From the Department of Microbiology and Molecular Genetics,
University of Texas Medical School, Houston, Texas 77030
Single cysteine substitutions were introduced
into three positions of otherwise cysteineless HtrI, a phototaxis
transducer found in Halobacterium salinarum that transmits
signals from the photoreceptor sensory rhodopsin I (SRI) to a
cytoplasmic pathway controlling the cell's motility. Oxidative
cross-linking of the monocysteine HtrI mutants in membrane suspensions
resulted in dimer forms evident in SDS-polyacrylamide gels. The rate of
cross-linking of I64C on the cytoplasmic side of HtrI was accelerated
by SRI binding in the dark and further increased by SRI
photoactivation. Several residue replacements of His-166 in SRI
accelerated the cross-linking rate of I64C in the dark and His-166
mutants that exhibit "inverted signaling" (mediating repellent
instead of the normally attractant response to orange light) inverted
the light effect on the cross-linking rate of I64C. Secondary structure prediction of HtrI indicates a coiled coil structure in the cytoplasmic region following TM2, a dimerization domain found in a diverse group of
proteins. We conclude that 1) HtrI exists as a dimer both in the
absence of SRI and in the SRI-HtrI complex, 2) binding of SRI in the
dark increases reactivity of the two cysteines at position 64 in the
dimer by increasing their proximity or mobility, 3) light activation of
wild-type SRI further increases their reactivity, 4) His-166
replacements in the SRI receptor have conformational effects on the
structure of HtrI at position 64, and 5) inverted signaling by His-166
mutants likely results from an inverted conformational change at this
region induced by SRI photoactivation.
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INTRODUCTION |
HtrI is a transducer protein found in the archaeon
Halobacterium salinarum (1, 2), which is homologous to
eubacterial methyl-accepting chemotaxis proteins
(MCPs),1 such as the
aspartate receptor (Tar) in Escherichia coli (3-5). It
contains two transmembrane segments, connected by a 220-residue portion
to methylation regions and a His-kinase binding domain that share high
sequence identity with those of the MCP family. Together with its
membrane partner, sensory rhodopsin I (SRI), HtrI mediates phototaxis.
HtrI and SRI physically interact and form a tight complex in the
membrane (6-8). Signaling by the complex starts from the
photoactivation of SRI, which is structurally similar to the visual
pigment rhodopsin.
The E. coli Tar also contains two transmembrane segments and
exists as a dimer both in its ligand-free state and activated, ligand-occupied state (9). Extensive site-directed disulfide cross-linking studies of Tar and related MCPs have revealed a four-helix bundle of the transmembrane helices in the membrane and
details of helix packing (10-12). Activation of these types of
receptors occurs when a new conformation is assumed either within a
single subunit (13-15) or between subunits within a dimer (11, 12,
16-18).
In this study, we applied site-directed disulfide cross-linking to
monocysteine HtrI mutants to examine whether HtrI exists as a dimer, to
detect a conformational change in HtrI during SRI activation, and to
assess the effects of SRI signaling mutants on the HtrI conformational
change. The results indicate that HtrI is a dimer both in the presence
and absence of SRI and both in the dark and in the light. The
conformation of HtrI, as probed by the cross-linking behavior of I64C,
is sensitive to SRI binding, photoactivation of SRI, and mutations in
SRI. Secondary structure analysis predicts the region in the
cytoplasmic part following TM2 to be coiled coil, a motif responsible
for dimerization in many other proteins.
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MATERIALS AND METHODS |
Chemical Reagents and Enzymes--
Polyethylene glycol 600, 1,10-phenanthroline, formamide, and N-ethylmaleimide were
purchased from Sigma; AG 501-X8 and Bio-Rex MSZ 501 (D) mixed bed resin
(20-50-mesh) were from Bio-Rad; ECL Western blotting kit was from
Amersham Pharmacia Biotech; and Pfu DNA polymerase was from
Stratagene (La Jolla, CA).
Bacterial Strains, Culture Conditions, and
Transformation--
Halobacteria salinarum
strain Pho81Wr
(SRI
HtrI
) (1) and its
transformants were grown in the dark at 37 °C in flasks on a rotary
shaker at 240 RPM. Polyethylene glycol-mediated spheroplast transformation of halobacteria was performed as described (19) with the
following two modifications. Polyethylene glycol 600 was purified by
absorbing to ion exchange resin AG 501-X8 according to the instructions
provided by the manufacturer. Spheroplasts were made from freshly grown
cultures at A600 = 0.4; DNA (200 ng/µl in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was used
directly for transformation without first mixing with spheroplast
solution.
Site-directed Mutagenesis and Plasmid
Construction--
Site-specific mutagenesis was carried out by PCR
according to Chen and Przybyla (20). A 529-base pair
SpeI/SalI fragment from the native htrI gene (1),
which encodes the two transmembrane segments and a part of the
cytoplasmic portion, and a 630-base pair
BamHI/NotI fragment from the synthetic
sopI gene (21) were first cloned into pBluescript
KS
(Stratagene, La Jolla, CA) and used as the template
for the PCR reaction. T3 and T7 primers and synthetic oligonucleotides
(Bioserve, Laurel, MD) containing the desired mutations were used as
PCR primers. Reactions were performed in a Programmable Thermal
Controller-100 (MJ Research, Watertown, MA) at 94 °C for 1 min,
55 °C for 1 min, and 72 °C for 1 min for 31 cycles. To optimize
the reaction, 1-6% formamide was included in some of the PCR
reactions (22). PCR fragments were purified from agarose gel using a
glass powder method (23). After digestion by appropriate enzymes, the
fragment was replaced into pVJY1 (1) or pTR2 (6). The mutations were confirmed by sequencing. Escherichia coli strain DH5
(Stratagene) was used for plasmid manipulation and amplification.
Motion Analysis--
Motility responses to SRI photoactivation
were assayed by computer-assisted cell tracking and motion analysis as
described (21). Pulse durations were controlled by a Uniblitz
electronic shutter (Vincent Associates, Rochester, NY). Phototaxis
stimuli were delivered through an epiiluminator from a Nikon 100-W
Hg/Xe or from a 150-watt tungsten/halogen lamp.
Oxidation Procedure and Western Blot Analysis--
Membranes
were isolated from sonicated stationary phase cells as described (24)
and suspended in 4 M NaCl, 25 mM Tris-HCl, pH
6.8. Membrane samples in sonication buffer (4 M NaCl 25 mM Tris, pH 6.8); low salt membrane dilution buffer (250 mM KCl, 20 mM Tris-HCl, pH 8.0), which
previously has been shown to maintain HtrI and SRI in a molecular
complex as assessed by spectroscopic criteria (7); and all other
solutions were allowed to equilibrate to reaction temperature (10 or
25 °C) prior to mixing. The oxidation reaction was initiated by the
addition of 65 µl of membrane suspension at a protein concentration
of 4 mg/ml to 10 µl of 300 mM 1,10-phenanthroline (in
ethanol) and 10 µl of 150 mM CuSO4 (in
H2O) or other concentrations noted in the Fig. 5 legend,
diluted into 915 µl of low salt membrane dilution buffer. Oxidation
of the membrane by Cu(II)-(1,10-phenanthroline)3 in the
sonication buffer, which contains 4 M NaCl, is impractical due to the chelation of 1,10-phenanthroline by heavy metal ions present
as impurities in NaCl. To quench the reaction, 20-µl aliquots were
transferred to 60 µl of SDS sample buffer containing 10 mM N-ethylmaleimide and 5 mM EDTA
and left on ice. Samples were heated at 65 °C for 5 min before
loading on to a 7% SDS-PAGE gel for separation. Proteins were
electrotransferred to polyvinylidene difluoride membrane at 4 °C,
200 mA for 3 h. HtrI was detected using HtrI-specific multiclonal
antibody, and immunoblots were developed using the ECL Western blotting
kit. Linearity of the signals was tested by comparing bands of serially
diluted samples.
Reactions were performed in a removable sample chamber taken from an
SLM Aminco DW-2000 spectrophotometer (SLM Instruments, Urbana, IL)
connecting to a Haake K15 refrigerated water bath circulator (Haake
Mess-Technik GmbH u. Co., Karlsruhe, Germany) for temperature control.
A magnetic stirring bar was used throughout the experiment for
efficient mixing of reactants. Illumination was from a 100-watt
tungsten/halogen lamp focused on the sample after passing through a 5%
CuSO4 H2O solution (path length = 3 cm),
two heat-absorbing filters, and a long pass orange light filter (Corion
LG530, Corion Corp., Holliston, MA); The final light intensity was
1.5 × 106 ergs·cm
2·s
1
at the position of the sample. The temperature was continuously monitored with a thermocouple probe inserted in the sample and controlled to within ±0.25 °C.
 |
RESULTS |
Phototaxis Responses of Cysteine-substituted HtrI
Mutants--
Single cysteine substitutions were introduced into
cysteineless HtrI at three different positions. Ala-4 is near the
N-terminal side of the predicted TM1, Asn-33 is at the periplasmic end
of TM1, and Ile-64 is located in the cytoplasmic part following the predicted TM2 (Fig. 1). All three
mutants, like wild type, mediated attractant responses to orange light
and repellent responses to UV light (Fig.
2). However, HtrI cells carrying I64C
exhibited smaller responses to both 600-nm step down and 400-nm step up stimuli. The reduced responses were not due to a reduction in the
expression level of the mutant HtrI, since identical amounts of protein
were obtained for all the HtrI mutants as assayed by immunoblotting
(data not shown). Nor were the smaller responses due to reduction of
SRI expression: laser flash photolysis of SRI in membranes isolated
from these transformants revealed identical yields (±5%) of the
S373 photoproduct. These observations indicate that Ile-64
is in a sensitive position but not vital for signaling by the SRI-HtrI
complex.

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Fig. 1.
Positions of single cysteine substitutions in
HtrI. Cylinders represent -helical transmembrane
regions predicted by the PHDsec algorithm (50). N, N
terminus of HtrI; C, C terminus.
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Fig. 2.
Phototaxis responses of wild type and single
cysteine-containing HtrI mutants. Two stimuli were delivered: an
orange light step down stimulus, consisting of 4-s removal of 600 ± 20-nm light (open bars), and a near-UV
stimulus consisting of a 20-ms, 400-nm pulse under continuous orange
light background illumination (filled bars). The
phototaxis index is calculated in s 1 as the integral of
the swimming reversal frequency measured by motion analysis over the
first 2 s after the stimulus minus the integral over 2 s
starting from 6 s after the stimuli were initiated. Values plotted
represent the mean of at least two independent measurements, and the
error bar represents 1 S.D.
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Oxidative Cross-linking of Cysteine-substituted HtrI
Mutants--
To address the question of whether HtrI exists as a
homodimer, like its eubacterial counterparts, intermolecular disulfide bond formation between pairs of homologous cysteine residues
(i.e. from corresponding residues of the two monomers within
the same dimer) was studied by oxidative cross-linking. Since there are no cysteines in wild-type HtrI, any dimer formed will be derived from
the experimentally introduced substitutions. Samples were analyzed by
nonreducing SDS-polyacrylamide gel electrophoresis and immunoblotted
with anti-HtrI antibody. HtrI monomer, which has a molecular mass of 54 kDa, runs abnormally at a 97-kDa position, which has been attributed to
its low pI of 3.9 (1). All three cysteine-containing HtrI proteins
exhibited dimer forms evident in the presence of bands at ~200 kDa
before catalyst is added, while the wild-type HtrI migrated exclusively
as a monomer (Fig. 3A). The
extent of cross-linking without added catalyst was greater in the case
of A4C and N33C than in that of I64C. When catalyst was added to the
membrane suspensions and the reaction was incubated at room temperature
for 2 h, however, only I64C was found to cross-link completely
(Fig. 3A). Very little effect of the catalyst addition was
observed for A4C and N33C. Cross-linking of I64C was completed within
minutes at 10 °C (Fig. 3B). The complete cross-linking of
I64C indicates there is an even number of HtrI monomers in the tight
HtrI-SRI complex.

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Fig. 3.
Immunoblot analysis of membranes containing
wild type (WT) and single cysteine-substituted HtrI
mutants. A, oxidative cross-linking catalyzed by
Cu(II)-(1,10-phenanthroline)3 was performed with membrane
suspensions at room temperature for 2 h. Pho81Wr
membrane that does not contain HtrI was used as a control. In the case
of HtrI I64C, oxidized sample was reduced by the addition of 0.5 mM dithiothreitol and 10% -mercaptoethanol to the
SDS-polyacrylamide gel electrophoresis sample buffer. , no catalyst
added; +, catalyst added; R, reduced. B, time
course of the oxidative cross-linking reaction of cysteine-substituted
HtrI mutant membrane at 10 °C. Samples at time 0 were not treated
with catalyst.
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To rule out the possibility that random collisions of free monomers are
responsible for HtrI cross-linking, a plasmid that overexpresses the
I64C-HtrI-SRI complex was constructed and transformed into
Pho81Wr
. The cross-linking rate of the highly expressed
I64C in this membrane was found to be the same as that of the I64C
membrane in which HtrI was 4-fold less concentrated (data not
shown).
In the case of I64C, the cross-linked dimer can be reduced to monomer
by adding 0.5 mM dithiothreitol and 10%
-mercaptoethanol to the SDS-polyacrylamide gel electrophoresis
loading buffer (Fig. 3A), confirming the disulfide linkage.
However, extended reduction (room temperature, 2 h) did not give
complete dissociation to monomers. Incomplete reduction after oxidation
and preexisting dimers have been observed in other transducers
(25-27).
Effects of SRI on the Cross-linking Pattern of HtrI--
Previous
studies have shown that HtrI interacts with SRI in its native membrane
and changes SRI properties. This is based on the observations that
removal of HtrI (6-8), some deletion constructs of HtrI (28), and
mutations in HtrI (29) affect the kinetics of the SRI photocycle.
However, SRI effects on the conformation of HtrI have not been
reported. To test for a possible effect of SRI on the cross-linking
rate of I64C, plasmids were constructed to contain either only
I64C/htrI or the I64C/htrI-sopI pair and
transformed into strain Pho81Wr
. The kinetics of
cross-linking in the isolated membranes was monitored at 10 °C. HtrI
dimers were observed independent of the presence of SRI (Fig.
4A). However, complete
cross-linking of the I64C monomer occurred only in the presence of SRI
(Fig. 4A). When HtrI is expressed alone, the reaction is
slow (Fig. 4A) and does not reach completion even at room
temperature for 2 h (data not shown). Furthermore, in the absence
of SRI, more HtrI preexists as dimers than when it is complexed with
SRI (Fig. 4A). These data show that the cross-linking
reaction at position 64 is sensitive to the presence of SRI.

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Fig. 4.
Oxidative cross-linking of HtrI I64C in the
presence of SRI (A, +SRI; B,
a and b), absence of SRI (A,
SRI; B, c and d), and
in the dark (A; B, b and
c) and 600-nm light (B, a and
d). The amount of monomer at time 0 was taken as
100%. Reactions were done at 10 °C. Each point represents the
mean ± S.D. of at least three parallel measurements.
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Effects of Light on the Cross-linking Pattern of HtrI--
Orange
light converts SRI (
max = 587 nm) into its attractant
signaling conformation, which is believed to alter the conformation of
HtrI by protein-protein interaction (2). We tested whether the
cross-linking behavior of I64C is sensitive to the putative HtrI
conformational change induced by SRI photoexcitation. Orange light
moderately accelerated the reaction rate (Fig. 4B,
a and b), indicating that I64C is in a sensitive
position. Light accelerated the reaction rate only in the presence of
SRI (Fig. 4B, c and d) and was evident
at various catalyst concentrations (Fig.
5).

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Fig. 5.
Extent of oxidation of HtrI I64C at different
catalyst concentrations in the dark (filled
squares) and in 600-nm light (open
circles). The percentage of monomer remaining at
2 min after the initiation of the reaction, relative to the amount of
monomer at time 0 as 100%, was plotted as a function of catalyst
concentration. 1*, 6 min at 150 mM catalyst.
Reactions were at 10 °C.
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Cysteine Cross-linking as a Probe for the Conformational Changes
Introduced by SRI Mutations--
SRI mediates attractant phototaxis to
orange light stimulation. Previously, it was found that certain
substitutions at either of two positions in SRI, Asp-201 and His-166,
result in inverted (i.e. repellent) responses to orange
light (24, 30). Three SRI mutants that mediate inverted responses to
orange light (D201N, H166A, and H166Y) and one that eliminates
phototactic responses to orange light (H166R) were expressed together
with HtrI I64C in Pho81Wr
. In order to test for
correlation between the phototaxis signaling and the cross-linking
behavior, both the motion analysis and cross-linking reaction were
carried out at 25 °C. Wild type, H166R, H166A, and H166Y SRI
expressed with HtrI I64C mediate similar phototaxis responses as when
they are expressed together with wild type HtrI. However, D201N, which
has an inverted response phenotype when expressed with wild type HtrI
(24), had a normal response in the presence of the I64C mutation (Fig.
6A). Evidently, I64C is an
extragenic suppressor of D201N, which is an additional indication that
position 64 is a sensitive position in HtrI.

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Fig. 6.
A, phototaxis behavior of cells carrying
HtrI I64C expressed under different SRI mutant backgrounds at 25 °C.
Two stimuli were delivered: a 600-nm step down stimulus
(open bar) that is the same as in Fig. 2 and a
step up stimulus consisting of a 4-s pulse of 600 ± 20-nm light
(hatched bar). SRI H166A and SRI H166Y exhibit
inverted (i.e. repellent) orange light responses. The
dashed and dotted line
shows the level of detectable responses above the measurement noise.
B, in vitro cross-linking of membranes containing
HtrI I64C in complex with different SRI mutants at 25 °C. The
fraction of monomer remaining was plotted against time. The reactions
were done in complete darkness (filled circles)
or with orange light illumination (open circles).
The amount of monomer at time 0 was taken as 100%, and each
tick indicates 20%. Catalyst concentration for wild type
(WT), H166R, and D201N was 3 mM, and that for
H166A and H166Y was 0.03 mM.
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The cross-linking rates of I64C in HtrI complexed to the various SRI
mutants were compared. To obtain a measurable rate at 25 °C, the
catalyst was diluted to
of that used at 10 °C. Under
these conditions, HtrI I64C with wild type SRI maintained the same
dark/light relationship in terms of cross-linking rate, i.e.
light moderately accelerated the reaction. Two of the mutants, SRI
D201N-HtrI I64C and SRI H166R-HtrI I64C, exhibited cross-linking rates
comparable with that of the wild type in the dark (Fig. 6B),
and light accelerated the cross-linking rate of SRI D201N-HtrI I64C
(Fig. 6B), which mediated essentially wild type phototaxis
responses (Fig. 6A), but not of SRI H166R-HtrI I64C (Fig.
6B), which did not mediate phototaxis responses to orange
light (Fig. 6A). For the two inverted signaling mutants, SRI
H166A-HtrI I64C and SRI H166Y-HtrI I64C, greatly accelerated reaction
rates were observed in the dark (complete oxidation by 20 s). To
slow down the reaction to allow comparison of the rates in the dark and
light, catalyst was further diluted. Orange light was found to retard
the cross-linking reaction of I64C (Fig. 6B). Light retarded
the rate in the inverted mutant membranes also at 10 °C (data
not shown).
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DISCUSSION |
HtrI, an Archaeal Transducer, Like Its Eubacterial Counterparts,
Exists as a Dimer--
The dimeric nature of Tar was first suggested
from site-directed cysteine cross-linking experiments (9) and was
confirmed by observation of a dimeric ligand binding domain in Tar
crystals (31). The data presented here show that HtrI is also
oligomeric and are consistent with it being a dimer. All three
introduced cysteines cross-link HtrI into dimer forms that are
resistant to SDS in nonreducing electrophoresis. In the case of I64C,
complete oxidation within 20 s in some conditions was observed.
The unusual behavior of A4C and N33C, i.e. more preexisting
cross-linked dimer and slow reaction rate, resembles that of R4C in the
Tar homodimer (32). The high reactivity observed for I64C may reflect a
flexible structure in the cytoplasmic region following TM2 or proximity of the two cysteine residues in the dimer.
An Extended Coiled Coil Dimerization Domain in HtrI Is Identified
by Sequence Analysis--
In addition to the efficient cross-linking
of I64C, protein sequence analysis supports dimerization of the
cytoplasmic regions adjacent to TM2 in HtrI. A region of 71 residues
(Fig. 7A, from residue 90 to
160) corresponding to
6 in Tar is predicted to assume a coiled coil
structure by two prediction algorithms COILS (33) and PAIRECOIL (34).
From position 96 to 154, using a window of 21, the probability is
assessed as >99% by both algorithms, whereas the generally accepted
coiled coil methylation regions in Tar, K1, and R1 (33) are assessed at
about 70 and 80%, respectively, by these programs. Two-stranded
-helical coiled coil is found as a dimerization domain in a diverse
group of proteins (35) and is defined as two
-helices that are wound
into a superhelix through the "knobs-into-holes" packing of amino
acid side chains (36). A heptad repeat denoted as abcdefg is
characteristic, in which hydrophobic residues are found mainly at
a and d positions, while the other positions are
more hydrophilic. Frequently, opposite charged residues are found at
the e and g positions, which have been suggested
to stabilize the coiled coil structure (37). The coiled coil motif
is more evident in a helix wheel model representation of the 71 residues in HtrI (Fig. 7B); a and d
positions define hydrophobic faces, position e is highly
positively charged, position g is highly negatively charged,
and other positions are hydrophilic. The corresponding region in HtrIIs
from H. salinarum (38), Natronobacterium pharaonis (39), and Haloarcula vallismortis (39) and
three other unclassified MCPs from H. salinarum (40) are
also predicted to contain coiled coil
6 region with varied
lengths.

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Fig. 7.
A, sequence alignment of HtrI and eight
other methyl-accepting transducers. The predicted secondary structure
is represented by cylinders ( -helical), lines
(loop), or an open arrow ( -sheet). Predicted
transmembrane segments and conserved loop regions are boxed;
positions a and d in the predicted coiled coil
structure (see "Discussion") are shaded. The arrow from
residue 147 on HtrI shows the shortest region sufficient for SRI
interaction according to a deletion study (28), and HtrI/HtrII chimera
analysis indicates that only the first 60 residues of HtrI are required
(see "Discussion"). HtrI_H and
HtrII_H, HtrI and HtrII from H. salinarum; HtrII_N and
HtrII_V, HtrII from N. pharaonis and
H. vallismortis, respectively; Htc_H,
Htd_H, and Htf_H, putative
MCPs from H. salinarum; Tar_E and
Tar_S, Tar from E. coli and
Salmonella typhimurium, respectively. B, helix
wheel representation of the predicted coiled coil region in HtrI.
Positions in the heptad repeat are represented by circles
with hydrophobic a and d positions
shaded. The sequence at each position reads from the
center to the outside. Orientation of the helices
is as viewed from the periplasmic space. Clustered alanines and charged
residues in possible interacting faces of the helices (designated by
open double-headed arrows) are in a larger font
and boxed.
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Although coiled coil structure is not predicted for the corresponding
region of Tar by these two algorithms (probability < 2%), the
presence of a shortened version of this coiled coil region seems likely
when Tar.E and Tar.S are aligned with the seven halobacterial transducers (Fig. 7A). Indirect experimental evidence
supporting the existence and the importance of such a coiled coil
region in both Tar and HtrI is substantial. 1) A recent disulfide
cross-linking study of this region in Tar (residue 259-290) indicates
an
-helical conformation, a close juxtaposition, and a parallel
alignment at the interface between two subunits within a dimer,
consistent with it being a coiled coil (41). 2) Both biochemical and
genetic studies of hybrids consisting of a full-length and truncated
Tar proteins demonstrated that the
6 region 248-259 in Tar is
required for efficient signaling, suggesting its involvement in subunit interaction (13-15). 3) When a leucine zipper, an extensively studied coiled coil structure, was fused to the cytoplasmic portion (257-553) of Tar, CheA activation was observed (18, 42). 4) A deletion study with
HtrI showed that the N-terminal 147-residue fragment of HtrI interacts
with SRI, whereas the N-terminal 97-residue fragment that is lacking
most of the predicted coiled coil does not (28). Dimerization of HtrI
through this region may be important for its stability. Consistent with
this a chimeric transducer containing the cytoplasmic portion of HtrI
and the N-terminal 60 residues of HtrI is produced in amounts
comparable with wild type and fully interacts with
SRI.2
The 71-residue region in HtrI is unusually long for a coiled coil
domain, most known coiled coils spanning ~40 amino acids (36).
Possibly, the high salt concentrations found in H. salinarum, N. pharaonis, and H. vallismortis
cells may effectively shield electrostatic interactions between side
chains, and hence a longer coiled coil region and a more stringent
hydrophobic core are needed for stabilizing the dimer.
Implications for the Signaling Mechanism--
Light accelerates
the oxidation rate of I64C, suggesting that the reaction rate can be
used as a probe for the conformation of HtrI in the light-activated
SRI-HtrI complex. The effect of light on the reaction rate in several
mutants supports this suggestion. Light has little or no effect on the
rate in membranes from the double mutant SRI H166R-HtrI I64C, which
does not show phototaxis responses. The mutants SRI H166A-HtrI I64C and
SRI H166Y-HtrI I64C exhibit inverted (repellent) behavioral responses
to normally attractant orange light, which has been explained in terms
of an inverted conformational change in the SRI-HtrI complex (43). Consistent with this interpretation, the oxidation rates observed here
are inverted; the rate is much higher than that of wild type in the
dark, and the rate is decreased by light in the inverted mutant
membranes. The oxidation rate of I64C therefore correlates closely with
the conformational state of the complex deduced from behavioral
measurements.
The strong prediction of
6 as a long coiled coil structure has
implications for the transmission of the signal from the membrane to
the His-kinase binding domain, a current important question also for
eubacterial MCPs (44). Models involving significant sliding of one HtrI
monomer with respect to the other (e.g. piston-like, scissors-like, or see-saw movements) will be energetically unfavorable. Rotation of subunits in the plane of the lipid bilayer (45), which
would result in slight winding or unwinding (46) of the
6 coiled
coil, is an interesting possibility. The putative coiled coil of HtrI
(Fig. 7B), unlike the GCN4 leucine zipper in which the
hydrophobic core is exclusively formed by leucines (47), contains a
large number of small chain residues. In particular, the a
and d positions between residue 123 and residue 148 are exclusively occupied by alanine residues, which have been shown to
increase the flexibility of coiled coils (48). A similar feature has
been noted in CM-tropomyosin and has been suggested to facilitate
transmission of a conformational change along its long axis (49) as
might occur also in the HtrI dimer. The
6 coiled coil is bounded by
short flexible regions, which are present in all transducer sequences
so far examined (this study and Ref. 3). This feature might allow for
the divergence (viewed from the cytoplasm) of the two TM2 helices in
the membrane and of the methylation helices at the distal end of the
coiled coil.
We thank Elena Spudich and
Bastianella Perazzona for help with immunoblot analysis and
Kwang-Hwan Jung for stimulating discussions about the inverted
signaling mutants.