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J. Biol. Chem., Vol. 277, Issue 41, 38714-38722, October 11, 2002
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From the
Received for publication, April 15, 2002, and in revised form, June 26, 2002
The type III secretion system is used by
pathogenic Yersinia to translocate virulence factors
into the host cell. A key component is the multifunctional LcrV
protein, which is present on the bacterial surface prior to host cell
contact and up-regulates translocation by blocking the repressive
action of the LcrG protein on the cytosolic side of the secretion
apparatus. The functions of LcrV are proposed to involve
self-interactions (multimerization) and interactions with other
proteins including LcrG. Coiled-coil motifs predicted to be present are
thought to play a role in mediating these protein-protein interactions.
We have purified recombinant LcrV, LcrG, and site-directed mutants of
LcrV and demonstrated the structural integrity of these proteins using
circular dichroism spectroscopy. We show that LcrV interacts both with
itself and with LcrG and have obtained micromolar and nanomolar
affinities for these interactions, respectively. The effects of LcrV
mutations upon LcrG binding suggest that coiled-coil interactions
indeed play a significant role in complex formation. In addition,
comparisons of secretion patterns of effector proteins in
Yersinia, arising from wild type and mutants of LcrV,
support the proposed role of LcrG in titration of LcrV in
vivo but also suggest that other factors may be involved.
A number of Gram-negative bacteria use a conserved
type III secretion system to transport a range of virulence-associated proteins across their double membranes and into the host cell (1). This
mechanism plays a key role in the infection process of bacterial
pathogens including Chlamydia sp., enteropathogenic and enterohemorraghic Escherichia coli, Pseudomonas
aeruginosa, Shigella sp., and Yersinia sp.
(2). This system is a sec-independent pathway and is
composed of approximately 20 proteins, a number of which are
transiently assembled to form a "molecular syringe" that often
results from a signal released upon contact with the target cell (2).
This syringe structure is responsible for the translocation of protein
"effectors" that can cause a range of signaling and cytoskeletal
changes upon the target cell, ultimately leading to, for example,
intracellular invasion of a macrophage or apoptosis (3, 4).
The type III secretion system of Yersinia was the first to
be discovered and is the best characterized to date. Therefore, it is
considered to be the prototypical type III system. The genes encoding
these proteins are located on plasmid pYV, which is present in all
three human pathogenic Yersinia species, i.e.
Yersinia pseudotuberculosis, Yersinia
enterocolitica, and Yersinia pestis. These
Yersinia species resist the primary host immune defense by
inhibiting macrophage phagocytosis (2). This inhibition is mediated by
the type III secretion system and thus allows infection by
extracellular localization (5). The secretion machinery is principally
composed of the Yersinia outer
proteins (Yops),1
the Yersinia secretion apparatus (Ysc), and
specific Yop chaperones (Syc)
(6).
A key component of the Yersinia type III secretion apparatus
is the V antigen or LcrV, a 37-kDa protein, the expression of which is
transcriptionally up-regulated at 37 °C upon infection of the host
or in vitro in the absence of calcium (7). It is a
protective antigen of Y. pestis (8, 9) and is currently in
phase II trials as a principal component of a potential vaccine for
plague (caused by Y. pestis) (1). LcrV has several putative roles in Yersinia type III secretion and Yersinia
pathogenicity in general that are probably mediated by
self-interactions (multimerization) or interactions with other
proteins. LcrV has been proposed to assemble into a membrane-associated
extracellular structure, which facilitates the transfer of Yop effector
proteins into the eukaryotic cytosol, and is surface-located prior to
target cell contact (10). Thus, the extracellular localization of LcrV
on the bacterial membrane may provide a prime position from which to
control Yop targeting (10). LcrV further promotes protein secretion and targeting through an interaction with YopB and YopD, possibly enabling
their deployment for pore formation within the eukaryotic membrane
(11). In addition, it has recently been demonstrated that LcrV is both
extracellularly localized and translocated into the host cell cytosol
(12).
LcrV has been predicted to contain a coiled-coil motif (13) composed of
a repeating 7-residue signature sequence, indicative of the formation
of a superhelix from two or more In this context, elucidation of details of molecular interactions
involved in LcrV complex formation is fundamental to understanding secretion processes in Yersinia pathogenesis. However, a
lack of any quantitative protein interaction information has motivated us to produce pure folded recombinant forms of these proteins. Using
these materials, we report the relative affinities of LcrV for itself
and for LcrG. In addition, the availability of such materials has
afforded us the opportunity to produce site-directed mutants of LcrV
within the predicted coiled-coil motif and subsequently assess the
importance of this motif both in terms of LcrG interaction and Yop
secretion. Therefore, the present investigation provides the first
quantitative characterization of type III secretion protein-protein
interactions and a more detailed molecular description of LcrV and LcrG
folding and complex formation, which complements previous genetic approaches.
Materials--
Oligonucleotides were purchased from Sigma. Media
reagents Luria Broth (LB) and Brain-Heart infusion were obtained from
Merck. Isopropyl-1-thio- Construction of Plasmids and Site-directed Mutants--
Plasmids
pGEX-V and pGEX-G are pGEX-5X1 derivatives (Amersham Biosciences). Both
plasmids encode Schistozoma japonicum glutathione S-transferase (GST), a linker peptide containing a protease
cleavage site, and either the Y. pestis LcrV (pGEX-V) or
LcrG (pGEX-G) proteins. The genes encoding lcrV and
lcrG were ligated into the EcoRI-NotI
sites of the pGEX-5X-1 vector. pGEX-V was generated by a
Pfu-based PCR of lcrV using Primer1
(5'-ATCGAATTCATTAGAGCCTACGAACAAAAC-3'), which contains an
EcoRI restriction site upstream of the second codon (ATT),
and Primer2 (5'-ACATAGTATAGCGGCCGCGTGTCATTTACCAGACGT-3'), which
incorporates a unique NotI site downstream of the TCA
stop codon. pGEX-G was generated by a Pfu-based PCR of
lcrG using Primer3 (5'-ATAGAATTCACAATAAATATCAAGACAGACAGC-3'), which contains an
EcoRI restriction site upstream of the second codon (ACA),
and Primer4 (5'-ACATAGTATAGCGGCCGCATATTAAATAATTTGCCCTCGCAT-3'), which
contains a NotI restriction site downstream of the TAA stop
codon. pGEX-V and pGEX-G were subsequently transformed into the
E. coli strain BL21 (DE3) for overproduction of
recombinant GST-LcrV or GST-LcrG fusion proteins, respectively. Plasmid
pGEX-V was purified using the Qiagen QiaQuick Spin Miniprep kit and
then used as a template to amplify the Y. pestis lcrV gene
in a Pfu-based PCR. Primer1 was used with Primer5
(5'-CCCAAGCTTGTGTCATTTACCAGACGT-3'), which incorporates a unique
HindIII site downstream of the TGA stop codon. The 990-bp
lcrV fragment was ligated into the EcoRI and HindIII sites of the pTrc99A expression vector
(Amersham Biosciences) to produce plasmid pTrc-LcrV. Initial clones
were obtained by heat-shock transformation (21) into E. coli
strain BL21 (DE3).
PCR-based mutagenesis of lcrV was performed using the primer
overlap method. In all cases, Primer1 was used in conjunction with a
"mutagenic" primer containing a site-directed mutation to generate
a PCR fragment encoding from the 5' end of the gene to a few bases
downstream of the mutation site. A second PCR fragment was generated
from a few bases upstream of the mutation site using an overlapping
primer, complementary around the mutation site, in combination with
Primer2. Each PCR was carried out using pGEX-V as the template.
Fragments generated were gel-purified and subjected to a second round
of PCR using Primer1 and Primer2 to generate a fragment containing the
entire lcrV gene with the mutations of choice. Each fragment
was then ligated into the EcoRI and NotI sites of
the GST fusion vector pGEX-5X-1 and transformed (21) into E. coli strain BL21 (DE3). The resulting plasmids were
then used as templates for PCR using Primer1 and Primer5 as described above. The resulting PCR fragments were subsequently ligated into the
EcoRI and HindIII sites of the pTrc99A
expression vector. All mutants constructed in the lcrV gene
contained the CGT codon encoding an arginine residue at positions
within the DNA sequence predicted to encode the coiled-coil motif of
interest. The presence of desired mutations within the lcrV
gene was confirmed by sequencing the pGEX-5X-1-lcrV plasmids
with an automated Applied Biosystems 377 DNA Sequencer (Foster City, CA
using the dideoxy method with BigDye Terminator Ready Reaction Kits
(Applied Biosystems, Foster City, CA).
Identification of the putative coiled-coil motifs in LcrV and LcrG was
made using the COILS program (ulrec3.unil.ch/coils/COILS_doc.html) (22,
23). A 28-residue window size was used with weighting in favor of
hydrophobic residues at the a and d positions of
the heptad repeat. The MATCHER program
(cis.poly.edu/~jps/) (24) was also used to determine
whether a protein sequence contains the 7-residue periodicity
([aXXdXXX]n) associated with coiled-coils. Secondary
structure predictions were conducted using PHDsec from EMBL
(www.embl-heidelberg.de/Services/index.html).
Protein Purification--
GST, GST-LcrG, GST-LcrV, and GST-LcrV
mutant proteins were purified with minor modifications to a previously
described protocol (20). Two liters of LB containing 100 µg/ml
ampicillin were inoculated with 50 ml of an overnight culture of
E. coli BL21 (DE3) transformed with the
appropriate pGEX derivative. Cells were grown at 37 °C with shaking
for 2 h and then induced with isopropyl-1-thio-
6 ml of 50% (w/v) glutathione-Sepharose 4B in PBS were
added to the cell lysate supernatant (typically 12 ml) and incubated at
4 °C for 1 h with rotation. The material was transferred into a
plastic column and washed with 100 ml of PBS. GST and GST-LcrV proteins
were eluted after this stage by resuspending the glutathione-Sepharose in 6 ml of 10 mM glutathione in 100 mM Tris
HCl, pH 8.0, and collecting the flow-through from the column.
Otherwise, the glutathione-Sepharose with bound fusion proteins was
resuspended in 6 ml of PBS. Proteolytic cleavage to release LcrV or
LcrG was conducted by adding 200 µl of 100 µg/ml Factor Xa in PBS
followed by incubation at 4 °C for 16 h with rotation. Cleaved
protein was collected as flow-through from the column. The column was
then washed to retrieve all of the protein released. Proteins produced
from Factor Xa cleavage all contained five N-terminal residues (GIPEF)
prior to the second encoded residue of the predicted protein sequence.
Pooled samples of LcrV were concentrated in Centriprep-YM10
concentrators, whereas pooled LcrG samples required Centriprep-YM3 concentrators. Protein concentrations were determined by Bradford assay. Protein purity was estimated by 8-25% SDS-PAGE, and for LcrV
and LcrG by MALDI-MS at the Mass Spectrometry Facility, Department of
Biochemistry, University of Bristol (Bristol, United Kingdom).
Circular Dichroism Spectra--
Circular dichroism (CD)
measurements were performed on an AVIV 62DS spectropolarimeter. The
instrument was calibrated with camphor sulfonic acid for optical
rotation and benzene vapor for wavelength. Data were recorded at 0.2-nm
intervals and at 25 °C in a temperature-controlled chamber. Samples
of LcrV wild type and mutants (4 mg/ml in PBS), LcrG (9 mg/ml in PBS),
GST (6 mg/ml in PBS), GST-LcrV (4 mg/ml in PBS), or base lines
consisting of PBS were examined in 0.001-cm path length Suprasil
cuvettes. A minimum of five scans was collected for every sample.
Individual scans were smoothed with a Savitsky-Golay filter (25), and
the averaged base-line spectrum was subtracted from the averaged sample spectrum. Mean residue molecular weights of 114.2 for LcrV, 110.2 for
LcrG, 116.7 for GST, and 115.1 for GST-LcrV were used in the calculations of mean residue ellipticity.
Spectra were analyzed using the Dichroweb server (26). Reference data
sets 4 and 7 (27, 28), which were compatible with the wavelength range
obtainable in this study, were used. To test the reliability of the
determinations, a number of alternative algorithms were used for the
structure calculations: SELCON3 (29, 30), CONTIN (31, 32), and CDSSTR
(27). As a means of comparison of the goodness-of-fit of the various
methods, the NRMSD parameter (34) was calculated. NRMSD is defined as:
Gel Electrophoresis and Staining--
Purified protein samples
were analyzed using 8-25% polyacrylamide gradient Phast gels on a
Phast electrophoresis system (Amersham Biosciences). SDS and native
gels were stained with Coomassie Brilliant Blue using the Phast system
developer unit. Protein interaction studies involved the co-incubation
of LcrG with LcrV or GST-LcrV at an estimated 2:1 molar ratio at
4 °C for 30 min prior to visualization on a native 8-25% gel.
Identification of protein secretion patterns from Y. pseudotuberculosis was done using 14% homogeneous polyacrylamide
gels using a Novex Mini-gel system (Novex, San Diego, CA). 25 µl of
5× concentrated protein loading buffer (62.5 mM Tris-HCl,
pH 6.8, 2% SDS (w/v), 5% Western Blotting--
Proteins were separated on an 8-25%
gradient Phast gel and transferred onto a nitrocellulose Hybond-P
membrane, which was blocked overnight at 4 °C with 5% casein in
PBST (PBS containing 0.05% Tween 20 (v/v)). LcrV was detected using a
mouse-generated anti-LcrV antibody diluted 1:3000 in PBST + 5% casein
and incubated for 1 h at room temperature. After washing,
membranes were incubated with an anti-mouse horseradish
peroxidase-conjugated antibody (1:10,000 in PBST) for 1 h
at room temperature. Horseradish peroxidase was detected using ECL
reagents and visualized by exposure of Kodak X-Omat AR film.
Binding Assays--
ELISA methods were used to analyze
interactions between GST-LcrV and LcrV (self-association), GST-LcrV and
LcrG, and LcrV and LcrG based upon related studies of the
enteropathogenic E. coli EspA protein (36). The
self-association of LcrV was assayed by incubating 100 µl of aliquots
of LcrV at 20 µg/ml in carbonate/bicarbonate buffer (0.05 M carbonate/bicarbonate buffer, pH 9.6) in an ELISA plate
at 4 °C overnight. Wells were washed three times with 200 µl of
PBST and blocked with 200 µl of 2% (w/v) casein in PBST for
1 h at 37 °C. 70 µl of GST-LcrV at concentrations ranging from 0.2 to 2 mg/ml in PBST containing 1% casein (w/v) were added to
wells and incubated at 37 °C for 1.5 h. After washing, 70 µl of rabbit anti-GST IgG (1:5000 dilution in PBST) were added to each
well for 1 h at 37 °C. Further washing was followed by 70 µl
of alkaline phosphatase-conjugated anti-rabbit antibody (1:5000 dilution in PBST) being added to wells and incubated for 1 h at 37 °C. The reaction was visualized by adding 70 µl of 1 mg/ml
The LcrV·LcrG interaction was assayed by coating an ELISA plate with
100 µl of 20 µg/ml LcrG as described above. After washing, wells
were blocked with 1% bovine serum albumin (w/v) in PBST. 100 µl of
LcrV at concentrations between 0.07 and 80 µg/ml in PBST were added
to the wells for 2 h at 37 °C. Plates were washed and probed
with a mouse anti-LcrV monoclonal antibody (1:2000 dilution in PBST at
37 °C for 1 h) followed by alkaline phosphatase-conjugated anti-mouse antibody (1:2000 dilution, 37 °C for 1 h). The
reaction was visualized, and the KD was estimated
using an LcrV molecular mass of 37,000 as described above. LcrV binding
to the ELISA plate was used as a control.
The affinity of LcrV for LcrG was determined using surface plasmon
resonance (SPR) (37) in a BIAcore X instrument with dextran-coated CM5
sensor chips. LcrG at 15 µg/ml in a sodium acetate/acetic acid
buffer, pH 6.0, was immobilized to a CM5 chip by amine coupling (38).
The injection of 100 µl of 0.1-1.2 µM wild type and
mutant LcrVs in HEPES-buffered saline (10 mM HEPES, 150 mM NaCl, 0.01% (w/v) detergent P20) was performed at a
flow rate of 20 µl/min. The binding surface was regenerated with 30 µl of 10 mM HCl at 20 µl/min, which restored the
base-line response level without degradation of the sensor chip. The
binding of LcrV at all concentrations to the CM5 dextran chip alone was
subtracted from the experimental data as a control.
In addition, LcrV at 15 µg/ml in a sodium acetate/acetic acid buffer,
pH 6.0, was immobilized to a CM5 chip by amine coupling (38). The
injection of 100 µl of 0.7-8.0 µM wild-type LcrG in HEPES-buffered saline was performed at a flow rate of 20 µl/min. The
binding surface was regenerated with 30 µl of 10 mM HCl
at 20 µl/min. The binding of LcrG at all concentrations to the CM5 dextran chip alone was subtracted from the experimental data as a control.
Sensorgrams were analyzed using the BIAevaluation 3.0 package (BIAcore,
Uppsala Sweden) to derive values for association rate constants
(ka) and dissociation rate constants
(kd) by simultaneously fitting several data sets,
consisting of injections over a range of ligand concentrations using
simple 1:1 Langmuir binding models with the global fitting option. The
KD value was derived from
kd/ka. A second estimate of the
KD was derived from a plot of the response levels at
equilibrium against protein concentration. A steady-state affinity model was used to fit the curve.
Protein Secretion Assays--
Analyses of secretion patterns of
Yops in Y. pseudotuberculosis were conducted according to a
previously described method (10). For production of competent cells,
Y. pseudotuberculosis strain YPIII containing the pIB19
variant of the Yersinia pYV plasmid (lcrV
Identification of Coiled-coiled Motifs--
Protein sequences
along with secondary structure and coiled-coil motif predictions are
shown in Fig. 1 for regions of LcrV and
LcrG with the highest probabilities of containing a helix capable of
forming a coiled-coil. For LcrV, the COILS program (ulrec3.unil.ch/coils/COILS_doc.html) gave a probability of 0.988 that
the helical sequence (PHDsec) will adopt a coiled-coil conformation, whereas the MATCHER program and helical wheel plot
(marqusee9.berkeley.edu/kael/helical.htm) identified residues Leu-153,
Leu-157, Leu-160, and Leu-164 as being central within this region and
present in crucial a and d heptad repeat
positions. The probability of the Protein Production and Folding--
GST and GST-LcrV, purified
following elution from the glutathione-Sepharose 4B column, migrated as
single bands of ~27 and 64 kDa, respectively, using SDS-reducing
PAGE. LcrV and LcrG purified following Factor Xa cleavage from the GST
fusion tag also migrated as single bands at 37 and 11 kDa,
respectively. Using MALDI-MS, LcrV had a molecular mass of 37,720 Da
compared with 37,652 Da by calculation, whereas LcrG had a molecular
mass of 11,491 Da compared with 11,432 Da by calculation. Prior to
purification of the LcrV mutants, the presence of the desired mutations
in the pGEX-5X-1-lcrV plasmids used for expression was
confirmed. In all cases, the desired sequence was observed (compared
with the published entry in GenBankTM
(www.ncbi.nlm.nih.gov/)) with the required changes that are always present as a CGT codon encoding an arginine residue. Subsequent purification of LcrV mutants using the glutathione-Sepharose 4B column
yielded proteins with the same apparent molecular mass and purity
(judged by SDS-PAGE) as that observed for the wild-type protein.
The CD spectra of LcrG and LcrV cleaved from the GST moiety (Fig.
2A) exhibit similar features,
namely a maximum at 192 nm and minima at 208 and 222 nm that are
characteristic of proteins with a significant
The LcrV mutants displayed a range of spectral features. Many of these
proteins produce spectra that are indistinguishable from wild-type LcrV
(e.g. L157R, Fig. 2B), and consequently, their calculated secondary structures are identical (Table I). However, other
mutants produced slightly different spectra (e.g. L153R, Fig. 2C), which in some cases could appear similar to a
wild-type spectrum upon the addition of a second mutation
(e.g. L153R/L157R, Fig. 2D). When compared with
wild-type LcrV, all mutants had an equivalent 192-nm peak, but the
magnitudes and ratios of the 208- and 222-nm peaks were altered in some
cases (Table II). Consequently, the
calculated Native Gel Electrophoresis--
When analyzed by native PAGE,
purified LcrV appears to migrate as a mixture of forms (Fig.
3, lane 1). The lowest
migrating band is the predominant species, which corresponds to the
formation of a dimer with an estimated molecular mass of 74 kDa. Higher migrating bands, which can appear as doublets, represent higher order
oligomers that may adopt subtly different conformations depending upon
the mode of association perhaps arising from coiled-coil and other
inter-domain interactions. No LcrV monomer was visible on the native
gel, and no evidence of degradation was observable using SDS-reducing
PAGE as reported earlier. Native PAGE of the GST-LcrV fusion also
showed a mixture of forms, upshifted in comparison to Fig. 3 arising
from the presence of GST (data not shown). In contrast, LcrG appears as
a single species on the native gel (Fig. 3, lane 2) with an
estimated molecular mass corresponding to an 11-kDa monomer. The
addition of a 2:1 molar excess of LcrG to LcrV (Fig. 3, lane
3) results in a distinct downshift of the 74-kDa dimer band of
LcrV, suggesting the formation of a heterodimer between LcrV and LcrG.
The effects of the Leu to Arg substitutions upon the ability of
LcrV to multimerize were also analyzed by native PAGE analysis.
Variable mobility was observed between the different LcrV mutants. In
general, both single and double mutations appear to reduce the level of
multimerization of the protein with double substitutions doing so to a
greater extent (Table II).
Protein-Protein Interactions--
ELISA assays were carried out to
assess the binding of LcrV to LcrG and the interaction of LcrV with
itself. Fig. 4A shows the
results obtained using LcrG-coated microtiter wells, which were probed
with LcrV. Fitting to a single-site isotherm gave a good estimate for
the KD of 208 ± 20 nM. Using
GST-LcrV instead of LcrV, a similar dissociation constant was obtained (KD = 150 ± 12 nM). No appreciable
nonspecific binding to LcrG was observed using GST alone. This suggests
that the interaction observed with the GST-LcrV fusion is between the
LcrV domain and LcrG and is of a similar nature to that observed using
purified LcrV isolated after Factor Xa removal of GST. Fig.
4B shows the ELISA results obtained for the binding of
GST-LcrV to LcrV-coated microtiter wells. Fitting to a single-site
isotherm gave KD = 1.13 ± 0.11 µM. Low nonspecific binding was observed using GST alone,
suggesting that the interaction is specific for LcrV. Fig. 5 shows overlaid sensorgrams of LcrV
binding to LcrG immobilized to the CM5 dextran chip and then fitting of
the data using a simple 1:1 Langmuir binding model. This yielded
KD = 140 nM (
In most cases, the mutations of Leu to Arg in the predicted coiled-coil
motif of LcrV reduced binding to LcrG. ELISA assays (Fig. 4,
C and D) yielded KD values
ranging from ~400 nM to 2.2 µM (summarized
in Table II), and insignificant levels of binding (<15 response units)
were observed using SPR. The exceptions are two mutants, L153R and
L135R/L160R, which bound to LcrG with dissociation constants similar to
that of the wild-type LcrV measured using both ELISA and SPR methods
(Fig. 4E and Table II).
Secretion Profiles--
SDS-PAGE of culture filtrates of Y. pseudotuberculosis strain YPIII pIB19 (lacking the lcrV
gene) or the same strain transformed with plasmid pTrc99A
did not show significant levels of secreted Yops as expected.
Transformation with pTrc-LcrV (containing the wild-type lcrV
gene) restored Yop release, whereas the pTrc99A derivatives
containing lcrV coiled-coil mutants generated a range of
secretion patterns (Table II). Western blotting confirmed equal levels
of expression of LcrV from the pTrc-LcrV wild-type and mutant
constructs in each transformant (data not shown).
The secretion patterns can be roughly grouped into two sets. The
first set demonstrated greatly reduced levels of Yop secretion compared
with Y. pseudotuberculosis complemented with wild-type LcrV. This includes clones that were mutated in either of the two central leucine residues (L157R or L160R) and double mutants that
include at least one of these leucines (L157R/L160R, L157R/L164R, or
L160R/L164R). The second set demonstrated a secretion pattern that was
only slightly reduced compared with wild-type LcrV. This includes
clones that were mutated at either of the peripheral leucine residues
(L153R or L164R) and any double mutant that contained the L153R
mutation (L153R/L157R, L153R/L160R, or L153R/L164R).
Quantitative Analysis of LcrV Interactions--
This paper
describes the high level production of LcrV and LcrG wild-type proteins
and LcrV mutants, which have been used to establish a quantitative
characterization of the protein-protein interactions of LcrV with
itself and with LcrG. The virulence of Y. pestis
has been shown to be related to the presence of the LcrV protein, which
is required for the secretion of Yop effector proteins (41). The LcrG
protein is a negative regulator of secretion, and in the absence of
LcrV, LcrG protein blocks Yop secretion by interacting with the type
III secretion apparatus within the cytoplasm of the pathogen (18, 41,
42). Subsequent studies demonstrated the formation of an LcrV·LcrG
heterodimer identified in Y. pestis by chemical
cross-linking (17) and identified in Y. pestis and Y. enterocolitica by co-purification (11, 17). Recent yeast
two-hybrid analysis has also demonstrated an interaction between LcrV
and LcrG and that this interaction is required for Yop secretion
(19).
In these studies, a pure LcrV sample was shown to form multimers of
dimers and higher order species as reported recently (43), whereas LcrG
appears to be exclusively monomeric in contrast to previous reports
(44). This multimerization is dependent upon the concentration of the
protein with LcrV existing as a stable dimeric unit at low
concentrations (43) in the ELISA and SPR methods. LcrV·LcrG complex
formation was first observed using native gel electrophoresis, which
yielded a band consistent with heterodimer formation. Subsequently,
ELISA and SPR methods demonstrated similar nanomolar binding affinities
between LcrV and LcrG, thus suggesting that complex formation between
these two proteins is favored over dimer formation and perhaps over
other self-association interactions, the latter of which displays a
micromolar binding affinity. The close fit of a 1:1 binding model to
the data for LcrV binding to LcrG using SPR indicates that only a
single species of LcrV is binding immobilized LcrG. These results
support the proposal that in vivo LcrV titrates LcrG from
the type III secretion apparatus (17) and is in agreement with previous
data proposing that the ratio of LcrV to LcrG is important for type III
secretion activity (18, 19). Significantly, assuming a stable
LcrG·LcrV complex is essential for Yop secretion, the results
presented here predict that competing affinities between LcrG and the
type III secretion pore complex are less strong than the nanomolar affinity between LcrG and LcrV reported here.
Coiled-coil Interactions--
Pallen et al. (13)
proposed that complex formation in proteins involved in type III
secretion could be mediated by the formation of coiled-coil
interactions and identified the LcrV·LcrG complex as a possible
example based upon the prediction of a coiled-coil motif in both these
proteins. The specific residues identified in Fig. 1 as important for
coiled-coil formation are located within the regions reported
previously (i.e. residues 136-180 for LcrV and 7-34 for
LcrG) (13). In particular, Leu-153, Leu-157, Leu-160, and Leu-164 of
LcrV are located at critical a and d heptad
positions within the center of this region and should interdigitate
with the same residues via the formation of a coiled-coil in the LcrV dimer. In the case of LcrG, residues Leu-13, Ala-16, and Ile-20 of LcrG
are within the center of this region and are predicted to be important
for the same type of interactions in the LcrV·LcrG complex. Analyses
of the CD spectra of LcrV and LcrG produced very low values for the
NRMSD fit parameter (
To probe the potential importance of coiled-coil interactions, single
and all possible double mutants of the leucine residues in LcrV
identified in Fig. 1 were made by substituting arginine at these
positions. This strategy had previously been shown to be successful in
related studies of the EspA and EspD type III secretion pathway
proteins from enteropathogenic E. coli (36, 16). CD analysis
indicated that all LcrV mutants were produced as folded proteins and
still showed spectral features, suggestive of helices with possible
coiled-coil formation, which may relate to the multimer formation
observed in native gels (Table II). At low concentrations in the ELISA
and SPR assays, both LcrV wild-type and mutant proteins will be present
principally as a dimer. The six single and double mutants of Leu-157,
Leu-160, and Leu-164 all showed decreased levels of interaction with
LcrG, suggesting that these residues may be involved in coiled-coil
interactions with the hydrophobic residues identified in LcrG (Fig. 1).
These mutants have reduced on and off rates compared with wild-type LcrV and bind to LcrG in the equilibrium ELISA protocol but not in the
flow BIAcore experiments. The mutation of Leu-153 to Arg-, however,
resulted in a mutant with a higher affinity for LcrG than the wild-type
protein. Although this result does not discount Leu-153 from being
involved in a coiled-coil interaction, it is possible that an Arg at
this position could mediate other interactions with LcrG. Outside of
the heptad repeat region in LcrG, there are numerous aspartate residues
(7, 10, 24, 26) that could form a salt-bridge with the mutant Arg-153
side chain of LcrV and result in enhanced affinity between these two
proteins. Indeed, using ELISA methods, the interaction of LcrV L153R
with LcrG is affected by pH. Furthermore, double mutants involving residue 153 also displayed peculiar behavior. In combination with either the L157R or L164R mutations, double mutants showed the lowest
affinity for LcrG, whereas in combination with L160R, wild-type affinity was recovered. These data suggest that the high affinity character of the L153R mutant for LcrG can dominate in some cases depending upon the strength of the disruption caused by the second-site arginine mutation.
Yop Secretion--
The current model of the type III secretion
apparatus places LcrG on the cytosolic side of the Yersinia
inner membrane where it forms a plug and blocks the secretion of Yop
effector proteins (17). As already discussed, the relatively high
affinity between LcrV and LcrG compared with self-interaction of LcrV
drives the formation of a heterodimer complex, thus allowing secretion
to occur. Based upon this model of interaction, wild-type and the L153R
and L153R/L160R mutants, which all showed high affinity for LcrG, also
effectively complemented the Y. pseudotuberculosis lcrV
knock-out and showed predicted high levels of Yop secretion. In
addition, single mutants L157R and L160R and double mutants L157R/L160R, L157R/L164R, and L160R/L164R all showed reduced levels of
binding to LcrG and, predictably, very low levels of secretion. Therefore, these data suggest that coiled-coil interactions may indeed
play a significant role in mediating LcrV·LcrG complex formation.
Interestingly, not all secretion patterns observed follow predictions
based upon a titration model involving a discrete interaction between
LcrV and LcrG. First, the L164R mutant showed reduced levels of binding
to LcrG compared with wild-type LcrV protein, but Yop secretion was
only moderately reduced. It is possible that the effects of the Leu-164
mutant were not manifested as greatly as when other residues of the
coiled-coil (Leu-157 and Leu-160) were mutated. This is consistent with
the almost total loss of secretion when L164R is combined as a double
mutant with L157R or L160R (Table II). Although, more curious is the
presence of wild-type secretion for the L153R/L157R and L153R/L164R
double mutants, both of which display the weakest interactions with
LcrG based upon ELISA assays (Table II). One explanation is that
in vivo, the great increase in intracellular LcrV upon type
III secretion stimulation and the high affinity interaction of the
L153R mutation dominates, resulting in wild-type activity. However, it
is also tempting to speculate that recognition of LcrG by LcrV may not simply involve LcrG alone. In particular, the gating function of LcrG
implies that it forms an interaction with the Ysc apparatus located on
the cytosolic inner membrane of Yersinia. Given the small
size of LcrG (11 kDa), it is not unreasonable to consider that LcrV
could initially recognize an LcrG-type III secretion apparatus complex
prior to the formation of the LcrV·LcrG heterodimer. Although the
L153R/L160R and L153R/L164R double mutants show poor interactions with
LcrG, it is possible that these proteins may still promote dissociation
of LcrG from the type III secretion apparatus, especially if LcrV
initially recognizes a complex rather than LcrG on it own.
Taken together, these results lead us to propose a modified model of
LcrV·LcrG titration where stable and transient protein complexes are
formed and exchanged depending upon relative affinities as a means of
regulating type III secretion in Yersinia. Under non-secreting conditions, LcrG forms a complex with the Ysc apparatus located on the cytosolic side of the inner membrane (Fig.
6A) (17). Upon induction of
type III secretion, high levels of LcrV are produced predominantly as a
dimer (Fig. 6B). The high nanomolar affinity of LcrV for
LcrG disrupts LcrV dimer formation and interactions between LcrG and
the Ysc apparatus. The formation of an LcrV·LcrG complex mediated
through coiled-coil interactions as previously proposed (13) results in
the removal of the internal gating of the Ysc apparatus, thus enabling
Yop secretion to occur (Fig. 6C) (18). In addition, LcrV may
initially recognize LcrG as a bound complex near the inner membrane
(Fig. 6B) and promote LcrG dissociation through
conformational changes, altering the interactions between LcrG and
other type III secretion components. The use of the methods developed
here to further define the importance of LcrG complex formation and
other related pathway components is therefore underway. Furthermore,
although the type III secretion system from Yersinia is the
most studied to date, the system is also present in a number of other
pathogenic bacteria such as P. aeruginosa where PcrV, a
homologue of LcrV, was shown to mediate type III secretion in lung
infection (33). Sequence similarities among the different type III
components suggest that regulation of Ysc assembly and secretion may be
conserved across species. Hence, it is hoped that characterization of
individual protein-protein interactions between pathway components will
ultimately yield an improved understanding of the molecular basis of
type III secretion processes not only in Yersinia but in
other bacterial pathogens.
We thank Åke Forsberg (the Swedish Defense
Research Agency, FOI, Umeå, Sweden) for the kind gift of Y. pseudotuberculosis strain YPIII (pIB19).
*
This work was supported by the UK Biotechnology and
Biological Sciences Research Council, UK Ministry of Defense, and
Defense Science and Technology Laboratory.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.
**
To whom correspondence should be addressed. Tel.: 44-20-75945298;
Fax: 44-20-75945207; E-mail: k.brown@ic.ac.uk.
Published, JBC Papers in Press, July 9, 2002, DOI 10.1074/jbc.M203632200
The abbreviations used are:
Yop, Yersinia outer protein;
Ysc, Yop secretion;
ELISA, enzyme-linked immunosorbent assay;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
PBST, phosphate-buffered saline with Tween 20;
CD, circular dichroism;
SPR, surface plasmon resonance;
MALDI-MS, matrix-assisted laser desorption
ionization mass spectometry.
Interactions of the Type III Secretion Pathway Proteins LcrV and
LcrG from Yersinia pestis Are Mediated by Coiled-Coil
Domains*
,
,,, and**
Department of Biological Sciences, Centre
for Molecular Microbiology and Infection, Flowers Building, Imperial
College of Science, Technology and Medicine, London SW7 2AY, United
Kingdom, the § Department of Haematology, National Institute
for Biological Standards and Control, Blanche Lane, South Mimms,
Potters Bar, Hertfordshire EN6 3QG, United Kingdom, the ¶ School
of Crystallography, Birkbeck College, University of London, London WC1E
7HX, United Kingdom, and Defense Science and Technology
Laboratory, Chemical and Biological Sciences, Porton Down, Wiltshire
SP4 0JQ, United Kingdom
ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-helices (reviewed in Refs. 14 and
15). Coiled-coils have been suggested to represent a key structural
feature required for the formation of the multiprotein complexes
involved with the type III secretion apparatus (13), and it has been
suggested that the interaction of LcrV with the LcrG protein may be
mediated through the formation of a coiled-coil (13). Genetic
approaches such as the yeast-two hybrid system have been used to
identify strong interactions between type III pathway proteins such as
that observed between the EspA (filament-forming protein) and EspD
(translocation pore proteins) of enteropathogenic E. coli
(16). In Yersinia, similar experiments have demonstrated an
interaction between LcrV and an inhibitory gate protein to the Ysc
apparatus, LcrG (17). Overexpression of LcrG inhibits Yop secretion
even in the presence of LcrV (18). Hence a "titration model" has
been proposed whereby the up-regulation of LcrV during the low calcium
response allows binding to LcrG, thus removing the internal gating of
the Ysc apparatus (18). A site-directed mutant of LcrG was also
recently reported that failed to demonstrate an interaction with LcrV
using a yeast two-hybrid system. This variant was able to block
secretion of LcrV and Yops, further supporting the hypothesis
that the formation of an LcrV·LcrG complex is required for Yop
secretion (19).
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-D-galactopyranoside was from
Genesys (London, UK). Precast native and SDS-polyacrylamide
gels, protein molecular weight standards, Coomassie Brilliant Blue,
glutathione-Sepharose 4B, Hybond-P nitrocellulose membrane, ECL
detection reagents, and Factor Xa were from Amersham Biosciences. 30%
polyacrylamide solution for 14% PAGE was purchased from Severn Biotech
(Kidderminster, United Kingdom). DNA QiaQuick gel extraction and
Miniprep kits were from Qiagen. Restriction enzymes were purchased from
New England Biolabs Ltd. (Hitchin, United Kingdom). PCR reagents
including Pfu polymerase were obtained from Stratagene.
Protein Centriprep-concentrating devices were purchased from Millipore
(Bedford, MA). Bradford assay reagent and disposable plastic columns
were obtained from Bio-Rad. Kodak X-Omat AR was purchased from Eastman
Kodak Co. Dextran-coated CM5 sensor chips and detergent P20 were
purchased from BIAcore (Uppsala, Sweden). Suprasil cuvettes were from
Helma Kuvetten (Mulheim, Germany). All antibodies for enzyme-linked immunosorbent assay (ELISA) and Western blotting, apart from mouse anti-LcrV (Defence Science and Technology Laboratory, Wiltshire, United
Kingdom), were purchased from Sigma. Marvel dried skimmed milk powder
used as a source of casein was from Premier Beverages (Stafford, United
Kingdom). All other chemicals were purchased from Sigma.
-D-galactopyranoside to a final
concentration of 1 mM. Growth was allowed to continue for
another 4 h. Centrifugation of the culture yielded a cell pellet
that was resuspended in phosphate-buffered saline (PBS) (0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl, pH 7.4). Cells were lysed by sonication, and
insoluble material was removed by centrifugation at 12,000 × g for 45 min.
[(
exp-cal)2/(exp)2]1/2
summed over all wavelengths, where exp and cal are, respectively, the experimental ellipticities, and the ellipticities of the
back-calculated spectra for the derived structure. NRMSD values of
<0.1 mean that the back-calculated and experimental spectra are in
close agreement (35). A low NRSMD is not sufficient to indicate a
correct analysis, but a poor (high) NRMSD generally means the analysis
is not correct. In this study, all values were well below 0.1. Calculations of standard deviations between measurements were performed
at all wavelengths to determine the variation within a data set.
-mercaptoethanol (v/v)) were used to
resuspend protein samples, which were then boiled for 10 min.
10-µl samples were electrophoresed on gels in a Tris-glycine buffer
(21).
-nitrophenylphosphate in 100 mM Tris-HCl, pH
9.5, and observing the optical density measured at 405 nm after 20 min.
The mean optical density and mean ± S.D. were calculated from
triplicate experiments, and the KD was estimated
using a GST-LcrV molecular mass of 64,000 and a single-site ligand
binding model (Grafit v3.09, Erithacus Software Ltd., United Kingdom).
The interaction of GST with immobilized LcrV was measured as a control
for nonspecific binding. The association of GST-LcrV with LcrG was
assayed as just described with the exception that LcrG was immobilized
at 20 µg/ml to the ELISA plate and that subsequent incubation with GST-LcrV was performed at concentrations ranging from 0.19 to 100 µg/ml. The interaction of GST with immobilized LcrG was measured as a
control for nonspecific binding.
amino acids 10-313) (10) was grown in LB at 37 °C overnight
with 30 µg/ml kanamycin. Dilution 1:150 into 200 ml of LB with
30 µg/ml kanamycin was followed by growth at 37 °C to
A600 = 0.6. Cells were harvested, resuspended in
500 ml of distilled H20, centrifuged, resuspended in 5 ml
of 10% glycerol, centrifuged again, and finally resuspended in 400 µl of 10% glycerol. Plasmids pTrc99A, pTrc-LcrV, and
pTrc99A derivatives containing lcrV coiled-coil
mutants were transformed into competent Y. pseudotuberculosis strain YPIII (pIB19) by electroporation. For
Yop induction assays, single colonies of Y. pseudotuberculosis YPIII (pIB19) and the same strain transformed
with pTrc99A and its derivatives were each inoculated into 5 ml of calcium-depleted Brain-Heart infusion medium (BHI)
including 5 mM EGTA, 20 mM MgCl2,
and 0.1% Triton X-100 containing 30 µg/ml kanamycin and 100 µg/ml
ampicillin and grown overnight at 26 °C. A 1:20 dilution into 10 ml
of BHI was grown for 1 h at 26 °C and then for 3 h at
37 °C. Cells were harvested, and the supernatant passed through a
0.45-µm filter (Millipore). Supernatants were precipitated with 10%
trichloroacetic acid as described previously (39). The volumes of
5 × SDS sample buffer used to resuspend the protein samples
(typically ~25 µl) were calculated to normalize for small
variations in the final observed A600 values
measured from the original bacterial cultures. Proteins were separated
by 14% SDS-PAGE and visualized by Coomassie Brilliant Blue staining.
RESULTS
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DISCUSSION
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-helix in LcrG (PHDsec) forming
coiled-coil interactions was 0.806. Residues Leu-13, Ala-16, and Ile-20
were identified as being central within the region and at positions
indicative of the presence of a coiled-coil motif in this protein.
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Fig. 1.
Prediction of coiled-coil regions in both
LcrV and LcrG. The COILS program was used with a 28-residue window
size and a weighting of 2.5 toward the presence of hydrophobic residues
(boldface) at positions a and d. The
MATCHER program confirmed regions with 7-residue periodicity (indicated
by a and d), whereas the prediction of a helical
structure was carried out using PHDsec.
-helical content but
with other secondary structures present. The results reported in Table
I are for the CDSSTR secondary structure
method (27) with reference data base 4 (26), which gave the best fits
(lowest NRMSDs). However, in general, all calculations produced very
similar secondary structures. For wild-type LcrV, the calculated
secondary structure is 58% helix, 8% -sheet, 13% turn, and 22%
other compared with 56% helix, 8% -sheet, and 36% loop proposed
by secondary structure predictions (PHDsec). For wild-type LcrG, the
calculated secondary structure is 35% helix, 16% -sheet, 21%
turn, and 29% other compared with 60% helix, 0% -sheet, and 40%
loop proposed by secondary structure predictions (PHDsec). The CD
spectra of GST and GST-LcrV also exhibited features demonstrating
significant -helical content (data not shown). The former spectrum
is consistent with the known crystal structure of GST (40). The absence
of significant changes in non-helical features in the GST-LcrV spectrum
suggests that there is no gross disruption in the conformation of
either protein.
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Fig. 2.
CD spectra for wild-type LcrV, LcrG, and LcrV
mutant proteins cleaved from their GST 
moieties. Data are
represented as mean residue ellipticity and were recorded at 4 mg/ml
(LcrV, solid line) and 9 mg/ml (LcrG, dashed
line) at 0.2-nm scan steps (panel A). The error
bars in the data for repeated scans are represented only in
panel A for clarity but are representative of all data
collected. Panels B-D show a comparison of the spectra
between LcrV wild-type (solid line) and mutant (dashed
line) proteins (L157R, L153R, and L153R/L164R, respectively),
which were obtained using 4 mg/ml of each protein.
Secondary structure analysis from CD data
-helical content of these mutants tended to be lower than
that of the wild-type protein (Table I). These data provide evidence
for alterations in the secondary structure and perhaps the tertiary
structure (coiled-coil) interactions of the mutants. However, it should
be noted that none of the mutant structures was radically altered in
the sense of producing unfolded proteins as a consequence of the
replacement of leucine with arginine residues in the predicted
coiled-coil motif.
A summary of information obtained for LcrV wild type and mutant
proteins
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Fig. 3.
Native gel analysis of LcrV and LcrG.
LcrV migrates as a mixture of dimeric and multimeric species (1). Upon
the addition of LcrG (2), a downshift in the LcrV dimer band occurs
(3). An estimated 2:1 molar ratio of LcrG was added to 4 mg/ml LcrV,
incubated at 4 °C for 30 min, and run on an 8-25% native PAGE gel
using the Phast system. Native molecular weight markers (4) used were
thyroglobulin (669,000), ferritin (440,000), catalase (232,000),
lactate dehydrogenase (140,000), and albumin (66,000).
2 = 1.15;
ka = 4.38+3 1/ms, kd = 5.41
4 1/s), similar to that obtained using ELISA methods.
The reliability of the data was reinforced by plotting the
response unit values at equilibrium against ligand concentration, which
generated a binding curve reaching saturation at higher concentrations.
A fit to this data gave an estimated KD = 135 nM (2 = 0.0435). Inverting the system by
immobilizing LcrV to the CM5 dextran chip to follow LcrG binding was
also attempted. Although the small size of LcrG (11 kDa) meant that the
observed relative unit levels were only measurable at high
concentrations of LcrG, an estimated KD = 544 nM (2 = 0.154) was obtained. This value is
comparable with those obtained from ELISA assays and SPR methods as
already described.
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Fig. 4.
Binding assays of LcrV interactions.
A, the interaction of LcrV with LcrG. LcrG was immobilized
to the ELISA plate, and titration with LcrV was shown to bind using
mouse anti-LcrV monoclonal antibody and alkaline phosphatase-conjugated
anti-mouse antibody ( 
). The KD
for the interaction was determined as 208 ± 32 nM. The
binding of LcrV to the ELISA plate was used as a control (
).
B, the interaction of GST-LcrV with LcrV. LcrV was
immobilized to the ELISA plate, and titration with Gst-LcrV was shown
to bind using anti-GST IgG and alkaline phosphatase-conjugated
anti-rabbit antibody (). The KD
for the interaction was determined as 1.13 ± 0.11 µM. The binding of GST to immobilized LcrV was used as a
control (). C and D, binding of LcrV mutants
to immobilized LcrG. These mutants showed lower affinity for LcrG than
wild-type LcrV. E, binding of two anomalous LcrV mutants to
immobilized LcrG. These mutants have an affinity for LcrG equivalent to
or greater than that of wild-type LcrV.
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Fig. 5.
LcrV binding to LcrG demonstrated by surface
plasmon resonance. LcrG was immobilized to a CM5 dextran chip by
amine coupling. Inset, injection of 0.1-1.2
µM wild-type and mutant LcrVs in HEPES-buffered saline
was performed at a flow rate of 20 µl/min, and the binding was
measured as response units over time. A simple 1:1 Langmuir binding
model was used to fit the data and gave an estimated
KD of 1.40 × 10 7 M
(2 = 1.15). (main figure) Data reliability
was confirmed by plotting the response unit values at equilibrium
(Req) against ligand concentration. A steady-state affinity
fit to this data gave an estimated LcrV·LcrG KD of
1.35 × 107 M (2 = 0.0435).
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
0.1), which suggest that a reasonable
correspondence exists between calculated secondary structure and the
experimentally obtained spectra (Table I). Indeed these values for
predicted CD calculated structures are very close for LcrV. However,
the accuracy of any secondary structure determined is dependent upon
the reference databases used in the analyses being constructed from
proteins containing the structural features present in the protein
under investigation. As none of the existing reference databases
include examples of coiled-coiled structures, if LcrV does contain a
coiled-coil structure, the analyses may not be completely accurate in
absolute terms. It seems probable that a coiled-coil motif could be
present in the proteins studied based on one of the characteristics in
the spectra: the ratio of the 208-nm peak relative to 222-nm peak. In
the spectra of ordinary -helices, these two peaks are of roughly the
same intensity, but this is not true for the LcrV spectra in this study (Table II). It has been suggested that differences in the 208/222 ratio
could be the consequence of helix-helix interactions (45-47), so this
may provide some support for different extents of coiled-coil being
present in these proteins (Table II).
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Fig. 6.
Interactions inside the Ysc pore of
Yersinia. A, Yop secretion is repressed by
gating of the Ysc apparatus (light blue) externally by LcrE
(navy blue), and internally by LcrG (green). It
is postulated that LcrG associates with the Ysc components
(curved line). B, secretion is induced by the
loss of LcrE gating and an increase in the cytoplasmic concentration of
LcrV (red). The stable cytoplasmic LcrV dimer dissociates
and binds to LcrG with high affinity (solid line).
C, secretion is facilitated by the removal of LcrG because
of LcrV-LcrG heterodimer formation permitting the release of Yops
(purple).
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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