From the Department of Molecular Biology, Swedish
University of Agricultural Sciences, Box 590, Biomedical Center,
S-751 24 Uppsala, the ¶ Department of Cell and Molecular
Biology, Uppsala University, Box 596, S-751 24 Uppsala, Sweden, and
Howard Hughes Medical Institute, University of Texas,
Southwestern Medical Center, Dallas, Texas 75235-9050
Received for publication, October 20, 2000
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ABSTRACT |
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Three arginine residues of the binding site of
the Escherichia coli aspartate receptor contribute to its
high affinity for aspartate (Kd ~3
µM). Site-directed mutations at residue 64 had the
greatest effect on aspartate binding. No residue could substitute for
the native arginine; all changes resulted in an apparent
Kd of ~35 mM. These mutations had
little impact on maltose responses. At residue Arg-69, a lysine
substitution was least disruptive, conferring an apparent
Kd of 0.3 mM for aspartate. Results
obtained for an alanine mutant were similar to those with cysteine and
histidine mutants (Kd ~5 mM)
indicating that side chain size was not an important factor here.
Proline and aspartate caused more severe defects, presumably for
reasons related to conformation and charge. The impact of residue 69 mutations on the maltose response was small. Mutations at Arg-73 had
similar effects on aspartate binding (Kd 0.3-7
mM) but more severe consequences for maltose responses. Larger side chains resulted in the best aspartate binding, implying steric considerations are important here. Signaling in the
mutant proteins was surprisingly robust. Given aspartate binding,
signaling occurred with essentially wild-type efficiency. These results were evaluated in the context of available structural data.
Like all living creatures, bacteria need to keep track of things
happening around them and to respond appropriately to important events.
The proteins of the chemotaxis system allow the sensing of chemical
signals and the use of that information to direct appropriate swimming
behavior (for recent reviews see Refs. 1 and 2). Signal reception is
the task of receptors found in the cytoplasmic membrane that either
recognize a chemical of interest directly or indirectly through the
mediation of a periplasmic binding protein. The best characterized of
these proteins is the aspartate receptor, the product of the
tar gene. This 60-kDa membrane protein (3) can bind to
aspartate or glutamate directly (4). Although the
Tar1 receptors from
Escherichia coli and Salmonella typhimurium have 79% sequence identity, only E. coli Tar can recognize the
complex between maltose-binding protein (MBP) and maltose (5). It has been shown that aspartate and maltose responses are largely
independent, i.e. both attractants can be recognized
simultaneously or sequentially (6-8).
The structure of intact Tar is not yet known, although many studies
have combined to give a rather comprehensive picture of its structure.
The sequence (3) first suggested its functional organization. The
receptor has a short N-terminal segment (residues 1-6) on the
cytoplasmic side of the inner membrane. A short hydrophobic segment
(residues 7-30) then crosses to the periplasm, where a soluble domain
(residues 31-188) capable of binding aspartate is located. A second
membrane-spanning sequence (residues 189-212) brings the receptor back
to the cytoplasm. The remainder of the protein (amino acids 213-552)
composes a soluble cytoplasmic unit that possesses the signaling and
adaptation properties of the receptor. That the smallest functional
unit is a dimer was first established by cross-linking of mutant
cysteine-bearing receptors (9) and was later confirmed by the x-ray
structure of the ligand-binding domain of the receptor (Fig.
1A (10)). The four-helix
bundle of each periplasmic domain is connected to the two transmembrane segments through extensions of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
4. As found for the
periplasmic regions, the two
1 extensions of the dimer appear to be
in close proximity in the membrane, whereas the
4 extensions are
further apart (11). The structure of the greater part of the
cytoplasmic domain of the related serine receptor has been shown to
consist of a very long coiled coil in which two antiparallel helices
from each subunit are packed onto each other in a four-helix bundle; in
this portion of the receptor it is the extensions of
4 that form the
basis of inter-subunit contacts (12).
View larger version (34K):
[in a new window]
Fig. 1.
The Tar periplasmic domain and its
binding site for aspartate. A, ribbon drawing showing
the domain as observed in the 2LIG structure. Major helices are defined
as 1 (residues 33-75),
2 (residues 89-109),
3 (residues
117-143), and
4 (residues 154-178), with different coloring for
the two subunits. The short helix from residues 145 to 152 is shown as
a coil, as are the loops connecting the longer helices. The
A molecule is that with aspartate bound to Arg-64 and Thr-154, and the
B molecule contributes residues Arg-69' and Arg-73'. Bound aspartate is
shown as a ball-and-stick representation with atomic colors.
The figure was prepared using Molscript (53). B,
hydrogen-bonding interactions in the aspartate-binding site, using the
same coloring scheme for the residues as in part A.
Interactions requiring the side chains of Arg-64, Arg-69', and Arg-73'
(studied here) are shown in magenta, with other interactions
in red (if they have been tested elsewhere) or
green (those that have not yet been tested).
There are two sites for aspartate binding in the dimer, each incorporating residues from both subunits (Fig. 1). Binding of aspartate to one of the sites hinders binding of the second effector molecule through negative cooperativity (13). The effect is modest in the case of Salmonella Tar, but the affinity at the second site of E. coli Tar is reduced at least 50-fold (14). Responses resulting from aspartate binding require the action of a set of cytoplasmic and flagellar proteins that control swimming. Adaptation is associated with the reversible methylation of specific glutamates on the cytoplasmic domain of the receptor itself (15, 16).
Although more is presently known about this signaling system than any other, many mysteries remain, including the means by which the receptor conveys information about ligand binding into the cell. Tar is a dimer both in the presence and absence of ligand (9), precluding monomer-dimer equilibria as a mechanism for transmembrane signaling. Key changes within an existing dimer could include relative motions of the two subunits, as well as motions within a subunit. Most structural studies suggest the former (10, 17), whereas other evidence seems to support the latter (summarized in Refs. 18-20).
In the present paper, we describe a study of site-directed mutations in
the binding site of E. coli Tar. The residues chosen for
this work are Arg-64, Arg-69, and Arg-73, which are conserved in all
receptors that bind amino acids (21) and so are presumably central to
receptor function. We explore the significance of the results in the
light of available data on receptor structure and biology.
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EXPERIMENTAL PROCEDURES |
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Reagents, Strains, and Plasmids--
Maltose (PM grade) was
obtained from Merck, and aspartate (ultragrade) was from the Sigma.
Unlabeled Ado-Met (Sigma grade I, iodide salt) was purified using an
anion exchange resin (Dowex AG 1 × 8) to remove
S-adenosylhomocysteine, an inhibitor of methyltransferase, and then stored at 80 °C. Radiolabeled AdoMet (15 Ci/mmol) was obtained from Amersham Pharmacia Biotech. Strains RP437 (F
thi thr leu his met(am) eda strA), RP4080 (F
cheR217 thi thr leu his gal lac ara xyl
mtl strA recA nalA), and RP4372 (F
tsr518
(tar-tap)5201 thi thr leu his met(am) eda
strA) (22) were provided by J. S. Parkinson at the University
of Utah. Plasmid pME43 (23) was obtained from Jeff Stock (Princeton
University) and transformed into strain JM109 to provide a source of
methyltransferase (prepared as described earlier (24)). The final
buffer used in dialysis contained 1 mM
1,10-o-phenanthroline and 1 mM PMSF; dialyzed
cytosols were stored at
80 °C. Strains carrying other Tar mutants
(R64C, R69C, R69H, R73W, and R73Q (21)) were the generous gifts of J. S. Parkinson and C. Wolff. The plasmid pMK650, which bears E. coli Tar as well as ampicillin resistance, was described
previously (7).
Mutagenesis-- Oligonucleotides for mutagenesis were 21-mers generating changes of E. coli Tar residues Arg-64 and Arg-69 to Ala, Asp, Lys, Pro, and Ser and Arg-73 to Cys, Asp, Lys, Pro, and Ser by single- or double-base substitutions. Site-directed mutagenesis of pMK650 was carried out by standard methods. All mutants were confirmed by DNA sequencing.
Swarm Assays-- For in vivo chemotaxis assays, cells grown in Luria broth were inoculated with a sterile stick into the center of a Petri dish containing Vogel-Bonner citrate medium (25), 1% glycerol, 0.3% agar (Difco), and 50 µg/ml each His, Leu, Met, Thr, and thiamine, with or without attractant added, as specified, and with or without ampicillin (150 µg/ml) as needed. The radii of swarms were measured during growth at 30 °C, at 4-6 time points between 24 and 48 h. The lines fit to such data points had correlation coefficients ranging from 0.97 to 1.0.
Membrane Preparation and Methylation/Binding Assays--
Cell
growth and membrane preparation were as described earlier (26), except
that cells were routinely broken using a French press (2 passes at
5000-6000 pounds/square inch) rather than by sonication. Washed
membranes were stored at 80 °C in 50 mM Tris-HCl, pH
7.25, 10% (w/v) glycerol, 5 mM
1,10-o-phenanthroline, 1 mM PMSF. Expression of
mutant receptors was evaluated by SDS gel electrophoresis and Western
blots. In vitro methylation/binding assays (16) were
performed in a buffer containing 20 mM sodium phosphate, pH
7.0, 1 mM 1,10-o-phenanthroline, and 1 mM PMSF. Aspartate concentrations were chosen where
possible to represent multiple points both above and below the
Kd of the particular receptor (as determined from
preliminary experiments), with the highest concentration always at
least 60 mM. Samples were taken at 15 and 90 min to observe
both early and late stages of the modification process. The methylation
obtained for RP4080pEMBL19 was subtracted from each value to account
for methylation due to chromosomal receptor expression (less than 10%
of the total).
Analysis of Binding Data-- Methylation data were analyzed in terms of two aspartate-binding constants in the program Mathematica (Wolfram Research), using Equation 1,
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(Eq. 1) |
Structural Comparisons-- Table I lists the available x-ray structures of the periplasmic effector-binding region of Tar. Coordinates for most were obtained from the Brookhaven Protein Data Bank (27) and are identified here by their entry codes. Those for a form of E. coli Tar without bound ligands (28) were kindly provided by Prof. Sung-Hou Kim (University of California, Berkeley). All aspartate-bound structures have a dimer in the asymmetric unit. As all aspartate-free structures contain only a single subunit in the asymmetric unit, the second subunit of the dimer was first generated using crystallographic symmetry. The dimeric structures were compared with the lsq options of the graphics program O (29, 30), initially matching residues 40-74 of the chain that carried aspartate bound at residue 64 to the same segment of the aspartate-free molecule (with two different possibilities for the 1VLT structure, which has two molecules of aspartate bound). A cut-off of 0.6 Å was used to improvement the alignment and to look for changes greater than the expected level of error in the structures. Distance diagonal plots were calculated with LSQMAN (31).
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RESULTS |
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Overproduction of Mutant Tar-- With the exception of R64P (which produced no detectable Tar), most mutant receptors were expressed at least half as well as the wild-type protein (Table II) and so represented 5-10% of the total membrane protein.
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Swarm Assays--
The behavior of bacteria can be assessed
qualitatively with an in vivo swarm assay, in which cells
are inoculated into a semi-solid agar medium with or without attractant
(at concentrations of 0.1 or 1 mM aspartate, or 0.1 mM maltose, as indicated). As the cells take up the
components of the medium, excrete wastes, and multiply, gradients are
built up which cause chemotaxis (32). For the present study, RP4372
(tar, tap
,
tsr
, cheB+,
cheR+) was transformed with pMK650 (containing
wild-type Tar), with mutated versions of the same plasmid, or tested by
itself as a control.
Representative swarm results are presented in Fig.
2. Although differences in the levels of
receptor expression, as well as any in transport or metabolism, make
quantitative assessments of receptor function unwise, a number of
patterns are clear. The background strain alone (RP4372) lacked Tar and
exhibited no chemotaxis to either aspartate or maltose; the rate of
swarming in the absence of added attractant is also low, due to the
inherent smooth-swimming bias of the strain. Overexpression of the
wild-type receptor (with pMK650) in the same strain improved swarming
somewhat, presumably by reducing that bias. The overproduced receptor
also enabled aspartate and maltose responses, with a characteristic
pattern of saturation of the response at higher aspartate
concentrations. Most of the mutant receptors also brought about a
similar improvement of the unstimulated rate, as well as allowing
aspartate responses. Mutants R64P and R73D were essentially blind to
both attractants, and indeed cells bearing them behave as though they
have no functional receptor. For R64P, this is consistent with the lack
of observable Tar on SDS gels; the location of residue 64 in the middle
of a helix makes this result unremarkable. As R73D was produced at normal levels, the swarm results presumably indicate severely impaired
function rather than grossly altered receptor structure. R64D, R64S,
and R69A generated modest increases in the unstimulated swimming rate,
which could reflect slightly lower levels of deamidation of these
mutant receptors and/or different levels of receptor expression (Table
II). R73P showed frequent back mutations and so was not tested
further.
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Most mutations generated behavioral patterns that correlated in a straightforward way with the altered residue. Mutations at residue 64 caused decreases in the aspartate response, with little or no effect on maltose chemotaxis. Mutations at residue 69 produced a similar pattern, although with more variation. R69A had slightly higher unstimulated rates and a large maltose response, suggesting that this Tar form may have some alterations in basic signaling properties. R69D also seemed to have some reduction of signaling to both aspartate and maltose. R69K was the least disruptive change; this mutant even showed saturation at the higher aspartate concentration, as observed for the wild-type Tar. Mutations at residue 73 gave rise to receptors with a relatively effective aspartate response but significantly impaired chemotaxis to maltose. The notable exception here was R73D, which had no apparent response to either ligand. These patterns confirm and extend the results of earlier studies (26, 33).
Methylation/Binding Assays--
Because of problems inherent in
measuring aspartate affinity to membrane-bound receptor when the
binding is weak, an indirect method (26) was used, which exploits the
proportional relationship between ligand binding and the modification
of a receptor by the chemotaxis methyltransferase (15). This method has
the additional advantage that it gives some measure of the
signaling/adaptation properties of the receptor. The results were first
analyzed with Scatchard plots, which consistently showed a concave
biphasic shape indicative of negative cooperativity. Nonlinear curve
fitting of the original data was then applied, as this method is
considered to be less vulnerable to distortions from systematic
experimental errors (34). Two sites were ultimately included in the
fitting of both wild-type and mutant receptors, as a single site did
not adequately reproduce the shape of the curves (despite suggesting a
very similar Kd1; Fig.
3). The assumption that aspartate binding
to the second site results in the same amount of methylation as binding
to the first site is implicit in this treatment, although no molecular
model for this exists. Assuming that a different amount of methylation
arose from binding at the second site did not improve the fit, and so
could not be justified, although it remains a very plausible physical
situation. The Hill equation (35) with n of the order of 0.8 (i.e. negative cooperativity) performed as well as the
two-site model. The values obtained from the two-site fitting are
summarized in Table II. Results with different time points did not
differ in any systematic way, and therefore averages are given, along
with the observed standard error. The apparent affinity of the second
site varied in response to a number of experimental factors (including
the properties of the different mutants), and so the estimated
Kd2 values are given only for the sake
of completeness, as their physical meaning is still unclear. To allow
comparison of the various receptors, extrapolation to the maximum
methylation expected appeared to be justified, as the methylation
generally covered a significant portion of the binding curve. The
largest degree of coupling observed in each case, i.e. the
increase in methylation found at the highest concentration of aspartate
tested, is also reported, although the size of this quantity varied
with the specific methyltransferase and AdoMet preparations. It should
be noted that the given values for coupling are underestimates of the
maximum, since the receptors were not usually fully saturated with
aspartate.
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The estimated affinity of the first site of the wild-type receptor for aspartate (2 µM) agrees well with earlier data obtained using a variety of methods (summarized in Ref. 2). Binding to the second site of E. coli Tar has previously been shown to be disfavored by negative cooperativity (14), although to an unknown extent. Our results suggest that the affinity of the second site is reduced at least 500-fold.
The strain RP4080, which has chromosomal expression levels of Tar and
methylesterase (CheB) but lacks methyltransferase (CheR), was used as
the background for the methylation studies here instead of RP4372
(tar, cheB+, cheR+) as used previously (26). During preparation of
most membranes, cells were broken in a French press, and previous
results were obtained with membranes prepared by sonication. To be
certain that no systematic errors arose from these changes, aspartate
binding to wild-type Tar was compared for membrane preparations that
differed in these respects. The results obtained were very similar. The
pair of experiments shown in Table II represents an extreme case, using
older radioactive AdoMet (2 years after purchase); the results
demonstrate that although the observed methylation and coupling under
these circumstances are lower, there is little impact on estimates of
binding to the first site. The Kd1
values (4-5 µM) are again in good agreement with
previously published estimates. As a further test, membranes for the
mutant R69C were also prepared using the RP4080 background and French
press. The Kd1 results were
indistinguishable from ones obtained with sonicated RP4372 membranes
(Table II). Other possible effects due, for instance, to changes in
ionic strength at high aspartate concentrations were ruled out by the fact that assays in which the 20 mM sodium phosphate buffer
was replaced with 100 mM Tris-HCl or 100 mM
phosphate gave equivalent results.
The Kd estimates for the different mutant proteins are shown in Table II, along with those derived using earlier data for mutants R64C, R69C, R69H, R73Q, and R73W overproduced in RP4372 (26), reprocessed with the nonlinear curve fitting. The values reported previously for the mutants are given for comparison and are consistent with those obtained here. As for the swarm assays, the results clearly fall into classes that are related to the site of mutation. The Kd1 of all of the residue 64 mutations was observed to be 20 mM or greater, with R64C having no detectable binding at aspartate concentrations as high as 60 mM. Residue 69 mutations had smaller effects on aspartate binding, with Kd1 values in the range of 2-8 mM; binding to the proline mutant receptor was 10-fold weaker and that to the lysine mutant was a factor of 10 stronger. At residue 73, replacing arginine with a large residue (glutamine, lysine, or tryptophan) was better than introducing a smaller one (cysteine or serine), by roughly a factor of 10, with Kd1 values clustered near 0.5 mM for the large residues versus 7 mM for the smaller ones. All of the mutant proteins gave methylation similar to that of the wild-type protein when saturated with aspartate, showing that once aspartate was bound all were competent for signaling.
Conformational Changes Caused by Aspartate Binding-- Interpretation of the mutational results must utilize available structural data on the mechanism of aspartate binding/signaling. As shown in Table I, structures of the periplasmic domain of the E. coli Tar are available only for the aspartate-free form. It is thus necessary to use the aspartate-bound and aspartate-free structures of the equivalent domain of the Salmonella protein (66% sequence identity in the periplasmic region). Three such pairs are presently available, of which only 1LIH/2LIG and 1VLS/1VLT were solved at sufficient resolution to study conformational changes with confidence. The 1LIH/2LIG pair represents Tar in which residue 36 is mutated to a cysteine, with the consequent formation of an inter-subunit disulfide bond. Such cross-linked receptors are still functional in aspartate binding (including negative cooperativity) and signaling (13, 14, 36); 2LIG has a single molecule of aspartate bound. Both members of the pair have a phenanthroline molecule located at the subunit interface; numerous studies, including this one, have shown that Tar responds normally to aspartate in the presence of phenanthroline. The 1VLS/1VLT pair represents the wild-type Salmonella receptor, without the disulfide cross-link and without bound phenanthroline. 1VLT has two molecules of aspartate bound. Both aspartate-free structures (1LIH and 1VLS) consist of two identical subunits, i.e. the dimer is symmetrical in the absence of bound aspartate. This symmetry is lost when the first molecule of aspartate enters 2LIG and is not regained when a second is bound to 1VLT.
The portions of the periplasmic domain that are most relevant to
transmembrane signaling are obviously those connected to the
membrane-spanning segments, i.e. 1 and
4 of each
subunit. Possible signaling mechanisms include intra-subunit (
1/
4
or
1'/
4') and inter-subunit (
1/
l' or
4/
4') changes.
In Fig. 4, a simplified distance-diagonal
plot illustrates the observed movements of these helices with respect
to each other in the two pairs of structures. Although somewhat
cumbersome to use, these plots have the advantage that they do not make
use of any prior model of what is actually equivalent, i.e.
which motions occur.
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In the 1LIH/2LIG pair, movement of 4 of subunit A (that bearing
aspartate bound to Arg-64) with respect to the rest of the dimer is
clearly the motion with the most potential significance for signaling
(Fig. 4, A and B). When the two structures are
aligned as described under "Experimental Procedures."
i.e. starting from
1 of the A subunit, 136 C-
atoms
(residues 51-76 of subunit A and residues 37-67, 92-134, 137-148,
and 150-173 of subunit B) match within a 0.6 Å cut-off, giving a root
mean square difference of 0.31 Å.
4 is seen to move about 1.5 Å toward the membrane relative to
1 and tilt slightly in a "swinging
piston" motion (18, 19). Smaller changes in
2 and
3 accompany
the movement of
4. The dimer interface, mainly a function of
1/
1' interactions, and the structure of the second subunit are
quite static. Negative cooperativity is explained by motions in and
near
2 which appear to make the second binding site smaller when the
first is occupied (14). The structure of this receptor domain in the
absence of phenanthroline is changed only locally (37).
For the 1VLS/1VLT pair, more complicated changes are observed in the
distance diagonal plots (Fig. 4, C and D). A
distinct rotation of the two subunits with respect to each other is
seen as 1/
1' movements. Motions of each
4 with respect to
1
of the same subunit are found as well, although with different
characteristics from that found in the 1LIH/2LIG pair. Both of the
4s, however, appear to move in the same manner within their
respective subunits. When the structures of the two dimers were aligned
as described under "Experimental Procedures," starting from
1 of
the A subunit, the root mean square difference was 0.32 Å with 83 atoms matching (residues 55-72, 89-104, 121-127, 130-140, and
142-166 from the A subunit and 50-55 from the B subunit). When
1
of the B subunit was the starting point for the alignment, it was 0.34 Å with 66 atoms matching (residues 54-74, 95-105, 116-123, 126-134
and 153-168, all from the B subunit). These results confirm that more
changes are found in this pair compared with the LIH/2LIG case, and
that
1/
1' motions are largest; changes within the individual
subunits are much smaller. The changes in both
1 and
4 are most
appreciable at the ends that would normally be attached to the
transmembrane segments (Fig. 1).
The two E. coli apo structures have additional, and
different, arrangements of the subunits, from each other, and from the Salmonella receptors. As there exist no structures for the
aspartate-bound forms of this Tar, a meaningful analysis of
conformational changes could not be carried out.
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DISCUSSION |
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Arg-64 is clearly established as most important for aspartate binding. Mutations here gave rise to a remarkably constant apparent Kd of ~35 mM. Although some changes (alanine, aspartate, and serine) reduced the expression of the protein (and presumably folding/stability), the binding result for each was the same. The only exception was a R64C mutant (26). Although it formed a stable receptor, R64C was totally defective in aspartate binding/signaling; since this could be explained by formation of an inter-subunit disulfide bond, the significance of this result as yet uncertain. Interestingly, the aspartate mutation (which might repel the ligand for electrostatic reasons) was not significantly worse than other substitutions. Nor did the lysine mutation, which should give a better match for the size and charge of the native arginine, result in better aspartate binding. The demands on this position in aspartate binding must, therefore, be so stringent that only arginine can fulfill them. Effects on unstimulated or maltose-induced swarming were not severe, indicating that most other aspects of Tar structure/function were intact.
Changes at residue 69 usually resulted in an apparent Kd of 2-8 mM. Binding to the proline mutant was 10-fold weaker, perhaps because Arg-73 (on the same helix) was also compromised. Binding to the R69D mutant was also poorer, which could be due to repulsion of the ligand by the negatively charged side chain or to the formation of a salt link with one of the remaining arginines. The swarm results, however, suggest that more general effects on receptor signaling are an issue for R69D; although its unstimulated swarm rate is normal, the maltose response for this mutant is weak. The relative efficiency of the lysine replacement at this position is interesting, given the inability of lysine to replace arginine at residue 64. In this respect, too, the role of Arg-69 appears to be less central for aspartate recognition. Effects on the maltose response were minimal for the residue 69 mutants.
Mutations at Arg-73 fell into two classes, apparently related to the size of the side chain introduced. Larger ones (regardless of their charge or hydrogen-bonding characteristics) gave rise to an apparent Kd of ~0.5 mM, whereas smaller ones resulted in a value of ~7 mM. Lysine was not significantly more effective than any other substitution. Thus, the interactions made by Arg-73 seem to be less critical than those of the other two arginine residues, provided that some space-filling role is satisfied. In contrast to the other sites, mutations at position 73 had a strong effect on the maltose response. The only exception to this pattern is the R73D mutant; the combined results suggest that although this receptor folds, it is defective in some aspects of signaling function.
Our results confirm earlier studies (21, 26, 33) and broaden the
available binding data sufficiently to allow conclusions to be drawn
from the patterns observed. The measured Kd of the
wild-type receptor for aspartate is ~3 µM. Binding was thus ~10,000, 1500, and 100-1500 weaker on mutating arginines 64, 69, and 73, respectively. As each arginine residue has bidentate interactions with the aspartate ligand, hydrogen bonding alone does not
offer an explanation for why their relative contributions to affinity
are different. The reasons must lie in the structural context of each
residue, including the nature of the aspartate-induced conformational
change. Unfortunately, the available crystallographic results
contradict each other in some respects. The wild-type structures show a
large relative movement of the two subunits on aspartate binding, and
the structures of Tar cross-linked at residues 36 suggest a motion of
4 within a single subunit as the significant change for
transmembrane signaling. A number of studies have shown that receptors
with cross-links across the
1/
1' interface are still functional
in signaling (reviewed in Ref. 19); these included cross-links
between residues 36 (36), as well as a case where two disulfide
bonds were introduced (18). As residues 36 are ~20 Å apart in the
1VLS structure, the relationship between this view of the
conformational change and that of the intact receptor is unclear. A
large number of genetic and spectroscopic studies (reviewed in Refs.
19, 20, and 38) have also suggested that intra-subunit (
1/
4)
changes are most important for receptor signaling. However, the
undeniable fact remains that inter-subunit changes are observed in the
wild-type structures. Although such differences could be due to crystal
packing in the different space groups or to the loss of the other
portions of the molecule, all new data should be measured against both
sets of structures.
Some aspects of the bound/free structures are consistent, notably the
nature of the interactions in the aspartate-binding sites (Fig.
1B). Arg-64 makes charge-charge interactions with the oxygen
atoms of the -carboxyl group of aspartate. This side chain appears
to be held in position by hydrogen bonds to those of Gln-155 and
Gln-158 in
4 of the same subunit, even before the introduction of
ligand, and so may be presumed to lose little entropy on binding. It
should be noted that arginine is the only residue that could make all
of these interactions. As Arg-64 is relatively buried, mutations here
may also disturb the local structure. In contrast, Arg-69 and Arg-73
interact with the side chain carboxyl group of the aspartate ligand and
are apparently not as central to its binding. These residues are more
exposed and so less constrained, so entropy may partly explain why they
contribute less. The effects of residue size at Arg-73 are probably
related to the observation that the loop just before
2 (near Ser-84)
folds down over this residue in occupied sites; this is the same change
that is thought to be the basis of negative cooperativity. Smaller
residues would leave a "hole" at this position of the
aspartate-bound form and so should destabilize it. The observed
affinities of residue 69 and 73 mutants for aspartate are at least as
good as that of wild-type Tar for glutamate (Kd
5 mM (4)), suggesting that either Arg-69 or Arg-73 (or both)
is not optimally used when glutamate is bound.
Perhaps the most surprising result is that the three arginine residues contribute so much to aspartate binding but so little to signaling. Each of the mutant receptors generates a chemotactic signal, once aspartate is bound. Apparently, none of the interactions contributed by these residues (magenta lines in Fig. 1B) is essential to that process. Therefore, the roles of the remaining interactions in binding and signaling must be inspected closely. Some have been explored elsewhere (Fig. 1B, red dashed lines). Mutations of Ser-68 generally have little effect on the affinity of Tar for aspartate, although they can change the cooperativity greatly (39); maltose responses were apparently not tested. Mutations of Thr-154 have only modest effects (10-100 fold) on aspartate binding with no impact on signaling (40); weaker binding was observed for a proline mutant, most likely resulting from changes in local main chain conformation. There is little disturbance of the maltose response. Other workers showed a role for Tyr-149 in signaling to both aspartate and maltose (33), concluding that some common signaling mechanism might be impaired. The effects on aspartate affinity specifically were not measured, but a Tyr-149 mutant did show some aspartate-induced swarming, suggesting that binding/signaling occurred at high enough concentrations. More detailed studies of the aspartate affinity and signaling competence of Tyr-149 and Thr-154 mutants of Tar would be helpful. It is also clear that in these and a number of other cases mentioned here, structural data for the mutant receptors would be a real asset in the interpretation.
The interactions of the binding sites that have not been explicitly
tested are shown as green dashed lines in Fig.
1B. Two things seem to be significant about them. First,
they are all main chain and water-mediated interactions (that cannot be
investigated by mutagenesis). Second, they represent links between the
backbones of helices 1 and
4 of the same subunit that are made
via the aspartate ligand. The presumably critical connection involving the main chain oxygen atom of Arg-64 and water would be expected to be
altered in the R64P mutation, but unfortunately this mutant receptor
could not be expressed and tested. Any of the many available Ser-68,
Arg-69, or Arg-73 mutations would be expected to weaken links between
1 and
1', and yet such mutants are able to signal effectively
once ligand is bound. The fact that the side chain of Arg-64, which
also mediates
1/
4 connections, is so important for aspartate
binding would also seem consistent with the idea that
4 motions are
involved in signaling, as is the fact that mutation of Tyr-149 affects
both aspartate and maltose responses. Thus, the mutational results
appear to be more consistent with a model of signaling in which the
relative movement of
4 within a subunit is most critical. Important
links in the chain of interactions whereby aspartate links
1 and
4 include ordered waters in the crystal structures. That such waters
can have as large a contribution as protein atoms is supported by a
number of structural studies (e.g. Refs. 41 and 42). An
alternate interpretation is that none of the interactions is by itself
essential, but rather that aspartate fills some appropriate space and
so positions the important helices correctly. This argument would seem
to be weakened by the fact that the "appropriate space" has a very
different size and shape in the various mutant receptors, and yet
signaling occurs with a similar final efficiency for each.
Also pertinent are recent studies showing that under physiological conditions, receptors form a noncovalent complex with the intracellular signaling proteins CheA and CheW (43, 44); together they form large aggregates that are necessary for maximal activity of CheA (45). It has been demonstrated further (46, 47) that the affinity of Tar, as well as of the related serine receptor, is modulated by its interactions with CheA and CheW (and in that complex by the state of receptor methylation). The feedback effects of receptor methylation on ligand binding appear to be small (e.g. Refs. 4 and 48) when CheA and CheW concentrations are low (discussed in Ref. 46). Both receptor overproduction and the high-salt washes used in the membrane preparation here would effectively reduce the amounts CheA and CheW. The methyltransferase preparation, although not highly purified, is also overproduced and thus will not add large amounts of CheA and CheW. Although the binding studies reported here can only claim to address intrinsic properties of the receptor itself, these must underlie the behavior of the receptor in the larger complexes with CheA and CheW.
Signaling of E. coli Tar in response to maltose is, in
agreement with earlier studies (21, 33, 49), affected by changes at
Arg-73 but not generally by ones at Arg-64 and Arg-69. Interactions with maltose-bound MBP seem to be dependent on having an arginine at
position 73 of Tar, as no other amino acid, even lysine, could replace
it efficiently. As this residue is not important to the structural
integrity of Tar, direct involvement in interactions with MBP must be
suspected. A model for the MBP/Tar interaction has been proposed (50)
in which MBP interacts simultaneously and asymmetrically with the two
subunits of a Tar dimer. Inspection of this model suggests to us the
possibility of an interaction between residue Asp-41 or Glu-45 of MBP
and Arg-73 of Tar; mutations of these MBP residues have both been
reported to affect chemotaxis (50, 51). Although the loss of a single
charge-charge interaction (even a bidentate one such as arginine can
form with acidic side chains) would not ordinarily destroy function,
the binding of MBP and Tar is already weak (52), and it may be very
sensitive to such disruptions. The location of these residues of MBP
just above 4 of Tar in the proposed complex may also be significant. A movement of
4 downward toward the membrane, as suggested above for
the aspartate signal, could bring this portion of MBP into a more
optimal position for interactions with Arg-73 of Tar and so support its
role in signaling. The second aspartate-binding site would appear to be
free to accept ligand, in a relatively independent signaling event (8).
Our results suggest that the second site of Tar is indeed active under
appropriate conditions.
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ACKNOWLEDGEMENTS |
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We thank Quang A. Vu and Vahid Feiz for invaluable experimental assistance, Sandy Parkinson for strains, Jeff Stock for plasmids, Andrew Kolodziej and Steve Chervitz for helpful discussions, Sung-Hou Kim for coordinates, and Mike Manson and Paul Gardina for the model of the Tar MBP interaction and as well as other valuable help.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Natural Science Research Council and the Howard Hughes Medical Institutes (to S. M.).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.
§ Current address: Roche Research Center, Hoffmann-La Roche, Inc., Nutley, NJ 07110.
** Current address: Dept. of Pediatric Nephrology, Southwestern Medical Center, Dallas, TX 75390.
To whom correspondence should be addressed. Tel.: 46 18 471 49 90; Fax: 46 18 53 69 71; E-mail: mowbray@xray.bmc.uu.se.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M009593200
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ABBREVIATIONS |
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The abbreviations used are: Tar, aspartate receptor from E. coli; MBP, maltose/maltodextrin-binding protein; AdoMet, S-adenosylmethionine; PMSF, phenylmethylsulfonyl fluoride.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Djordjevic, S., and Stock, A. M. (1998) J. Struct. Biol. 124, 189-200[CrossRef][Medline] [Order article via Infotrieve] |
2. | Mowbray, S. L., and Sandgren, M. O. J. (1998) J. Struct. Biol. 124, 257-275[CrossRef][Medline] [Order article via Infotrieve] |
3. | Russo, A. F., and Koshland, D. E., Jr. (1983) Science 220, 1016-1020[Medline] [Order article via Infotrieve] |
4. | Clarke, S., and Koshland, D. E., Jr. (1979) J. Biol. Chem. 254, 9695-9702[Abstract] |
5. | Mizuno, T., Mutoh, N., Panasenko, S. M., and Imae, Y. (1986) J. Bacteriol. 165, 890-895[Medline] [Order article via Infotrieve] |
6. | Wolff, C. (1983) Genetic and Biochemical Studies of Maltose Chemotaxis in Escherichia coli.M.Sc. thesis , University of Konstanz, Germany |
7. | Mowbray, S. L., and Koshland, D. E., Jr. (1987) Cell 50, 171-180[Medline] [Order article via Infotrieve] |
8. | Gardina, P. J., Bormans, A. F., and Manson, M. D. (1998) Mol. Microbiol. 29, 1147-1154[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Milligan, D. L.,
and Koshland, D. E., Jr.
(1988)
J. Biol. Chem.
263,
6268-6275 |
10. | Milburn, M., Privé, G. G., Milligan, D. L., Scott, W. G., Yeh, J., Jancarik, J., Koshland, D. E., Jr., and Kim, S.-H. (1991) Science 254, 1342-1347[Medline] [Order article via Infotrieve] |
11. | Lynch, B. A., and Koshland, D. E., Jr. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10402-10406[Abstract] |
12. | Kim, K. K., Yokota, H., and Kim, S. H. (1999) Nature 400, 787-792[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Milligan, D. L.,
and Koshland, D. E., Jr.
(1993)
J. Biol. Chem.
268,
19991-19997 |
14. | Biemann, H.-P., and Koshland, D. E., Jr. (1994) Biochemistry 33, 629-634[Medline] [Order article via Infotrieve] |
15. | Springer, M. S., Goy, M. F., and Adler, J. (1979) Nature 280, 279-284[Medline] [Order article via Infotrieve] |
16. |
Terwilliger, T. C.,
Bogonez, E.,
Wang, E. A.,
and Koshland, D. E., Jr.
(1983)
J. Biol. Chem.
258,
9608-9611 |
17. |
Yeh, J. I.,
Biemann, H.-P.,
Pandit, J.,
Koshland, D. E., Jr.,
and Kim, S.-H.
(1993)
J. Biol. Chem.
268,
9787-9792 |
18. | Scott, W. G., and Stoddard, B. L. (1994) Structure 2, 877-887[Medline] [Order article via Infotrieve] |
19. |
Chervitz, S. A.,
and Falke, J. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2545-2550 |
20. |
Ottemann, K. M.,
Xiao, W.,
Shin, Y. K.,
and Koshland, D. E., Jr.
(1999)
Science
285,
1751-1754 |
21. | Wolff, C., and Parkinson, J. S. (1988) J. Bacteriol. 170, 4509-4515[Medline] [Order article via Infotrieve] |
22. | Parkinson, J. S., and Houts, S. E. (1982) J. Bacteriol. 151, 106-113[Medline] [Order article via Infotrieve] |
23. |
Simms, S. A.,
Stock, A. M.,
and Stock, J. B.
(1987)
J. Biol. Chem.
262,
8537-8543 |
24. | Bogonez, E., and Koshland, D. E., Jr. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4891-4895[Abstract] |
25. |
Vogel, H. J.,
and Bonner, D. M.
(1956)
J. Biol. Chem.
218,
97-106 |
26. |
Mowbray, S. L.,
and Koshland, D. E., Jr.
(1990)
J. Biol. Chem.
265,
15638-15643 |
27. | Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. T., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977) J. Mol. Biol. 112, 535-542[Medline] [Order article via Infotrieve] |
28. | Chi, Y. I., Yokota, H., and Kim, S. H. (1997) FEBS Lett. 414, 327-332[CrossRef][Medline] [Order article via Infotrieve] |
29. | Jones, T. A., and Kjeldgaard, M. O. (1997) Methods Enzymol. 277, 173-208[CrossRef] |
30. | Kleywegt, G. J., and Jones, T. A. (1997) Methods Enzymol. 277, 525-545 |
31. | Kleywegt, G. J. (1996) Acta Crystallogr. Sect. D Biol. Crystallogr. 52, 842-857[CrossRef] |
32. | Parkinson, J. S. (1978) J. Bacteriol. 135, 45-53[Medline] [Order article via Infotrieve] |
33. | Gardina, P., Conway, C., Kossman, M., and Manson, M. (1992) J. Bacteriol. 174, 1528-1536[Abstract] |
34. | Kermode, J. C. (1989) Biochem. Pharmacol. 38, 2053-2060[CrossRef][Medline] [Order article via Infotrieve] |
35. | Hill, A. V. (1910) J. Physiol. (Lond.) 90, iv-vii |
36. |
Falke, J. J.,
Dernburg, A. F.,
Sternberg, D. A.,
Zalkin, N.,
Milligan, D. L.,
and Koshland, D. E., Jr.
(1988)
J. Biol. Chem.
263,
14850-14858 |
37. | Scott, W. G., Milligan, D. L., Milburn, M. V., Privé, G. G., Yeh, J., Koshland, D. E., Jr., and Kim, S.-H. (1993) J. Mol. Biol. 232, 555-573[CrossRef][Medline] [Order article via Infotrieve] |
38. | Ottemann, K. M., Thorgeirsson, T. E., Kolodziej, A. F., Shin, Y. K., and Koshland, D. E., Jr. (1998) Biochemistry 37, 7062-7069[CrossRef][Medline] [Order article via Infotrieve] |
39. | Kolodziej, A. F., Tan, T., and Koshland, D. E., Jr. (1996) Biochemistry 35, 14782-14792[CrossRef][Medline] [Order article via Infotrieve] |
40. | Lee, L., and Imae, Y. (1990) J. Bacteriol. 172, 377-382[Medline] [Order article via Infotrieve] |
41. | Quiocho, F. A., Wilson, D. K., and Vyas, N. K. (1989) Nature 340, 404-407[CrossRef][Medline] [Order article via Infotrieve] |
42. | Shakked, Z., Guzikevich-Guerstein, G., Frolow, F., Rabinovich, D., Joachimiak, A., and Sigler, P. B. (1994) Nature 368, 469-473[CrossRef][Medline] [Order article via Infotrieve] |
43. | Gegner, J. A., Graham, D. R., Roth, A. F., and Dahlquist, F. W. (1992) Cell 70, 975-982[Medline] [Order article via Infotrieve] |
44. | Schuster, S. C., Swanson, R. V., Alex, L. A., Bourret, R. B., and Simon, M. I. (1993) Nature 365, 343-347[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Liu, Y.,
Levit, M.,
Lurz, R.,
Surette, M. G.,
and Stock, J. B.
(1997)
EMBO J.
16,
7231-7240 |
46. | Li, G., and Weis, R. M. (2000) Cell 100, 357-365[Medline] [Order article via Infotrieve] |
47. | Bornhorst, J. A., and Falke, J. J. (2000) Biochemistry 39, 9486-9493[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Dunten, P.,
and Koshland, D. E., Jr.
(1991)
J. Biol. Chem.
266,
1491-1496 |
49. | Kossman, M., Wolff, C., and Manson, M. D. (1988) J. Bacteriol. 170, 4516-4521[Medline] [Order article via Infotrieve] |
50. |
Zhang, Y.,
Gardina, P. J.,
Kuebler, A. S.,
Kang, H. S.,
Christopher, J. A.,
and Manson, M. D.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
939-944 |
51. |
Zhang, Y.,
Conway, C.,
Rosato, M.,
Suh, Y.,
and Manson, M.
(1992)
J. Biol. Chem.
267,
22813-22820 |
52. |
Manson, M. D.,
Boos, W.,
Bassford, P. J., Jr.,
and Rasmussen, B. A.
(1985)
J. Biol. Chem.
260,
9727-9733 |
53. | Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |
54. | Yeh, J. I., Biemann, H. P., Privé, G. G., Pandit, J., Koshland, D. E., Jr., and Kim, S. H. (1996) J. Mol. Biol. 262, 186-201[CrossRef][Medline] [Order article via Infotrieve] |
55. | Bowie, J. U., Pakula, A. A., and Simon, M. I. (1995) Acta Crystallogr. Sect. D Biol. Crystallogr. 51, 145-154[CrossRef] |