Yersinia
pestis
TIR-domain
protein
forms
dimers that interact with the human adaptor
protein MyD88
Rohini R. Rana1, Peter Simpson1, Minghao Zhang1, Matthew Jennions3,
Chimaka Ukegbu1, Abigail M. Spear2, Yilmaz Alguel1, Stephen J.
Matthews1, Helen S. Atkins2 and Bernadette Byrne1*
Division of Molecular Biosciences, Imperial College London, London SW7 2AZ,
UK1, Department of Biomedical Sciences, Defence Science and Technology
Laboratory, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK2 and Membrane Protein
Laboratory, Diamond Light Source, Harwell Science and Innovation Campus,
Chilton, Didcot, Oxfordshire OX11 0DE, UK3.
*Corresponding
Dr Bernadette Byrne,
Division of Molecular Biosciences
Imperial College London
South Kensington
London
SW7 2AZ.
Fax: +44 20 7594 3022
E-mail: b.byrne@imperial.ac.uk
1
@ Crown Copyright 2010. Published with the permission of the Defence Science and
Technology Laboratory on behalf of the Controller of HMSO.
Abstract
Recent research has highlighted the presence of Toll/Interleukin 1 receptor (TIR)domain proteins (Tdps) in a range of bacteria, suggested to form interactions with the
human adaptor protein MyD88 and inhibit intracellular signaling from Toll-like
receptors (TLRs). A Tdp has been identified in Yersinia pestis (YpTdp), a highly
pathogenic bacterium responsible for plague. Expression of a number of YpTIR
constructs of differing lengths (YpTIR1, S130-A285; YpTIR2, I137-I273; YpTIR3,
I137-246; YpTIR4, D107-S281) as fusions with an N-terminal GB1 tag (the B1
immunoglobulin domain of Streptococcal protein G) yielded high levels of soluble
protein. Subsequent purification yielded 4-6 mg/L pure, folded protein. Thrombin
cleavage allowed separation of the GB1 tag from YpTIR4 resulting in folded protein
after
cleavage.
Nuclear
magnetic
resonance
spectroscopy,
size
exclusion
chromatography, SDS-PAGE analysis and static light scattering all indicate that the
YpTIR forms dimers. Generation of a double Cys-less mutant resulted in an unstable
protein containing mainly monomers indicating the importance of disulphide bonds in
dimer formation. In addition, the YpTIR constructs have been shown to interact with
the human adaptor protein MyD88 using 2D NMR and GST pull down. YpTIR is an
excellent candidate for further study of the mechanism of action of pathogenic
bacterial Tdps.
2
Keywords: Innate immune evasion; TIR domain protein; Yersinia pestis; pathogenic;
MyD88
1. Introduction
Toll-like receptors (TLRs) are key components of the innate immune system, which
detect a range of pathogen associated molecular patterns (PAMPs) including
lipopolysaccharide (LPS), bacterial cell wall components and nucleic acids [1], and
initiate the first line of host defence against infection. TLRs have a conserved domain
architecture comprised of a large extracellularly located leucine rich repeat (LRR)
domain [2] and an intracellular Toll/Interleukin 1 receptor (TIR) domain [3] linked by
a single pass transmembrane region. TLRs are suggested to exist as dimers, which can
be either heterotypic, homotypic or both depending on the receptor [4,5]. Upon
interaction with a PAMP the TLR dimer is thought to undergo a molecular
rearrangement of the intracellular TIR domains to generate an active interaction
domain [4,5,6] allowing recruitment of intracellular adaptor proteins; MyD88,
MyD88 adaptor like (MAL, also called TIRAP), TIR-domain-containing adaptor
protein inducing IFN- (TRIF), TRIF-related adaptor molecule (TRAM) and sterile
- and armadillo-motif containing protein (SARM) [7]. All of the adaptors also
contain TIR domains and in some cases other protein-protein interaction domains
important in signal transduction [8].
Broadly there are two signal transduction pathways, the MyD88 dependent
pathway utilized by all TLRs except TLR3 and the MyD88 independent pathway
utilized by TLR3 and also TLR4 [7,9]. The recruitment of adaptor proteins initiates
the complex signaling cascades which upregulate gene expression of nuclear factor
3
kappa B (NF-B) and interferon response factor (IRF) controlled genes. This
ultimately initiates
an
innate
immune
response
through
the
release
of
proinflammatory cytokines [7].
Key components of the signaling cascade are the heterotypic interactions
between the TIR domains upon receptor activation and adaptor recruitment. The
importance of the TIR domains in the innate immune response has made them the
subject of intense study. The structures of the monomeric forms of the TIR domains
of TLR1 and TLR2 [10] revealed a conserved architecture comprised of a central
five-stranded parallel -sheet surrounded by five -helices. These structures
highlighted the presence of a flexible region, the BB loop connecting strand B and
helix B, which projects away from the main TIR domain. Mutational analysis
revealed that several residues within this loop have important roles in signal
transduction [10], most notably a proline residue. Mutation of the equivalent residue,
Pro712, to histidine in TLR4-TIR results in mice unable to initiate the innate immune
response upon stimulation with LPS [11]. The BB loop also plays a role in mediating
the interactions between the monomers of the dimer of TLR10-TIR [12]. The recent
NMR structure of the adaptor MyD88-TIR domain confirmed the conserved
architecture of the TIR domain family and the flexible nature of the BB loop [13].
Bioinformatics analysis has identified TIR containing proteins in a range of
both pathogenic and non-pathogenic bacteria. The first studied example of such a
protein was the TIR-like protein A (TlpA) identified in Salmonella enterica serovar
Enteridis [14]. In vitro analysis showed that this protein was able to reduce the ability
of TLR4 and MyD88 to stimulate NF-B activity. Furthermore bacteria with
disrupted TlpA genes exhibited reduced pathogenicity in mice, suggesting that TlpA is
4
an important virulence factor. These data indicated that the bacterial TIR-like proteins
were likely to have a role in evasion of the innate immune system. Studies on
equivalent proteins, from Brucella abortus (Btp1), the uropathogenic Escherichia coli
strain CFT073 (TcpC) and Brucella melintensis (TcpB) have supported a role in
immune system evasion [15,16,17].
A recent in-depth bioinformatic analysis [18] identified 922 TIR-domain
proteins in a range of fungi, archaea, viruses and pathogenic and non-pathogenic
bacteria. The high incidence of these proteins in non-pathogenic species suggests that
these proteins may have a range of functions, including evasion of the innate immune
system, depending on the organism in question. However it has been shown that the
TIR-domain protein from the non-pathogenic thermophilic bacterium, Paracoccus
dentrificans, PdTLP interacts with the human adaptor protein MyD88 in vitro [19,20].
In addition, the crystal structure of PdTIR displays a similar fold to the known human
TIR domains [20]. Given the benign nature of this organism it would seem unlikely
that this protein has a role in innate immune system evasion, therefore making it of
limited use as a model system for studying the interactions between bacterial TIR
proteins and human TIR proteins. Further work is required to reveal the precise
function of PdTIR.
Our bioinformatic analysis [18] identified a TIR domain protein in the
pathogenic bacterium, Yersina pestis (YpTdp). Y. pestis is a facultative intracellular
bacterium that causes the zoonotic diseases bubonic and pneumonic plague [21]. The
main reservoirs of the disease are rodents, birds, farm animals and their associated
fleas and the disease is transmitted to humans via flea or animal bites. Most famously
associated with the Black Death pandemics of the Middle Ages, Y. pestis continues to
cause a significant number of deaths every year mainly in Africa and Asia [22,23].
5
YpTdp is a 41 kDa protein containing five cysteines. Other work in our group has
shown that when over-expressed in vitro YpTdp is able to disrupt immune
signalling pathways but that its removal has no obvious effect on virulence and
instead affects the characteristics of Yersinia pestis growth (Spear at al,
manuscript submitted). Additionally, microarray studies have shown that the gene
encoding YpTdp is expressed in vitro in various conditions [24,25]. In order to
understand more about the role of YpTdp in immune system evasion, we have
attempted to produce the TIR domain of YpTdp (YpTIR) with a view to obtaining
high quality samples for functional and structural studies. Here, we have expressed
and purified YpTIR as a fusion with an N-terminal GB1 tag. The resulting protein is
folded and exists in solution as dimer that interacts with the TIR domain of human
adaptor protein MyD88.
2. Results
2.1 Expression and purification of GB1-tagged YpTIR constructs
The region of the YpTdp containing the TIR domain (YpTIR1; residues S130 to
A285) was estimated based on sequence alignments with TIR domains of known
structure (Figure 1). A construct based on this region and two shorter YpTIR
constructs, corresponding to residues I137-I273 (YpTIR2) and I137-N246 (YpTIR3),
were generated. YpTIR3 lacks the region of the protein corresponding to the Box 3
motif (Figure 1). All three gene fragments were cloned into the GEV2 vector as a
fusion with an N-terminal GB1 tag (the B1 immunoglobulin domain of Streptococcal
protein G) [26] and a C-terminal His tag. High-level expression was achieved for
GB1-YpTIR1 and GB1-YpTIR2 but not for GB1-YpTIR3. Affinity chromatography
followed by size exclusion chromatography (SEC) of all three constructs produced
6
highly pure protein although the final yield of GB1-YpTIR3 was extremely low
(Figure 2ab). The final yields of pure protein obtained were 5.7, 4.6 and 0.6 mg/L for
GB1-YpTIR1, GB1-YpTIR2 and GB1-YpTIR3 respectively. The size exclusion
profiles of GB1-YpTIR1 and GB1-YpTIR2 indicated that both proteins were
monodispersed (Figure 2a). However in both cases the protein had a lower retention
volume (~14 ml) than expected for a ~25 kDa protein suggesting that the protein was
present in a higher oligomeric form, possibly a dimer. The GB1-YpTIR3 eluted as a
broad peak from the SEC column suggesting that this protein was aggregating
possibly as the result of partial unfolding. The high yield, purity and quality (Figure
2ab) of GB1-YpTIR1 and GB1-YpTIR2 allowed further analysis of the proteins.
2.2 Removal of the GB1 tag
Whilst the expression and isolation of the GB1 tagged constructs is useful, it is
important to cleave the GB1 tag for certain downstream applications including some
structural studies. Attempts to cleave the GB1 tag from GB1-YpTIR1 and GB1YpTIR2 were unsuccessful even after extended periods of incubation with high
concentrations of thrombin protease, probably as the result of close association of the
GB1 and YpTIR domains. In order to allow efficient thrombin cleavage a further
longer YpTIR (GB1-YpTIR4, residues D107-S281) construct was designed. This was
also cloned into GEV2 and expressed and purified as described in Section 2.1 (Figure
2cd). Cleavage was followed by Co2+-IMAC to separate the His-tagged YpTIR4 from
the GB1 tag resulting in pure, cleaved protein with a yield of 2.5 mg/L.
2.3 YpTIR is a dimer in solution
In order to assess the folded state of the YpTIR constructs prior to further studies, the
GB1-YpTIR protein constructs were analysed using 1D NMR spectroscopy. All GB1YpTIR constructs displayed folded domains in addition to GB1 (Figure 3). YpTIR4
7
after thrombin cleavage also displayed folded domains. Resonance linewidths from
the 1D and 2D 1H-15N HSQC NMR spectra of 15N-GB1-YpTIR1 (Figure 3) strongly
suggest that the protein is dimeric, as suggested by SEC. This was further supported
by analysis using a SEC column together with static light scattering (SLS) which
indicated that the approximate molecular weights of the different YpTIR proteins
were ~40-54 kDa. The measured molecular weight of 40.9 kDa for YpTIR4
(predicted molecular weight of the monomer = 20.8 kDa) following removal of the
GB1 tag (Figure 4) strongly indicated that dimer formation is mediated through the
YpTIR domain. The YpTIR contains two Cys residues; Cys90 and Cys132. In order
to investigate the role of these residues in the formation of a stable dimeric protein we
generated both single and a double Cys-less mutant. All mutants were expressed and
isolated as GB1-tag fusion proteins and were shown to be folded by 1D NMR (data
not shown). SEC-SLS analysis of the Cys90Ser and Cys132Ser single mutants
indicated that both had the same molecular weight as the wild-type YpTIR (Fig 4) and
thus were in the dimeric form. The double Cys-less mutant resulted in a much less
stable protein. Although it proved impossible to obtain an accurate molecular weight
for this protein the SEC-SLS indicated that the protein sample contained mainly
monomers (data not shown).
2.4 Interaction of YpTIR with the human adaptor protein MyD88
The 2D NMR spectra of
15
N-GB1-YpTIR1 revealed the presence of peaks
corresponding to amino acid residues in addition to GB1 in the folded regions of the
spectrum (> 9 ppm). These additional peaks were observed to be significantly broader
than expected for a monomer of YpTIR1, presumably as a result of the protein
tumbling as a dimer (Figure 5a). Upon addition of unlabelled GB1-MyD88-TIR,
specific broadening of signals corresponding to structured regions of the YpTIR
8
domain was observed, whilst those from GB1 were unaffected (Figure 5b). This
strongly suggests that MyD88-TIR binds to the YpTIR domain, causing signal
broadening presumably as a result of the increased tumbling time and/or a binding
regime that is intermediate on the NMR timescale. The interaction between the
YpTIR constructs and MyD88 was also assessed by pull down using GST-MyD88 as
bait and GB1-YpTIR2 and YpTIR4 following removal of the GB1 tag as prey. Both
proteins specifically interact with GST-MyD88-TIR as shown in Figure 5cd.
Interestingly the uncleaved GB1-YpTIR4 interacts non-specifically with the
glutathione resin (Figure 5c) while the Cys132Ser mutant does not interact at all
(Figure 5e).
3. Discussion
This study describes a biophysical and functional characterization of a TIR domain
from the highly pathogenic bacterium Y. pestis. One of the key aims was to generate
constructs that formed a compact stable structure suitable for downstream structural
and functional studies. Expression of GB1-YpTIR1 and GB1-YpTIR2 yielded high
levels of soluble protein, which could be readily isolated to high homogeneity in a
folded and functional state. Interestingly the shortest construct, GB1-YpTIR3,
expressed poorly and yielded low levels of polydispersed protein. YpTIR3 is
truncated at residue N246 removing the TIR motif sequence Box 3 (Figure 1), which
is likely to be important for maintaining the overall fold of the YpTIR.
SEC analysis of the GB1-YpTIR1 and GB1-YpTIR2 suggested a shorter
retention time than would be expected for the ~25 kDa sized monomer and indicated
that the proteins were likely to be dimeric. This was supported by NMR spectra
displaying broader than expected peaks. The dimeric status of YpTIR was confirmed
9
by the SEC-SLS. This is a feature unique to YpTIR as the other TIR domains are
present as monomers in solution [13,19]. Mutating the two Cys residues, Cys90 and
Cys132, individually to Ser had no effect on the dimeric status of YpTIR. In contrast,
while it was difficult to obtain precise data on the molecular weight of the Cys-less
mutant, it was present mainly as monomers. These results suggest that mutation of
one Cys is tolerated however the mutation of both Cys residues results in the loss of
the stable quaternary structure and indicates that disulphide bridges have a key role in
mediating dimer formation.
It is not clear what the precise molecular arrangement of the Cys residues is
within the protein. If Cys132 forms disulphide bonds with Cys90 then it could be
expected that the mutation of one or the other Cys residue would result in the
monomeric form of the protein. However since both single mutants retain the dimeric
form this suggests that Cys90 of monomer 1 forms a disulphide bond with Cys90 of
monomer 2 and Cys132 of monomer 1 forms a disulpide bond with Cys132 of
monomer 2. The mutation of Cys132 to Ser however does result in a loss of
interaction between YpTIR and MyD88. This suggests that whilst one disulphide
bridge may be sufficient to maintain the dimer, both are required to maintain the
functional conformation of the protein. A high resolution structure of YpTIR will
shed light on these issues.
Although YpTIR is the only TIR domain so far reported to form dimers in
solution [13,19] there are several crystallographic structures of TIR domains in the
form of dimers. The structure of the TIR domain of TLR10 revealed that the BB loop
is key in mediating the interaction between monomers [12]. In contrast, the structure
of the TIR domain from Paracoccus denitrificans [20] revealed that residues from the
both DD and EE loops (Figure 1) mediate the interaction between monomers. In
10
addition, the crystal structures of the TIR domains of TLR 1 and 2 [10] reveal the
formation of dimers stabilized by disulphide bridges. Initially it was suggested that
these dimers were artifacts of the crystallization process however the subsequent data
from the crystal structures of the Cys713Ser mutant of the TLR2-TIR and IL-1RAPL
also revealed the formation of disulphide linkages potentially important in dimer
formation [27,28]. The data presented here on YpTIR, together with the known
structures, indicates that the dimeric form may be a common feature for TIR proteins.
Functional characterization of the GB1-YpTIR1 was carried out by 2D NMR
analysis using
15
N-GB1-YpTIR1 in the presence and absence of unlabeled GB1-
MyD88-TIR. The observed peak broadening for certain residues of the YpTIR
domain indicates an interaction between the two proteins. This is supported by GSTpull down assays demonstrating that the GB1-YpTIR2 specifically interacts with
human GST-MyD88-TIR. However NMR titration studies were complicated by the
fact that the YpTIR exists as a dimer, precluding facile resonance assignment for
chemical shift mapping. GST pull down analysis also revealed an interaction between
GST-MyD88-TIR and YpTIR4 following thrombin cleavage indicating that the
interaction is not mediated by the GB1 tag.
In conclusion we have generated a series of highly expressing soluble, stable
and folded YpTIR constructs. We have obtained functional YpTIR, which is uniquely
dimeric in solution compared to the known TIR domains. YpTIR is an excellent
candidate for further study of the mechanism of action of pathogenic bacterial Tdps.
4 Materials and Methods
4.1 Materials
11
Oligonucleotide primers were obtained from MWG Biotech, the GEV2 expression
vector was obtained from Addgene. Co2+-IMAC resin was obtained from Clontech
Laboratories. The Superdex 200 10/300 GL column, thrombin protease and ECF
reagent were purchased from GE Life Sciences. Protease inhibitor tablets were
obtained from Roche. -mercaptoethanol (BME) and anti-His antibodies from Sigma.
Anti-GST antibodies were obtained from Merck. The Bradford Protein Assay kit for
protein concentration determination and the Imperial Protein Stain for Coomassie
staining were purchased from Pierce. SDS-PAGE gels and BL21 (DE3) Star™ cells
were purchased from Invitrogen. Molecular weight cut-off (MWCO) filters were
purchased from Millipore. The QuikChange kit was obtained from Stratagene.
4.2 Generation of expression constructs
The oligonucleotide primers used for generating the different constructs are detailed
in Table 1 (need to add in the mutagenic oligos). In each case a 5’ XhoI site and a 3’
BamHI site were incorporated to the target gene sequence. The region of the YpTdp
containing the TIR domain (YpTIR1; residues S130 to A285) was estimated based on
sequence alignments with TIR domains of known structure (Figure 1). For this work,
the putative YpTdp gene in Y. pestis strain CO92 (YPO1883) was selected. Two
shorter YpTIR constructs were also generated, corresponding to residues I137-I273
(YpTIR2) and I137-N246 (YpTIR3) and a longer construct corresponding to residues
D107-S281 (YpTIR4). YpTIR3 lacks the region of the protein corresponding to the
Box 3 motif (Figure 1). All four constructs were cloned into GEV2 [26]. The region
of the human MyD88 gene encoding for the core TIR domain ([13], residues F163P296) was also cloned into GEV2. The GB1-YpTIR2 Cys90Ser, Cys132Ser and
double mutants were generated using the QuikChange mutagenesis kit according to
12
manufacturers instructions. The presence of the mutations was confirmed by DNA
sequencing and the proteins expressed and purified as described above. The GSTMyD88 expression vector was generated by cloning the region of the human MyD88
gene encoding for the core TIR domain ([13], residues F163-P296) into pGEX5.
4.3 Expression and purification of YpTIR and MyD88 constructs
The YpTIR and MyD88-TIR constructs were individually expressed in BL21 (DE3)
Star™ E. coli cells using optimized induction conditions (1 mM IPTG at 25 °C for 4
hr) in 1 L cultures of LB. The cells were lysed by sonication and following
centrifugation to removal the insoluble material, the soluble fraction was applied to
Co2+-IMAC resin (1 ml/ 1 L prep) equilibrated with Buffer A [20 mM Tris-HCl, pH
7.0, 250 mM NaCl, 2 mM BME, protease inhibitors]. The protein was batch bound
for 1 hr at 4 C. The resin was washed with 10 CVs of Buffer A and the bound
proteins eluted with Buffer A supplemented with 150 mM imidazole. The eluted
protein was exchanged into Buffer B [20mM Tris-HCl, 150 mM NaCl, pH 7.0] on a
Superdex 200 10/300 GL column. Buffer B was supplemented with 10 mM DTT for
GB1-MyD88-TIR in order keep the sample in its reduced monomeric form. The GB1tagged proteins were concentrated to 10 mg/ml using a 10 kDa MWCO filter. GB1YpTIR4 was isolated as described above and the pure protein incubated with 2.5 units
of thrombin protease per 100 g for 16 hr at 18 °C. The digested protein was then
separated from the GB1 tag by a further Co2+-IMAC step as described above. All
proteins were analysed by SDS-PAGE by separation on a 4-12% Bis-Tris NuPAGE
gels. The protein bands were either directly visualised using Imperial Protein Stain or
13
detected by ECF reagent following transfer to an immobilized membrane and
incubation with either anti-His or anti-GST primary antibodies.
4.4 Protein concentration determination
Protein concentration was determined using the Bradford Protein Assay and the UV
absorption method [29]. Bovine serum albumin (BSA) was used as a protein standard
for the Bradford Assay.
4.5 Determination of the oligomeric state of YpTIR
The oligomeric state of the GB1-YpTIR constructs at a detected protein concentration
of 1.4 mg/ml was assessed with a Malvern Viscotek TDAmax Tetra detection system,
including static light scattering, UV and refractive index detectors, connected
downstream of a Superdex-200 10/30 gel-filtration column previously equilibrated in
Buffer B. The data was analysed with the Omnisec software (Malvern) following the
manufacturers protocols.
4.5 1D/2D NMR analysis of pure protein
10% D2O was added to all the unlabeled and labeled pure protein samples prior to
recording NMR spectra for spectrometer field lock. Experiments were recorded using
either a Bruker Avance III 600 (at 600 MHz) spectrometer at 303 K. Experiments
were performed using the TopSpin software package. For 2D NMR analysis GB1YpTIR1 was labeled with the 15N isotope by expressing the protein in minimal (M9)
media supplemented with 15N-labeled NH4Cl (0.07%). Expression and purification of
the labeled protein was as described for the unlabeled proteins. All samples were
14
prepared in 20mM Tris-HCl, 150 mM NaCl, 10 mM DTT, pH 7.0 for 2D NMR. 2D
spectra were processed with NMRPipe [30]. Spectral manipulation and layout was
attained using NMRView (One Moon Scientific).
5. Acknowledgements
We thank Dr Tom Monie and Prof Nick Gay, University of Cambridge for helpful
discussions and advice. This work was funded by the UK Ministry of Defence.
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Figure legends
Figure 1 Alignment of YpTIR1 (residues S130-A285) with other TIR domains of
known structure. The regions corresponding to the TIR motifs, Box1, 2 and 3, are
denoted by the solid rectangles above the alignment. The regions of the sequence of
TLR1-TIR corresponding to secondary structure elements as determined by X-ray
crystallographic analysis (Pdb accession code: 1FYV) are indicated below the
alignment. The alignment was generated using ClustalW and Seaview [31].
Figure 2 (a) SEC of the GB1-YpTIR1, 2 and 3 constructs and (b) SDS-PAGE
analysis of the protein following SEC. M indicates molecular weight markers and
lanes 1, 2 and 3 contain GB1-YpTIR1, GB1-YpTIR2 and GB1-YpTIR3 respectively.
SEC (c) and SDS-PAGE analysis (d) of the GB1-YpTIR4 before (lane 4) and after
(lane 5) thrombin cleavage.
Figure 3 1D NMR analysis of (a) GB1-YpTIR1 (b) GB1-YpTIR2 (c) GB1-YpTIR4
and (d) YpTIR4 (after thrombin cleavage). The arrows indicate the peaks
corresponding to folded YpTIR protein.
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Figure 4 Representative static light scattering measurements of GB1-YpTIR1 and
cleaved YpTIR4. The data from all constructs tested; GB1-YpTIR1, GB1-YpTIR2,
GB1-YpTIR4 and cleaved YpTIR4, indicate the presence of dimers (table inset). The
measurements from the refractive index (RI) detector for GB1-YpTIR1 and cleaved
YpTIR4 are indicated in dotted and dashed lines respectively. The data for the
measured weight-average molecular weights for GB1-YpTIR1 and cleaved YpTIR4
are shown in the solid and broken lines respectively. The measured molecular weights
for GB1-YpTIR1 and cleaved YpTIR4 are 50.7 and 40.9 kDa respectively. The
predicted molecular weights of the monomeric forms of GB1-YpTIR1 and cleaved
YpTIR4 are 25.3 and 20.8 kDa respectively.
Figure 5 Analysis of the interaction between YpTIR and MyD88 using 2D (1H/15N)
HSQC NMR spectroscopy and GST pull down. The 2D (1H/15N) TROSY-type HSQC
spectra of 15N-GB1-YpTIR1 were recorded (a) in the absence and (b) presence of 1 M
equivalent of unlabeled GB1-MyD88-TIR. Peaks arising from GB1 are boxed (as
judged from the spectrum of the free domain, data not shown) and some structured
amide peaks from GB1-YpTIR1, which are broadened upon GB1-MyD88-TIR
addition are indicated with arrows. Western blot analysis of samples obtained from
GST pull down experiments revealed (c-d) a positive and specific interaction of GB1YpTIR2-His and cleaved GB1-YpTIR4-His with GST-MyD88-TIR and (c,e) a lack of
specific interaction of GB1-YpTIR4-His and the C132S mutant form of GB1YpTIR2-His with GST-MyD88-TIR. GST-MyD88-TIR in the eluate fraction from
one of the GST pull down experiments with GB1-YpTIR2-His is indicated in (f).
Samples were probed with (c-e) anti-His antibody in order to detect the wild-type and
mutant forms of GB1-YpTIR-His and (f) anti-GST antibody in order to detect GSTMyD88-TIR.
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Figure 1
Figure 2
Figure 3
Figure 4
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Figure 5
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