(Received for publication, June 15, 1995; and in revised form, July 21, 1995)
From the
The aspartate receptor of the bacterial chemotaxis pathway
regulates the autophosphorylation rate of a cytoplasmic histidine
kinase in response to ligand binding. The transmembrane signal, which
is transmitted from the periplasmic aspartate-binding domain to the
cytoplasmic regulatory domain, is carried by an intramolecular
conformational change within the homodimeric receptor structure. The
present work uses engineered cysteines and disulfide bonds to probe the
nature of this conformational change, focusing in particular on the
role of the second transmembrane -helix. Altogether 26
modifications, consisting of 13 cysteine pairs and the corresponding
disulfide bonds, have been introduced into the contacts between the
second transmembrane helix and adjacent helices. The effects of these
modifications on the transmembrane signal have been quantified by in vitro assays which measure (i) ligand binding, (ii)
receptor-mediated regulation of kinase activity, and (iii) receptor
methylation. All three parameters are observed to be highly sensitive
to perturbations of the second transmembrane helix. In particular, 13
of the 26 modifications (6 cysteine pairs and 7 disulfides)
significantly increase or decrease aspartate affinity, while 15 of the
26 modifications (5 cysteine pairs and 10 disulfides) destroy
transmembrane kinase regulation. Importantly, 3 of the perturbing
disulfides are found to lock the receptor in the ``on'' or
``off'' signaling state by covalently constraining the second
transmembrane helix, demonstrating that it is possible to use
engineered disulfides to lock the signaling function of a receptor
protein. A separate aspect of the study probes the thermal motions of
the second transmembrane helix: 4 disulfides designed to trap large
amplitude twisting motions are observed to disrupt function but form
readily, suggesting that the helix is mobile. Together the results
support a model in which the second transmembrane helix is a mobile
signaling element responsible for communicating the transmembrane
signal.
The ability to sense and respond adaptively to changes in the
environment is fundamental to all organisms. Bacteria such as Escherichia coli and Salmonella typhimurium acquire
chemical information about their environment through a family of
homologous transmembrane chemotaxis receptors (reviewed by Stock and
Surette(1995), Swanson et al.(1994), Parkinson(1993),
Hazelbauer(1992), and Bourret et al.(1991)). The aspartate
receptor, a representative of this family, monitors the concentration
of aspartate in the periplasmic compartment and transmits this
information to the cytoplasm, where it ultimately guides the
chemotactic behavior of the cell. The aspartate receptor is a
homodimer, in which each 60-kDa subunit possesses two membrane spanning
-helices linking the 18-kDa periplasmic domain to the 36-kDa
cytoplasmic domain. The cytoplasmic domain associates with a soluble
histidine kinase (CheA) and a coupling protein (CheW), together forming
a kinetically stable ternary complex (Schuster et al., 1993;
Gegner et al., 1992). This complex carries out an
autophosphorylation reaction that is inhibited by aspartate binding to
the receptor (Ninfa et al., 1991; Borkovich et al.,
1989). Following autophosphorylation, the phosphate moiety is
transferred from the ternary complex to a soluble signaling protein
(CheY) which diffuses to the flagellar motor, binds, and regulates the
transition between two propulsion states.
The present study addresses the mechanism utilized by the aspartate receptor to send a transmembrane signal from the periplasmic ligand-binding site to the cytoplasmic ternary complex. The same signaling mechanism is almost certainly employed by the other chemotaxis receptors, which regulate the same CheA histidine kinase and share extensive sequence identities in their cytoplasmic domains. Moreover, this same mechanism may also be utilized by an even broader class of receptors that includes the chemotaxis receptors as a subset: all members of this class exist as elements of histidine kinase pathways and exhibit nearly identical transmembrane topologies. It would appear that these receptors represent an ancient, highly adaptable signaling motif distributed throughout the prokaryotic world (reviewed by Parkinson(1993) and Spudich(1993)) and recently discovered in eukaryotes as well (Swanson et al., 1994; Alex and Simon, 1994; Ota and Varshavsky, 1993; Chang et al., 1993).
Although a molecular picture of the
transmembrane signal remains to be elucidated for the aspartate
receptor and its relatives, previous studies have revealed important
features. The aspartate receptor signals via an intramolecular
conformational change within the dimer, since (i) numerous covalently
linked dimers containing an engineered inter-subunit disulfide have
been shown to retain transmembrane signaling (Chervitz et al.,
1995; Scott and Stoddard, 1994; Stoddard et al., 1992; Lynch
and Koshland, 1991; Falke and Koshland, 1987), and (ii) the oligomeric
state of the receptor does not change upon addition of ligand (Yeh et al., 1993; Milligan and Koshland, 1988). ()Since
the signal is intramolecular, it must be carried by the transmembrane
helices. The first transmembrane helix located at the subunit interface
has been shown to play a structural role and does not appear to carry
the signal (Chervitz et al., 1995); a similar picture has
emerged for the closely related ribose and galactose chemoreceptor (Lee et al., 1994, 1995). Here the goal is to test the hypothesis
that the second transmembrane helix is the critical signaling element.
Further mechanistic studies of the aspartate receptor are
facilitated by the extensive structural information available for its
periplasmic and transmembrane domains, which together generate the
transmembrane signal. X-ray crystallography has revealed the structure
of the isolated periplasmic domain in both its apo and ligand-occupied
conformations (Scott et al., 1993; Yeh et al., 1993;
Milburn et al., 1991), in which each subunit of the
homodimeric domain is observed to be an elongated four-helix bundle. A
structural model has also been developed for the transmembrane region
using disulfide mapping (Scott and Stoddard, 1994; Stoddard et
al., 1992; Pakula and Simon, 1992; Lynch and Koshland, 1991; Falke et al., 1988), which has revealed the -helical secondary
structure and packing arrangement of the two transmembrane helices
provided by each subunit. The first transmembrane helix begins near the
receptor N terminus and is believed to be continuous with the first
helix (
1) of the periplasmic domain. Similarly, the last helix
(
4) of the periplasmic domain is thought continue across the
bilayer, yielding the second transmembrane helix. The two transmembrane
helices of each subunit are therefore referred to as
1/TM1 and
4/TM2, respectively.
The periplasmic and membrane-spanning
regions of the dimer interface are stabilized by extensive contacts
between the N-terminal first transmembrane helices of the two subunits
(1/TM1 and
1`/TM1`) (Scott et al., 1993; Pakula and
Simon, 1992; Falke et al., 1988). Disulfide bonds covalently
linking these helices over a large fraction of their contact surfaces
retain transmembrane signaling, suggesting that structural changes
involving the subunit interface and the first transmembrane helix are
not required for signal transmission (Chervitz et al., 1995)
(also Lee et al.(1995) for the ribose and galactose
chemoreceptor).
To probe the role of the second transmembrane helix in signaling, the present study introduces cysteine pairs and disulfides at 17 locations in the periplasmic and transmembrane domains of the receptor, which possesses no intrinsic cysteines. These engineered cysteines and disulfides are designed to (i) perturb or covalently lock the interface between the second transmembrane helix and its adjacent helices, or (ii) force translational or twisting movements of the second transmembrane helix. If this helix serves as a critical signaling element, such perturbations should alter the signal, or even lock the receptor in the ``on'' or ``off'' signaling state. To quantitate the effects of engineered cysteines and disulfides on signaling, the receptor-regulated phosphorylation pathway is reconstituted in vitro, providing a direct measure of receptor-kinase coupling. The effects of receptor engineering on ligand binding and in vitro receptor methylation are examined as well. The results indicate that the transmembrane signal is highly sensitive to perturbation of the second transmembrane helix. Importantly, three engineered disulfides are found to lock the receptor in the on or off state, in each case by covalently cross-linking the second transmembrane helix to the adjacent first helix of the same subunit. The implications of these results for the mechanism of transmembrane signaling are discussed.
where [B] was the total binding site concentration. Control binding experiments using membranes lacking receptors yielded no detectable aspartate binding (data not shown).
Figure 1:
Schematic structure of the aspartate
receptor, including the locations of the engineered cysteines. A, top view of the periplasmic -helices, looking down
their long axes toward the cytoplasm (Milburn et al., 1991).
The periplasmic domain consists of two subunits distinguished by shading and primes, each forming a four-helix bundle.
The
1 and
1` helices at the subunit interface form a
coiled-coil pair represented by 7-fold helical wheels (Scott et
al., 1993), while the remaining standard helices are shown as
18-fold helical wheels. Positions of the engineered cysteines on
helices
1 and
4 are indicated; those designed to trap
twisting motions of the
4/TM2 helix are underlined. B,
model for the packing of the transmembrane helices (Scott and Stoddard,
1994; Pakula and Simon, 1992; Lynch and Koshland, 1991; Falke et
al., 1988), illustrating the positions of engineered cysteines on
TM1 and TM2. C, schematic representation of the periplasmic
and transmembrane helices of each subunit, illustrating the positions
of the engineered cysteine pairs placed at the interfaces of helix
4/TM2 with helices
1/TM1 and
3. For simplicity, the
cysteine pairs designed to trap
4/TM2 twisting motions are
omitted. Given the 2-fold symmetry of the homodimer, both subunits
possess the same cysteine pair or disulfide bond. Symbols indicate the degree of motion required for disulfide formation
between a given cysteine pair: no motion required (filled
square);
1.5-Å translation required (open
square); undetermined (open
circle).
To simplify the presentation, the following sections focus
initially on the 13 interfacial disulfide pairs and disulfide bonds
targeted to 4/TM2 and its adjacent helices. Subsequently, the four
disulfides designed to trap
4/TM2 twisting motions are examined
separately.
Turning to the transmembrane region of the receptor, five engineered
cysteine pairs were placed at positions previously demonstrated to
yield efficient intra-subunit disulfide formation (Pakula and Simon,
1992). These cysteine pairs are predicted to lie within the bilayer, at
or near the interface between the two transmembrane helices 4/TM2
and
1/TM1 (S25C,L197C; L21C,L201C; L11C,G211C; M10C,G211C;
V7C,G211C; Fig. 1, B and C).
The resulting
receptors displayed varying degrees of spontaneous intra-subunit
disulfide cross-linking when grown under normal conditions in E.
coli, as summarized in Table 2. Of the eight interfacial
cysteine pairs located in the periplasm, four were nearly fully
disulfide-linked in vivo (90-100%: A119C,Y168C;
G39C,T179C; G39C,S183C; N36C,S183C). Three periplasmic cysteine pairs
were partially cross-linked in vivo (50-60%: S43C,Y176C;
S43C,T179C; G39C,Y176C). By contrast, all five disulfide pairs located
in the membrane spanning helices were predominantly reduced in vivo (<5% disulfide: S25C,L197C; L21C,L201C; L11C,G211C; M10C,G211C;
V7C,G211C), as was the remaining periplasmic pair (Y130C,L161C). These
results suggest that, in general, the engineered cysteine pairs within
the periplasmic region of the 4/TM2 helix are exposed to the
oxidizing environment of the periplasm (Zapun et al., 1995),
unlike the protected cysteine pairs at the subunit interface (Chervitz et al., 1995) or those within the bilayer.
Each of the 13
engineered receptors possessing an interfacial cysteine pair was tested
for the ability to mediate chemotaxis toward aspartate in vivo by means of a chemotaxis swarm plate assay (Weis and Koshland,
1988; Adler, 1966), as quantitated in Table 2. These assays were
carried out under normal growth conditions, and no attempt was made to
alter the extent of disulfide formation during the assay. Engineered
receptors exhibited in vivo activities ranging from no
aspartate swarming up to 180% of the native swarm rate. Of the four
receptors exhibiting extensive in vivo disulfide formation,
two retained significant aspartate chemotaxis (>20% native:
A119C,Y168C; N36C,S183C), while one was severely defective in aspartate
chemotaxis (10% native, G39C,T179C). The remaining nine engineered
receptors, which exhibited low levels of in vivo disulfide
formation, retained significant aspartate chemotaxis (
20% of
native).
Receptors were prepared for in vitro studies by isolating E. coli membranes
(Foster et al., 1985) containing a given engineered receptor,
then reducing or oxidizing the membrane-bound receptor to generate
either the free cysteine pair or disulfide bond, respectively.
Reduction was carried out using dithiothreitol, while two different
oxidation systems were used to drive disulfide formation: (i) oxidation
by ambient oxygen catalyzed by
Cu(II)-(1,10-phenanthroline), or (ii) oxidation by iodine
(Chervitz et al., 1995; Pakula and Simon, 1991; Falke and
Koshland, 1987; Kobashi, 1968). The system providing the most complete
disulfide formation for a given disulfide pair was determined
empirically and used in subsequent studies. Disulfide formation
reactions proceeded to 50-100% completion, with most reactions
generating over 90% desired product. For reactions yielding greater
than 70% disulfide formation, the majority of the final receptor
population contained two intra-chain disulfides, one in each subunit of
the homodimer.
Aspartate binding was found to be perturbed by certain
cysteine substitutions targeted to 4/TM2 and its adjacent helices,
as summarized in Table 3. Of the 13 interfacial cysteine pairs
examined, only two, Y130C,L161C and L11C,G211C, significantly weakened
aspartate affinity (approximately 4-fold and 10-fold, respectively). In
contrast, four cysteine pairs yielded aspartate affinities
3-5-fold greater than the native receptor: S43C,Y176C;
S43C,T179C; G39C,S183C; and S25C,L197C. (One engineered receptor,
N36C,S183C, formed its disulfide in vivo and could not be
completely reduced unless it was first denatured; thus the aspartate
affinity of this receptor in its reduced state could not be accurately
determined.)
Aspartate binding was also measurably altered by
specific engineered disulfides constraining the 4/TM2 helix. One
such disulfide which cross-links
4/TM2 to the adjacent
1/TM1
helix, S25C-L197C, decreased the aspartate affinity 9-fold
relative to the reduced state, or 2.8-fold relative to the native
receptor. More commonly, disulfides cross-linking the
4/TM2-
1/TM1 interface tended to enhance aspartate binding; in
particular, seven interfacial disulfides provided a 2-6-fold
increase in the aspartate affinity relative to the native receptor
(G39C-Y176C; G39C-T179C; G39C-S183C;
S43C-T176C; S43C-T179C; M10C-G211C; V7C-G211C).
These results indicate that the aspartate-binding site can be easily
perturbed, but is not easily disrupted, by engineered cysteines and
disulfides in the periplasmic and transmembrane domains which constrain
the second transmembrane helix. The basis of this unusual coupling
between the putative signaling helix and the ligand-binding site is
analyzed further under ``Discussion.''
Figure 2: Effect of engineered cysteines and disulfides on the in vitro phosphorylation reaction. Shown is the time course of CheY phosphorylation by the reconstituted receptor-kinase complex, illustrating the activities of the wild-type receptor and three classes of engineered receptors. Isolated E. coli membranes containing the wild-type (WT) and indicated engineered cysteine receptors were reduced (red) to eliminate preexisting disulfide bonds, or oxidized (ox) to drive disulfide bond formation linking engineered cysteine pairs. Subsequently, purified CheA, CheW, and CheY were added to reconstitute the receptor-kinase complex, and the effects of 1 mM aspartate (Asp) on CheY phosphorylation were quantitated (see ``Experimental Procedures''). The illustrated reactions are representative of the wild-type receptor (A), or receptors in which an engineered disulfide retains measurable transmembrane kinase regulation (B), locks transmembrane kinase regulation in the off state (C), or locks transmembrane kinase regulation in the on state (D).
Periplasmic and transmembrane surfaces of the 4/TM2
helix critical for kinase regulation were identified by investigating
the effects of the interfacial cysteine substitutions themselves. The
observed phospho-signaling activities of the reduced receptors are
compared in Table 4. The
4/TM2
1/TM1 interface (Fig. 1) was found to be highly sensitive to cysteine
substitution. Five of the 11 cysteine pairs at this interface
essentially destroyed kinase activation or aspartate regulation
(
10% of native: S43C,T179C; S25C,L197C; L21C,L201C; M10C,G211C; and
V7C,G211C). Two cysteine pairs caused moderate inhibition of both
kinase activation and aspartate regulation (20-40% of native:
S43C,Y176C and G39C,Y176C). Only three cysteine pairs retained
relatively normal kinase activity (>40% of native: G39C,T179C;
G39C,S183C; and L11C,G211C). Thus, altogether 7 of the 11 cysteine
pairs along the
4/TM2-
1/TM1 interface yielded moderate to
complete inhibition of transmembrane regulation of the kinase,
suggesting that the interface is tightly coupled to the transmembrane
signal. (The activity of the N36C,S183C receptor could not be
accurately assessed due to incomplete reduction.)
Cysteine
substitution along the periplasmic interface between the 4/TM2 and
3 helices was somewhat less inhibitory (Fig. 1, Table 4). The Y130C,L161C pair caused moderate inhibition of both
kinase activation and aspartate regulation (20% of native). In
contrast, the receptor containing the A119C,Y168C pair was essentially
normal in both measures of kinase signaling (
70% of native). Based
on this limited data, the
3-
4/TM2 interface may be less
tightly coupled to the transmembrane signal than the
1/TM1-
4/TM2 interface.
The two engineered disulfides covalently
linking the 4/TM2
3 interface both produced significant
changes in signaling (Fig. 1, Table 4). The
Y130C-L161C disulfide restored normal phospho-signaling to this
receptor, which was substantially inhibited in the reduced state. In
contrast, the A119C-Y168C disulfide substantially reduced
regulation of the kinase by aspartate, although significant ligand
regulation still remained (30% of native).
Of particular interest
were three perturbing disulfides, all cross-linking 4/TM2 helix to
the adjacent
1/TM1 helix, which appeared to lock the receptor
signaling state. The S25C-L197C disulfide both restored and
locked the signal in the fully on state. As expected for a lock on
disulfide, S25C-L197C substantially decreased the aspartate
affinity (10-fold relative to the reduced receptor, or 3-fold relative
to native; Table 3), and maintained full kinase activation even
in the presence of saturating aspartate (Table 4, Fig. 2).
Such behavior is expected for a disulfide which traps the
apo-conformation of the receptor, since it is this conformation which
activates the kinase. The trapped conformation appears to be somewhat
perturbed, however, since it exhibits a higher than normal in vitro methylation rate (see below). The G39C-T179C disulfide, by
contrast, prevented kinase activation and increased the aspartate
affinity 4-fold relative to native, as expected for a receptor locked
in the off or ligand-occupied conformation. A number of other
disulfides exhibited less dramatic lock off behavior: S43C-Y176C
is the next best example.
In principle, the effects of lock on and off disulfides can be reversed by reduction. Such reversibility was confirmed for the periplasmic G39C-T179C and S43C-Y176C lock off disulfides, for which kinase activation was restored by reduction with dithiothreitol (data not shown). For the S25C-L197C lock on disulfide; however, reduction could not be accomplished except under denaturing conditions. This resistance to reduction likely stems from the buried location of this disulfide within the bilayer, which could decrease accessibility to the polar dithiothreitol molecule, or increase the energy barrier for the ionic disulfide exchange reaction.
Altogether, the engineered disulfide
results provide additional evidence for the critical coupling between
the 4/TM2 helix and the transmembrane signal, which is
particularly sensitive to disulfide linkages along the
4/TM2-
1/TM1 interface. Such sensitivity appears to be
specific to interfaces involving
4/TM2, since disulfides linking
the
1/TM1-
1`/TM1` helix contacts at the subunit interface
are, by comparison, relatively nonperturbing (Chervitz et al.,
1995).
Figure 3: Effects of engineered cysteines and disulfides on the in vitro phosphorylation and methylation reactions: summary. The left panels plot the maximum initial phosphorylation and methylation rates produced by each engineered receptor in its reduced and oxidized states, relative to the activity of the wild-type receptor. The maximum rate of CheY phosphorylation or receptor methylation was obtained in the absence or presence of ligand, respectively. The right panels summarize the ability of aspartate (1.0 mM) to down-regulate CheY phosphorylation, or stimulate receptor methylation, where each aspartate effect is relative to reactions possessing the wild-type receptor. (See Methods and Table 4and Table 5for further details.)
In most cases, the
effects of interfacial cysteine pairs and disulfides on receptor
methylation mirrored the effects seen on in vitro kinase
regulation. The majority of cysteine pairs and disulfides engineered
into the 4/TM2-
1/TM1 interface led to substantial inhibition
of methylation parameters (Table 5). One of the eleven cysteine
pairs, as well as six of the interfacial disulfides, essentially
destroyed aspartate control of methylation (
10% of native:
M10C,G211C; S43C-Y176C; S43C-T179C; G39C-Y176C;
G39C-S183C; L11C-G211C; V7C-G211C). Moreover, eight
additional cysteine pairs and five disulfides yielded moderate
inhibition of aspartate regulation (20-40% of native: S43C,Y176C;
S43C,T179C; G39C,Y176C; G39C,S183C; N36C,S183C; S25C,L197C; L21C,L201C;
L11C,G211C; G39C-Y176C; N36C-S183C; S25C-L197C;
L21C-L201C; M10C-G211C). Overall, 9 cysteine pairs and all
11 disulfides caused moderate to complete inhibition of
aspartate's ability to stimulate methylation, despite the
addition of sufficient aspartate to saturate the ligand-binding site.
Interestingly, most of the perturbations that damaged the aspartate
response retained maximum methylation rates between 0.6- and 4.1-fold
that of the native receptor (16 of 20 examples), suggesting that these
perturbations interfered with communication between the ligand-binding
and methylation sites, rather than distorting the receptor into a
methylation-inaccessible structure.
Notably, five of the disulfides
at the 4/TM2-
1/TM1 interface caused receptor overmethylation
both in the absence and presence of aspartate: G39C-T179C,
S25C-L197C, L21C-L201C, L11C-G211C, and
M10C-G211C (Table 5). As discussed above, the
G39C-T179C disulfide was especially interesting because it
appears to lock the receptor in its off state, which is normally
stabilized by ligand binding. In the presence of aspartate the maximum
methylation rate of this receptor was 1.3-fold faster than native,
while in the absence of aspartate its rate was 4.1-fold that of the
apo-native receptor, supporting the conclusion that this disulfide
locks a conformation resembling the ligand-induced off state. Another
lock off disulfide, S43C-Y176C, failed to yield overmethylation
but its maximum methylation rate, which was 0.6-fold that of native,
did not respond to aspartate: such methylation parameters are
consistent with a locked conformation close to a native signaling
state. In contrast, the 1.5-10-fold overmethylation caused by the
S25C-L197C, L21C-L201C, L11C-G211C, and
M10C-G211C disulfides appeared to stem from perturbations of the
receptor structure, since none of these disulfides caused the increase
in ligand affinity characteristic of the locked off receptor (Table 3).
Engineered cysteine pairs and disulfides at the
4/TM2-
3 interface exhibited considerably smaller effects on
receptor methylation. Only the A119,Y168C cysteine pair generated a
significant perturbation, increasing the maximum methylation rate
2.4-fold (Table 5). Formation of a disulfide bond between this
cysteine pair largely restored the normal methylation parameters.
Although usually in agreement, the methylation and phosphorylation
assays yielded conflicting results in several cases ( Table 4and Table 5and Fig. 3). For example, three engineered cysteine
pairs or disulfides (S43C,T179C; V7C,G211C; and L21C-L201C) which
severely inhibited kinase activation or regulation (10% of native)
were observed to retain significant signaling in the methylation assay
(40-150% native methylation rate, and 30-80% native
aspartate regulation). These results confirm the previous observation
(Chervitz et al., 1995) that the in vitro methylation
assay occasionally detects conformational changes which fail to trigger
transmembrane kinase regulation.
All four of the
engineered cysteine pairs were efficiently converted to disulfide
bonds, indicating that the 4/TM2 helix possesses significant
thermal mobility. Analysis of the four receptors in vivo revealed that all were nearly fully disulfide-linked in the native
environment (90-100%, Table 2), where disulfide formation
is facilitated by several periplasmic proteins during or after protein
synthesis and assembly (Darby and Creighton, 1995). Similarly, when
disulfide formation was driven by oxidation of receptor-containing
membranes in vitro, all four reactions approached completion (Table 4). The latter observation demonstrates that
large-amplitude motions of the
4/TM2 helix are features of the
fully folded and assembled receptor.
Despite the efficiency with
which they were formed, the rotational disulfides caused significant
perturbation of receptor activity. In the native cellular environment,
where all four cysteine pairs were primarily in their disulfide-linked
form, the corresponding engineered receptors were each defective in
mediating aspartate chemotaxis during the in vivo swarm assay
(0-20% native activity, Table 2). When the effects of these
disulfides were further analyzed in vitro, all four were
observed to (i) increase aspartate affinity (4-7-fold native, Table 3), (ii) essentially destroy both maximum kinase activation
and its regulation by aspartate (10% of native, Table 4), and
(iii) severely reduce either the maximum methylation rate or its
regulation by ligand, or both (
10% of native, Table 5). It
follows that the receptor conformations trapped by these disulfides
represented significant deviations from normal signaling states.
The present study has characterized the transmembrane
signaling behavior of seventeen separate intra-subunit cysteine pairs
and their corresponding disulfides engineered into the bacterial
chemotaxis aspartate receptor, a representative of a growing class of
receptors which regulate histidine kinase pathways. The effects of
these cysteines and disulfides on ligand binding and transmembrane
kinase regulation support the model presented in Fig. 4, in
which the second transmembrane helix, designated 4/TM2, plays a
critical role in transmembrane signaling. The model proposes that
4/TM2 is a mobile signaling element and that movements of this
helix carry the transmembrane signal. Earlier evidence for this picture
was provided by
F NMR studies of the isolated
ligand-binding domain, which revealed perturbations of the
4 helix
triggered by aspartate binding (Danielson et al., 1994).
Similar conclusions have been reached in independent studies examining
signaling perturbations caused by (i) specific mutations in the
4/TM2 helix of the aspartate receptor (Jeffery and Koshland,
1994), and (ii) engineered disulfides constraining the
4/TM2 helix
of the related ribose and galactose receptor (Lee et al.,
1995). Further evidence obtained in the full-length aspartate receptor
can now be summarized as follows.
Figure 4:
Locations of engineered disulfides which
retain, restore, or lock the transmembrane signal. Shown is a schematic
model of the periplasmic and transmembrane regions of the receptor
dimer (note that perspectives have been altered for clarity). The
indicated disulfide bonds retain or restore aspartate-triggered
transmembrane regulation of kinase activity (solid bar;
20% native) or lock the receptor in the on (open bar) or
off (stippled bar) signaling state. Formation of a disulfide
between the remaining cysteine pairs essentially destroys transmembrane
kinase regulation (fine bars;
10% native). Only disulfides
located at the interfaces of adjacent helical faces are shown; for
simplicity, disulfides are shown in just one of the two symmetric
subunits, and disulfides designed to trap
4/TM2 twisting motions
are omitted. The model proposes that the transmembrane signal
originating in the ligand-binding site is generated by an undefined
movement of the
4/TM2 helix relative to the subunit interface,
which communicates the ligand-binding event to the cytoplasmic domain
and its associated histidine kinase.
As expected for a crucial
signaling element, most engineered cysteines and disulfides involving
helix 4/TM2 are observed in the present study to perturb ligand
binding and/or transmembrane kinase regulation. The packing interface
between
4/TM2 and the adjacent first transmembrane helix,
1/TM1, was probed by eleven engineered cysteine pairs and the
corresponding disulfides, as illustrated in Fig. 1C and Fig. 4. The majority of the resulting modifications (5 cysteine
pairs and 7 disulfides) were observed to significantly alter
ligand-binding affinity. Moreover, most of the modifications (5
cysteine pairs and 10 disulfides) essentially destroyed either
transmembrane kinase activation or regulation of the kinase by
saturating concentrations of aspartate, or both of these measures of
the transmembrane signal. Thus, the
4/TM2-
1/TM1 packing
interface appears to be tightly coupled to receptor function. In
contrast, a previous study of the
1/TM1-
1`/TM1` packing
interface within the dimer contact region has indicated that this
interface is more weakly coupled to kinase regulation (Chervitz et
al., 1995). Finally, the
4/TM2-
3 interface examined in
the present study exhibited an intermediate susceptibility to
perturbation: half of the cysteine pairs and disulfides tested within
this interface yielded moderate effects on ligand binding or kinase
regulation (only 2 cysteine pairs and 2 disulfides were tested,
however). Altogether, the available evidence indicates that the
4/TM2 helix is tightly coupled to the transmembrane signal, while
the
1/TM1 helix plays a simple structural role in dimer
stabilization.
Perhaps the strongest evidence that the 4/TM2
helix is the critical signaling element is provided by disulfides
observed to lock the signaling state on or off. Three disulfides were
observed to lock the signaling state of the receptor, in each case by
covalently cross-linking the
4/TM2 signaling helix to the adjacent
1/TM1 structural helix of the same subunit. The S25C-L197C
disulfide appeared to stabilize a conformation similar to the
apo-conformation or on state, yielding decreased ligand affinity as
well as maximum kinase activation both in the absence and presence of
saturating ligand. In contrast, the G39C-T179C and
S43C-Y176C disulfides exhibited enhanced ligand affinity, as well
as ligand-insensitive kinase inhibition and in vitro methylation, as expected for receptors locked in the
ligand-occupied or off state. These disulfides appear to trap
conformations resembling true receptor signaling states. Moreover, the
locked state can be restored to normal ligand regulation by simple
reduction of the disulfide. One disulfide cross-linking
4/TM2 to
1/TM1, namely N36C-S183C, retained kinase activation and
aspartate regulation, suggesting that this disulfide lies at a special
location which is able to accommodate the aspartate-induced movement of
4/TM2 via the modest flexibility of the disulfide linkage.
(Protein disulfide bonds exhibit a range of
-carbon separations
spanning 1.2 Å (Careaga and Falke, 1992a, 1992b; Srinivasan et al., 1990; Balaji et al., 1989).)
Evidence that
the 4/TM2 helix is a mobile element within the receptor structure
is provided by disulfide trapping results. In the present work, four
disulfide bonds designed to trap
4/TM2 rotations about its long
axis were generated. These disulfides, which all disrupted kinase
regulation, were observed to form rapidly and with high efficiency,
despite the fact that helix
4/TM2 must twist about its long axis
approximately 90° to 180° relative to the
1/TM1 helix to
bring the engineered disulfides into sufficient proximity for disulfide
formation (Fig. 1A). Moreover, previous disulfide
trapping studies detected long-range, intramolecular collisions between
the
4/TM2 and
4`/TM2` helices within the same receptor dimer
(Pakula and Simon, 1992; Falke and Koshland, 1987). Such dramatic
movements indicate that the
4/TM2 helix is highly mobile in the
plane of the bilayer, or that this helix spontaneously unravels at a
rate sufficient to yield the observed long-range collisions. It is not
yet clear whether this mobility is essential for signaling, or simply
represents random fluctuations away from the important signaling
conformations.
Interestingly, the coupling between the
ligand-binding site and the 4/TM2 signaling helix appears to be
remarkably plastic. At first glance, the structure of the periplasmic
domain suggests that the ligand-binding site would be tightly coupled
to the
4/TM2 helix, since nearly 60% of the contacts between the
receptor and the bound aspartate involve residues at the N terminus of
4/TM2 (positions Y-149-T-154; the remainder of the binding
pocket consists of three arginine side chains located at the C-terminal
end of
1/TM1 and
1`/TM1`) (Milburn et al., 1991).
Yet in all cases where engineered cysteine pairs or disulfides were
observed to severely disrupt kinase regulation by perturbing the
4/TM2 signaling helix (a total of 20 examples in the present
study), the inhibition arose from blockage of the transmembrane signal
to the kinase, not from failure to bind aspartate. Surprisingly, most
of these perturbations actually enhanced aspartate binding. For
instance, the four disulfides designed to trap extreme twisting motions
of
4/TM2 all disrupted the transmembrane signal but yielded
4-7-fold increases in aspartate affinity. Such ``negative
coupling'' between ligand binding and signaling suggests that
aspartate binding must carry out thermodynamic work to move the
4/TM2 helix into a different signaling position. In such a
picture, perturbations which uncouple the
4/TM2 helix from the
ligand-binding site would increase the aspartate-binding affinity.
At least two molecular explanations can be proposed for the observed
plasticity of the coupling between aspartate binding and the signaling
helix. (i) The linkage between the aspartate-binding site and the
distal regions of 4/TM2 may contain a flexible hinge which allows
dissipation of certain helix perturbations without destroying the
affinity of the site. Such a hinge would need to be sufficiently rigid
in the motional coordinate triggered by ligand binding, but could be
flexible in other coordinates. Structural features which might yield
such flexibility include a local distortion of the
4/TM2 helix
generated by Pro-153 within the binding site, or three Gly residues
near the site which presumably weaken the helix (Gly-157, Gly-162, and
Gly-166). Alternatively, (ii) the aspartate affinity may be dominated
by electrostatic interactions with the three arginines on
1/TM1
and
1`/TM1` (Arg-64, Arg-69`, and Arg-73`). According to the
latter model, the contacts observed between
4/TM2 and the bound
aspartate are of secondary importance for aspartate affinity but would
be required for generating the conformational work resulting in the
transmembrane signal. There is genetic and biochemical evidence
demonstrating that the aforementioned arginines on
1/TM1 and
1`/TM1` are critical for aspartate affinity (Wolff and Parkinson,
1988; Mowbray and Koshland 1990) and that the N terminus of
4/TM2
plays a key role in aspartate chemotaxis (Lee and Imae, 1990). However,
the importance of the N-terminal end of
4/TM2 for aspartate
affinity has not been studied directly.
Altogether, in vitro and in vivo studies of the aspartate receptor and its
relatives strongly implicate the second transmembrane helix as the
element which carries the transmembrane signal across the bilayer (this
study; Chervitz et al., 1995; Lee et al., 1995;
Jeffery and Koshland, 1994; Danielson et al., 1994). This
signal could be a simple movement of the 4/TM2 helix induced by
ligand binding, which could serve to ``switch'' the
cytoplasmic domain between its kinase activating and inactivating
conformations. The nature of the helix movement triggered by ligand
binding remains to be elucidated.