutions (class 1 suppressors) almost completely reverse thedefects caused by A19K, and this effect can be exerted between
the two subunits of the Tar dimer. In contrast, the W192R and30114

Transmembrane Helices of the Aspartate Receptor

FIG. 7. Expression levels and methylation patterns of Asp- or
Glu-substituted Tar-A19K proteins. RP4372recA cells expressing
wild-type or mutant Tar receptors were incubated with (1) or without(2) 10 mM aspartate (Asp), and whole cell lysates were subjected to
SDS-polyacrylamide gel electrophoresis and immunoblotted with antiTsr serum. None, no Tar; WT, wild-type Tar; A19K, Tar-A19K; A198D
etc., Tar-A19KzA198D etc.; Nonsp., a band cross-reacting nonspecificallywith the serum.

FIG. 8. Trans suppression of A19K by Asp or Glu substitutionsin TM2. RP4372recA cells co-expressing Tar-A19K and Tar-T154P withan Asp or Glu substitution from compatible plasmids were examined fortheir swarming ability as described in the legend to Fig. 2. The Tarproteins present are: a, wild-type Tar; b, no Tar; c, Tar-A19K; d, TarT154P; e, Tar-A19K 1 Tar-T154P; f, Tar-A19K 1 Tar-T154PzA198D; g,
Tar-A19K 1 Tar-T154PzV201D; h, Tar-A19K 1 Tar-T154PzI204D; i,
Tar-A19K 1 Tar-T154PzL205D; j, Tar-A19K 1 Tar-T154PzA208D; k,
Tar-A19K 1 Tar-T154PzA198E; l, Tar-A19K 1 Tar-T154PzV201E; m,
Tar-A19K 1 Tar-T154PzI204E; n, Tar-A19K 1 Tar-T154PzL205E; o,
Tar-A19K 1 Tar-T154PzA208E; p, Tar-T154PzA198D; q, TarT154PzV201D; r, Tar-T154PzI204D; s, Tar-T154PzL205D; t, TarT154PzA208D; u, Tar-T154PzA198E; v, Tar-T154PzV201E; w, TarT154PzI204E; x, Tar-T154PzL205E; y, Tar-T154PzA208E.

V202L substitutions (class 2 suppressors) fail to suppress somesignaling defects, and suppressions did not occur between subunits. This finding supports the validity of the experimental
design for intersubunit suppression, because it excludes thepossibility that apparent intersubunit suppression results fromcompensation of smooth swimming biased signaling of the TarA19K homodimer by tumbling biased signaling of the TarT154PzSup homodimer.
Disulfide-crosslinking studies predict that residues 198 and201 of TM2 point toward residue 19 of TM19 (Lys-199) (Fig. 4).

Therefore, the g-carboxyl groups of the glutamate residues of
the class 1 suppressors are likely to form an intersubunit salt
bridge with the e-amino group of Lys-199. Consistent with this,all of the same-site pseudorevertants isolated from A19K are to
introduce uncharged residues (Ile, Thr, and Gln) (16).
The possibility of salt bridge formation might be tested bychanging ionic strength. However, such in vivo experiments
should be difficult, because E. coli cells show abnormal responses to higher salt concentrations without chemoreceptors
or any other chemotactic signaling proteins (termed pseudotumbling) (37). The hypothesis might also be examined byintroducing a negative charge in TM1 and a positive charge inTM2. Although either of these mutations might be detrimental
and suppressed by a positive charge in TM2 or a negative
charge in TM1, respectively, there is no obvious candidate forsuch mutation (it should be noted that W192R is harmless).
Perhaps the most realistic way to test the salt bridge formation is to systematically introduce Asp or Glu substitutions on
the face of the TM2 helix on which residues 198 and 201 arelocated. Indeed, some of these mutations (V201D, L205D, andL205E) reversed the defects caused by A19K, whereas others(A198D, I204D, I204E, A208D, and A208E) produced little orno suppression. The effective suppressors result from substitutions for residues 198, 201, and 205 that are predicted to faceLys-199 (Fig. 4). On the other hand, substitutions that do notsuppress, with the exception of A198D, are predicted to belocated further from Lys-199. The positively charged side chainof Lys-19 probably disturbs normal packing of the four TM
helices (TM1, TM19, TM2, and TM29), and introduction of anegatively charged residue in TM29 may restore this packing
by creating a salt bridge between TM1 and TM29.
It is striking that A198D and A198E had such different
effects. An Asp residue at position 198 might not extend far
enough to form a salt bridge with Lys-199 or, if a salt bridge
does form, it might affect the receptor structure and function.
Hydrophobic interaction between TM1 and TM29 might beimportant for the receptor architecture, and a charge-neutralizing salt bridge might restore such interactions. A similar salt
bridge between TM helices has been implicated in the structural stabilization of wild-type lactose permease (38, 39).
The interhelical salt bridges have another, perhaps more
important, implication for signal transduction. They would
probably be maintained upon binding and release of a small
molecule-like aspartate. The enthalpies (DH) of formation of
the various salt bridges cannot be calculated precisely, but theyshould be larger than the enthalpy (218 kcal/mol) reported forserine binding to Tsr (40). Therefore, the salt bridges presumably restrict the potential ability of TM1 and TM29 to move
relative to one another during the signal-transduction cycle.
Recent studies have provided good evidence that TM1 andTM19 do not have to move relative to one another for effective
TM signaling to take place after ligand binding (10 –15).
Rather, vertical displacement, tilting, or rotation of TM2 relative to TM1 seems to be the critical element in signaling. Thesefindings predict that ligand binding may cause both intra- andintersubunit displacement of TM2. The multiple interhelicalsalt bridges that are compatible with receptor function argue
that such a displacement cannot be too large. These small
movements of TM2, however, may trigger structural changes in
the cytoplasmic domains that are essential for signal production. Such changes have been suggested to occur between subunits (41– 45) as well as within a subunit (or between dimers)(30, 46, 47).
The mode(s) of suppression by W192R and V202L are still
unknown. These mutations might exert their effects within asubunit or they might be effective only when they are placed inTransmembrane Helices of the Aspartate Receptor
both subunits of a dimer. Moreover, A19K can also be suppressed by substitutions in the cytoplasmic “linker” region,
which is contiguous from TM2 (16). Further investigation ofthese suppressors should help elucidate the mechanism of receptor signaling.
Acknowledgments—We thank Dr. K. Oosawa for strains, plasmids,
and helpful discussion and Dr. M. D. Manson (Texas A & M University)
for critically reading the manuscript.
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