Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten
Trang 1is a natively partially folded protein
Daizo Hamada1, Tomoaki Kato1,2, Takahisa Ikegami3, Kayo N Suzuki1, Makoto Hayashi2,
Yoshikatsu Murooka2, Takeshi Honda4and Itaru Yanagihara1
1 Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, Japan
2 Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan
3 Laboratory of Structural Proteomics, Institute for Protein Research, Osaka University, Japan
4 Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Japan
Several bacteria, including enterohaemorrhagic and
enteropathogenic Escherichia coli (EHEC and EPEC,
respectively), express type III secretion systems [1]
consisting of various proteins encoded at the genetic
locus of enterocyte effacement [2–5] To date, type III
secretion systems have been identified in more than
20 pathogenic bacterial species [6] Type III secretion
systems are multiprotein complexes that span the
bacterial and host membranes, permitting the direct delivery of effector proteins, such as the EPEC pro-teins [7], Tir [8–10], EspF [11,12], EspG [13] and Orf19 [14] In the case of EHEC and EPEC, such complexes are formed by proteins including EspA, EspB and EspD [15,16] Thus, the type III system regulates effector secretion and delivery into host cells
Keywords
circular dichroism; natively partially folded
proteins; nuclear magnetic resonance;
fluorescence quenching; multiangle laser
light scattering
Correspondence
I Yanagihara, Department of Developmental
Infectious Diseases, Research Institute,
Osaka Medical Center for Maternal and
Child Health, 840 Murodo, Izumi, Osaka
594-1011, Japan
Fax: +81 725 57 3021
Tel: +81 725 56 1220 (ext 5302)
E-mail: itaruy@mch.pref.osaka.jp
(Received 20 August 2004, revised 17
November 2004, accepted 2 December
2004)
doi:10.1111/j.1742-4658.2004.04513.x
The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized Far-UV and near-UV
CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes a-helical structures and that some tyrosine tertiary contacts may exist All tyrosine side-chains are exposed to water, as determined by acryl-amide fluorescence quenching spectroscopy An increase in the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at
pH 7.0 These data suggest the formation of a molten globule state at
pH 2.0 Destabilization of EspB at low pH was shown by urea-unfolding transitions, monitored by far-UV CD spectroscopy The result from a sedi-mentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation The chemical shift patterns of EspB
15N-1H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea-unfolded EspB Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules This structural feature of EspB may be advan-tageous when EspB interacts with various biomolecules during the bacterial infection of host cells
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; HSQC, heteronuclear single quantum correlation; LB, Luria–Bertani.
Trang 2EspB is an E coli type III system protein that
inter-acts with various biomolecules For example, EspB
binds to EspD, forming a pore complex of 3–5 nm
diameter in the host cell membrane [17] The
N-ter-minal region of EspB also binds to host cell a-catenin
and inhibits F-actin accumulation at adherence sites
[18] It has been recently shown that a1-antitrypsin, a
host cellular protein, binds to and interferes with the
function of EspB [19] Moreover, EspB may bind to
the external end of the filamentous apparatus formed
by EspA proteins [20] The filamentous apparatus is
characteristic of type III secretion systems [21,22]
Fila-mentous EspA may form a conduit for translocation
of bacterial effector proteins into host cells [23] It has
been suggested that EspA filaments attach to host cells
via EspB⁄ D pore complexes and that the pore complex
also interacts specifically with the host protein,
a-cate-nin [16] However, other studies have demonstrated
that EspB is not required for the interaction of EspA
with host cells [20]
Although the precise functions of EspB during
bac-terial infection are still somewhat ambiguous, the
information discussed above indicates that EspB is a
multifunctional protein with the potential to interact
with various biological molecules Knowledge of the
conformational properties of EspB may clarify the role
of EspB in bacterial attachment, but no information
about the structural properties of EspB is currently
available
In this study, we characterized the conformational
properties of EspB in solution by using several
spectro-scopic and hydrodynamic techniques, including CD,
8-anilinonaphthalene-1-sulfonate (ANS) binding,
ultra-centrifugation, multiangle laser light scattering and
heteronuclear NMR The results of our analyses allow
us to understand the conformational property of EspB
and predict its role in bacterial infection to the host cell
Results
CD
The secondary structure of EspB, predicted from its
amino acid sequence by using the PredictProtein server
[24–26], indicates that the protein is predominantly
a-helical (Fig 1) As stated in the Experimental
proce-dures, the recombinant EspB was purified from both
soluble and insoluble fractions of cell lysates At
pH 7.0 and at a temperature of 20C, recombinant
EspB prepared from the insoluble fraction showed a
far-UV CD spectrum equivalent to EspB prepared
from the soluble fraction This suggests that both
purification procedures adequately yielded the native
conformation of EspB The CD spectra are typical for the presence of a-helices (Fig 2) However, the a-heli-cal content estimated from far-UV CD data is 23%,
Fig 1 Secondary structure prediction of EspB derived from its amino acid sequence H and E refer to a-helical and b-strand struc-tures, respectively The data were obtained by using the Predict-Protein server [24,25].
Fig 2 CD spectra of EspB (A) Far-UV and (B) near-UV CD spectra
of recombinant EspB purified from the insoluble fraction at pH 2.0 (dashed lines) and 7.0 (solid lines), and from the soluble fraction at
pH 7.0 (s) (C) The dependence of the ellipticity, at 222 nm, on pH.
Trang 3which is substantially less than the predicted amount
(76.3%; Table 1)
The near-UV CD spectrum of EspB at pH 7.0 and
20C shows a minimum at around 280 nm It is in the
near-UV spectrum that aromatic residues display
opti-cal activity EspB contains three tyrosines at positions
66, 75 and 212, and no tryptophans Therefore, the
shape of the near-UV CD spectrum of EspB implies
the presence of some tertiary contacts involving at
least one of the tyrosines, although the intensity of
each peak is not very high
To gain further insight into the conformational
prop-erties of EspB, we recorded the far-UV CD spectrum
of recombinant EspB prepared by different protocols
between pH 1.0 and pH 7.0 (Fig 2C) Interestingly,
irrespective of the preparation procedures and pH
con-ditions, these far-UV CD spectra are almost identical
Therefore, the amount of secondary structure seems to
be virtually same at each pH value (Fig 2 and
Table 1) On the other hand, the near-UV CD
spec-trum at pH 2.0 showed a less intense signal at 280 nm
relative to the spectrum at pH 7.0, suggesting
destabili-zation of tertiary interactions upon decreasing pH
Owing to the small difference observed here between
the recombinant proteins prepared by the different
procedures, we mostly used the EspB prepared from
insoluble fraction because the purification yield was
much higher
Quenching of protein tyrosine fluorescence
by acrylamide
A fluorescence spectrum of intrinsic tryptophan and
tyrosine residues in proteins can be a good
conforma-tional probe In particular, the fluorescence quenching
effect by small chemicals such as acrylamide provides
information on the solvent-exposure of aromatic side-chains in proteins
As mentioned above, EspB contains only three tyro-sines and no tryptophan The quenching effect of acryl-amide on the fluorescence of these EspB tyrosine side-chains at pH 7.0 was analyzed Interestingly, a plot of F0⁄ Fobs vs [Q] (Stern–Volmer plot [27]), where
F0 and Fobs are the fluorescence intensities in the absence and presence of quencher, respectively, and [Q]
is the concentration of quencher, shows a positive devi-ation from linearity at high acrylamide concentrdevi-ations (Fig 3) Therefore, the quenching behavior does not follow the simple Stern–Volmer equation (F0⁄ Fobs¼
1 + Ksv [Q]) This finding suggests that the tyrosine residues in EspB behave as independent fluorophores, each having their own Ksv value Additional informa-tion was obtained by analyzing the data using the following modified Stern–Volmer equation [28]:
Table 1 Secondary structure content of EspB at various pH values.
Values were calculated using the data from Fig 2A in conjunction
with the CDPRO package [70,71] Predicted values are calculated
from the results of secondary structure prediction (Fig 1) using the
PHDsec algorithm available at the PredictProtein server (http://
cubic.bioc.columbia.edu/predictprotein/) [24,25].
Fig 3 Fluorescence quenching of intrinsic tyrosine (A) Stern–Volmer plot (B) Modified Stern–Volmer plot (Eqn 1).
Trang 4F0=ðF0 FobsÞ ¼ 1=ðfaKsv½QÞ þ 1=fa ð1Þ
where fais the fraction of accessible tyrosines The plot
of F0⁄ (F0 – Fobs) vs 1⁄ [Q] (Fig 3) shows a linear
cor-relation between F0⁄ (F0 – Fobs) and 1⁄ [Q] The values
for Ksv and fa are calculated as 32.7 ± 1.5Æm)1 and
1.05 ± 0.01, respectively An fa value close to 1
sug-gests that the three tyrosine residues are likely to be
solvent-exposed at neutral pH
ANS binding
ANS binds to solvent-accessible hydrophobic surfaces
and, when bound, an increase in ANS fluorescence
intensity near 500 nm occurs This property of ANS is
often used to detect the presence of partially folded
protein intermediates, e.g molten globules [29] Molten
globule is originally defined as the partially folded state
of protein that assumes a significant amount of
native-like secondary structures but disrupted in tertiary
struc-tures [30–38] We used ANS fluorescence spectroscopy
to probe the hydrophobic surface accessibility of EspB
At pH 4 and 7, the ANS fluorescence is low
(Fig 4), suggesting that hydrophobic surfaces are not
exposed to solvent On the other hand, ANS
fluores-cence increases as the pH is decreased to 2.0 (Fig 4)
This observation suggests that hydrophobic surfaces
are solvent-exposed at more acidic pH values Under
the same conditions, the protein assumes an a-helical
conformation according to the far-UV CD spectrum at
pH 2.0 (Fig 2) The results obtained by CD and ANS
fluorescence suggest the formation of a typical molten
globule structure for EspB at acidic pH 2.0
Below pH 2.0, the ANS fluorescence decreased For these experiments, the pH of the solution was adjusted
by the addition of HCl The decreased fluorescence intensity may be caused by the quenching effect of chloride ions on ANS fluorescence, rather than reflect-ing additional conformational changes in EspB
Urea unfolding Urea-induced unfolding transitions of EspB were monitored by far-UV CD spectroscopy Plots of [h]
at 222 nm vs urea concentration show co-operative unfolding transitions throughout the pH range of 1.0–7.3 (Fig 5) Between pH 3.0 and 7.3, unfolding
Fig 4 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence at
500 nm as a function of pH Data were taken at 20 C in the
pres-ence of 0.1 mgÆmL)1EspB Circles represent the raw data The line
is drawn only for visual assistance and is not a mathematical fit.
Fig 5 Urea unfolding of EspB at various pH values and at 20 C (A) The far-UV CD spectra obtained in the presence and absence
of urea The numbers refer to the concentration of added urea (B) The urea-unfolding transition curves obtained at pH 2.0 (s),
pH 5.4 (h) and pH 7.3 (n) Continuous lines are theoretical curves The dotted and dashed lines correspond to the baselines for unfolded and folded states.
Trang 5transitions occur between 2.5 and 4.5 m urea, but shift
to lower urea concentrations of 1.0–3.5 m at pH 2.0
The urea-induced unfolding curves (Fig 5) were
analyzed assuming a linear relationship between DG
and urea concentration and assuming a two-state
fold-ing mechanism, although there is no direct evidence
that the transitions are two-state in nature The
derived parameters, DGwater and m, are summarized in
Table 2
Compared to the conformational state at pH 2.0,
those at higher pH values are stabilized by 3–10 kJÆ
mol)1 However, their m-values, which probably
cor-relate with changes in the solvent-exposed surface
area associated with unfolding (DASA), are similar regardless of pH In our experiments, DASA largely reflects structural changes in the folded species There-fore, given the m-values, EspB, at pH 2.0, which pos-sesses molten globule-like properties, has a similar accessible surface area as EspB conformations existing
at higher pH Therefore, EspB at around neutral pH should be a less compact structure than typical glob-ular proteins
Hydrodynamic property of EspB The hydrodymanic property of EspB has been ana-lyzed by multiangle dynamic scattering and ultracen-trifugation
Multiangle laser light scattering experiments for EspB at pH 2.0 and pH 7.0 revealed the presence of
a single species with a Stokes radius of 3.7 and 3.1 nm, respectively (Fig 6) A similar value was obtained at pH 4.0 and pH 6.0 (3.4 and 3.5 nm, respectively) This size is larger than the expected value for a globular protein of 32 kDa molecular mass, and corresponds to the value of globular pro-teins, of 70 kDa From its amino acid sequence, the molecular mass of the recombinant EspB is
calcu-Table 2 Values of DG water and m for urea-induced unfolding of
EspB.
pH
(kJÆmol)1)
DG water
Fig 6 Hydrodynamic property of EspB at
20 C Multiangle laser light scattering of EspB at pH 2.0 (A) and pH 7.0 (B) (C) Sedi-mentation equilibrium of EspB at pH 7.0 The data were analyzed assuming a single species in solution In the lower panel, raw data are shown by circles, and the line is the theoretical curve The upper panel shows the difference between raw data and theoretical values.
Trang 6lated to be 32 kDa Thus, if EspB assumes a rigid
globular conformation at these conditions, the protein
should assume a dimeric structure
The result of the sedimentation equilibrium study at
pH 7.0 also indicated the presence of only a single
spe-cies (Fig 6C) The molecular mass estimated from this
experiment is, however, 34 kDa, which is similar to the
expected value for the monomeric EspB
From these results, we concluded that EspB at
pH 7.0 assumes a relatively expanded monomeric
con-formation whose Stokes radius is approximately twice
as large as expected for a globular protein with a
molecular weight similar to that of EspB
Importantly, the Stokes radius of EspB estimated
from light scattering was almost independent of
pro-tein concentration or pH value This suggests that only
a single monomeric species is present in each protein
solution at pH 2.0–7.0
Heteronuclear NMR spectroscopy
To further probe the structural properties of EspB, we
recorded its15N-1H heteronuclear single quantum
cor-relation (HSQC) spectra at pH 2.0 in the absence of
urea and at pH 7.0 in the presence and absence of
urea
At pH 2.0, in the absence of urea, the 15N-1H
HSQC spectrum shows little chemical shift dispersion
(Fig 7) Although the resolution is poor owing to the
overlapping of peaks, the number of peaks that
cor-respond to the main-chain 1H-15N crosspeaks was
estimated to be 120 These peaks are relatively
sharp and may reflect the amino acid residues that
rapidly fluctuate with a timescale of nanosecond
order The recombinant EspB used in this study
contains 333 amino acid residues Thus, 64% of
main-chain 1H-15N crosspeaks are missing A similar
phenomenon is often found for the molten globule
state, reflecting the slow fluctuation of a particular
region of protein molecules with a timescale of
micro-second to millimicro-second order This is highly consistent
with our other spectroscopic studies, which show that
the protein, at pH 2.0, is in partially folded
confor-mation, similar to that of molten globules
Interest-ingly, the NMR spectrum of EspB at pH 7.0 in the
absence of urea also shows little chemical shift
disper-sion, with 110 possible main-chain 1H-15N
cros-speaks Thus, about 67% of main-chain 1H-15N
crosspeaks are probably slowly fluctuating Both of
the aforementioned NMR spectra are similar to the
spectrum obtained for urea-unfolded EspB at pH 7.0
In this case, the number of peaks that correspond to
main-chain 1H-15N crosspeaks slightly increased to
about 140 This implies the significant overlapping of crosspeaks or the presence of some residual struc-tures, even in the presence of 8.0 m urea
The result of little chemical shift dispersions with the small number of observable crosspeaks in the 15N-1H HSQC spectrum of EspB at pH 7.0 in the absence of urea is inconsistent with the previous data obtained by
CD and fluorescence spectroscopies showing the pres-ence of well-ordered conformations This discrepancy suggests that, at neutral pH, EspB assumes a natively partially folded conformation without exposed hydro-phobic clusters accessible to ANS molecules
Fig 7. 15N-1H Heteronuclear single quantum correlation (HSQC) spectra of EspB taken at 15 C (A) pH 7.0 in the absence of urea (B) pH 2.0 in the absence of urea (C) pH 7.0 in the presence of 8.0 M urea.
Trang 7Conformational properties of EspB
We analyzed the conformational properties of EspB by
using three spectroscopic techniques The spectral
results suggest that EspB assumes intrinsically partially
folded conformations [39–44] under various conditions
The different conformational states of EspB, found
under different pH conditions, are summarized in
Table 3 The shapes of the far-UV CD spectra suggest
the presence of a substantial amount of secondary
structure for EspB throughout the pH range of 1.0–
7.0 On the other hand, ANS fluorescence spectroscopy
indicates that EspB shows a conformational transition
involving the exposure of hydrophobic clusters when
the pH of the protein solution is decreased to 2.0
Thus, at pH 2.0, the structure of EspB is consistent
with the traditional definition of a molten globule, i.e
a compact partially folded state with a significant
amount of native-like secondary structures, but
disrup-ted in tertiary contacts [30–38]
Importantly, all EspB NMR spectra had chemical
shift signals that were less dispersed than those of
globular proteins, even at pH 7.0 in the absence of
de-naturant The NMR spectra reported here are
qualita-tively similar to those observed for proteins that are
unstructured when in the presence of denaturants On
the other hand, when analyzed by far-UV CD
spectro-scopy, EspB showed the presence of secondary
structures EspB is therefore in a partially folded
conformation, even at near-physiological conditions
However, ANS fluorescence spectroscopy suggests the
presence of a negligible amount of exposed
hydropho-bic surface for EspB at pH 7.0 This is a very unusual
result because partially folded proteins generally have
exposed hydrophobic clusters that are detected by
increases of ANS fluorescence intensity One possible
explanation for the discrepancy could be that the
hydrophobic clusters, found for the EspB molten
glob-ule at pH 2.0, are disrupted in the structure found at
pH 7.0 A similar situation is, indeed, often found
for a-helical polypeptides in alcohol⁄ water solvents [45–51] However, this explanation can be ruled out as EspB is more stable at higher pH, which would prob-ably be inconsistent with the loss of intramolecular hydrophobic contacts Thus, most EspB hydrophobic clusters should be buried at neutral pH Variations in the conformational and thermodynamic properties of molten globules have been characterized For example, the thermal unfolding experiments on the molten glob-ule state of a-lactalbumin shows a gradual transition, which suggests less organized hydrophobic contacts [34] However, the cytochrome c molten globule state
is highly ordered and the thermal unfolding transition
of this species is co-operative with a clear enthalpy change upon unfolding [52,53] This indicates that some organized hydrophobic contacts exist in the mol-ten globule state of cytochrome c Furthermore, the presence of tertiary contacts in the molten globule states are shown by apomyoglobin and cytochrome c [54,55], and EspB also showed the presence of weak, but distinctive, peaks in the near-UV CD spectrum Therefore, EspB, at neutral pH, may have the charac-ter of a highly ordered molten globule [56] with dis-tinct and ordered regions probably stabilized by the interactions between hydrophobic clusters On the other hand, the NMR data also indicate the presence
of highly fluctuating regions in EspB As the data from ultracentrifugation and laser light scattering suggest that EspB assumes an expanded monomeric form, EspB may assume a partially folded structure with well-ordered regions and highly fluctuating regions under near-physiological conditions
Uversky et al [41] proposed that natively unfolded proteins tend to have a low mean hydrophobicity and a relatively high net charge, and provided the following expression of inequality, <H> < (<R> +1.151)⁄ 2.785, between the hydrophobicity value
<H> and the mean net charge <R> for this class
of proteins According to the amino acid sequence
of EspB, its <H> and <R> values are 0.478 and 0.013, respectively These values actually do not satisfy the above criteria
Several www servers, which predict the disordered regions in a protein from its amino acid sequence, are currently available We used GlobPlot (http://globplot embl.de/cgiDict.py), DisEMBL (http://dis.embl.de/ cgiDict.py) and PONDR (http://www.pondr.com) [57–60] In the case of DisEMBL, some disordered regions are predicted and, according to Remark-465 definition, residues at 12–27, 124–145, 157–188, 246–
257 and 303–312 are disordered On the other hand, GlobPlot, when using the Russel⁄ Linding definition, does not show a high probability for EspB to be largely
Table 3 The conformational properties of EspB at pH 2.0 and 7.0,
at a temperature of 20 C ANS, 8-anilinonaphthalene-1-sulfonate;
HSQC, heteronuclear single quantum correlation.
pH
Far-UV
CD
Near-UV
CD
Hydrophobic exposure
by ANS
15 N- 1 H HSQC
Urea unfolding 7.0 Folded Folded Less exposed Unfolded Co-operative
2.0 Folded Partially
folded
Highly exposed Unfolded Co-operative
Trang 8disordered The results from PONDR suggest that
the amino acid residues at 1–53, 128–230 and 247–287
may be disordered This is relatively consistent with the
prediction of DisEMBL If the prediction from
PONDR is correct, only the amino acid sequences at
residues 54–127 and 288–325 of EspB, i.e one-third of
the EspB sequence, assume ordered conformations
This amount may overestimate the disordered regions
as the CD spectum indicates that about 50% of the
EspB sequence should be, at least, partially folded
Of various approaches, only PONDR and
DisEM-BL indicated that EspB may be natively partially
folded Incidentially, both prediction methods are
based on artificial neural networks, whereas GlobPlot
or the category shown by Uversky et al [41] rely on
the physicochemical propensities of amino acids to
favor the disordered or globular structures These
results may not be surprising as, in contrast to natively
unfolded proteins in general, EspB has a relatively
ordered conformation Importantly, human
a-lactalbu-min, in the absence of Ca2+[34], adopts a typical
mol-ten globule structure at neutral pH However, none of
the algorithims predict such a property of human
a-lactalbumin Thus, the prediction of natively
parti-ally folded protein from its amino acid sequence
should still be difficult compared with the prediction
of natively unfolded proteins
Implications for the function of EspB
It is well established that proteins fold to their unique
native conformations, as determined by their amino
acid sequences [61] However, it is also clear that some
proteins are unable to maintain well-defined structures,
even under physiological conditions [39–44] These
proteins are often called natively unfolded or
intrinsic-ally disordered proteins and assume either partiintrinsic-ally
folded or completely unfolded conformations in an
aqueous environment at neutral pH and, ideally, under
near-physiological conditions
Our results clearly indicate that the structural
char-acteristics of EspB are those of a natively partially
folded protein The far-UV CD spectra of IpaC, a
homolog of EspB from Shigella flexneri, revealed an
absence of significant amounts of secondary structure
at neutral pH [62] Thus, the intrinsically less
organ-ized conformations of EspB and IpaC may be a
com-mon property for this class of proteins
Importantly, some proteins that are natively
unfol-ded show dramatic conformational changes into
well-ordered structures when bound to their target
molecules [40–44] Therefore, it will be important to
characterize the conformational state of EspB when
bound to its target molecules, e.g EspA, EspD, a-cate-nin and a1-antitrypsin
Using the genomic sequence of E coli, Dunker and co-workers predicted that 8% of all proteins will have intrinsically disordered segments of greater than 50 res-idues in length [62] Interestingly, the same predictions indicated that this percentage increases to 41% for Drosophila melanogaster proteins Thus, intrinsically structural protein disorder is probably a common occurrence in vivo It is unclear why structural disorder would confer a physiological advantage to the function
of a protein function Several possible reasons have been proposed to answer this question [39–44] For example, if a protein is highly flexible, its association with various targets of different molecular dimensions and binding surfaces would be facilitated as different conformations might be assumed Indeed, EspB prob-ably associates with various biological molecules, e.g EspA [20], EspD [17], a-catenin [18] and a1-antitrypsin [19] The molecular weights, physicochemical proper-ties and functions of EspA, EspD, a-catenin and a1-antitrypsin differ significantly It is probable that different areas of EspB bind different target molecules However, while EHEC and EPEC invade a variety of animals with target molecules of varying amino acid sequences, EspB should still specifically recognize isoforms of the target molecules at the same binding surfaces Therefore, the conformational flexibility of a virulence factor should provide a mechanism that enables bacteria to infect various host species via the same infection system
Interestingly, exogenously added IpaC, an EspB homolog from S flexneri, enhanced the invasion activ-ity of this bacterium into host cells [63] As discussed above, IpaC assumes an almost fully unstructured con-formation near physiological conditions in vitro [64] Such a property may also facilitate the penetration of this molecule into host cells Thus, the conformational flexibility of EspB may also be advantageous for effi-cient penetration into host cell membranes This idea
is consistent with the concept that partial unfolding may be required for the insertion of protein toxins into host membranes [65]
Experimental procedures
Expression and purification of EspB
The cDNA, encoding EspB, was amplified from an EHEC
E coli O157:H7 cosmid library (RIMD 0509890, Sakai strain) [66,67] by PCR and cloned into a pT7 vector (Novagen) The full-length espB gene was subcloned into the expression vector pET28a (Novagen, Madison, WI,
Trang 9USA) The recombinant EspB has 20 amino acids, MGSS
HHHHHHSSGLVPRGSH, added at the N terminus of the
original sequence
Recombinant EspB was expressed in E coli BL21(DE3)
transformed with the afore mentioned plasmid Cultures of
Luria–Bertani (LB) broth, supplemented with 50 lgÆmL)1of
kanamycin, were inoculated with colonies and grown
over-night at 37C with shaking Then, a portion of each culture
was diluted 100-fold into 1 L of fresh LB medium and
incu-bated at 37C with shaking Protein expression was induced
by the addition of isopropyl thio-b-d-galactoside (at a final
concentration of 1 mm) when cultures reached an attenuance
(D) of 0.6 at 600 nm For the expression of protein
uni-formly labeled with 15N, M9 medium supplemented with
15
NH4Cl (Nippon Sanso Co., Kanagawa, Japan) was used
instead of LB medium After 4 h of further shaking at
37C, the cells were harvested by centrifugation (10 min,
10 000 g, 4C) and placed on ice Protein was expressed as
both soluble and insoluble fractions when the cells were
dis-rupted by 20 mm sodium phosphate, pH 7.0, containing
0.1% (v⁄ v) Triton X-100 However, the solubility was quite
low On the other hand, EspB cannot be extracted into the
soluble fraction when the cells are disrupted by 20 mm
sodium phosphate, pH 7.0 The purifications were therefore
performed from either the soluble fraction obtained by
dis-ruption of the cells in the presence of Triton X-100 or from
the insoluble fraction obtained by cell disruption in the
absence of detergent For preparation from the soluble
frac-tion, the cells were suspended with 20 mm sodium
phos-phate, pH 7.0, containing 0.1% (v⁄ v) Triton X-100 and the
solution was separated by centrifugation (15 000 g, 10 min,
4C) The solution was loaded onto Chelating Sepharose
Fast Flow (Amersham Biosciences, Corp., Piscataway, NJ,
USA) supplemented with NiCl2 in 20 mm sodium
phos-phate, pH 7.0, washed with the same buffer and eluted using
a 0–1.0 m imidazole gradient The eluted protein was further
purified with size-exclusion chromatography (S-300; 20 mm
sodium phosphate, pH 7.0; Amersham Biosciences, Corp.)
For preparation from the insoluble fraction, the cells were
suspended in 20 mm sodium phosphate, pH 7.0, and lysed
by sonication The solution was centrifuged (15 000 g,
10 min, 4C) to separate the soluble and pellet fractions
The protein was extracted from the pellet by the addition of
20 mm sodium phosphate, pH 7.0, containing 8.0 m urea
This solution was clarified by centrifugation and diluted
100-fold by dropwise addition into 20 mm sodium
phos-phate, pH 7.0, at 4C The solution was then purified by
Chelating Sepharose Fast Flow supplemented with NiCl2
and further purified by size-exclusion chromatography
(S-300) as in the case of preparation from the soluble
frac-tions The purification yields from soluble and insoluble
fractions were 15 and 30 mg from 1 L of culture in LB
medium, respectively As judged by SDS⁄ PAGE, the purity
of recombinant EspB prepared from the insoluble fraction is
relatively higher than that of EspB purified from the soluble
fraction According to the CD spectrum, both purifications yielded the same conformational state of EspB (see text for details) Owing to the higher yields of purification,15N pro-tein was prepared from insoluble fractions
The protein concentration was determined by absorption
at 276 nm with the extinction coefficient of 4350 mÆcm)1 calculated from amino acid composition [68] The protein solution was stored at)20 C
CD spectroscopy
CD spectra were measured by using a J-600 spectropola-rimeter (Jasco, Tokyo, Japan) The temperature was held at
20C by using a thermostatically controlled cell holder in conjunction with a circulating waterbath For far-UV and near-UV CD spectra, cells of 1 mm and 1 cm path length were used, respectively Protein concentrations were 0.1 and
1 mgÆmL)1 for far-UV and near-UV CD measurements, respectively The data are expressed as molar residue ellip-ticity [69], [h], with [h]¼ 100 hobs(c l))1 The value, hobs, is the observed intensity, c is the concentration in residue moles per litre, and l is the path length in cm A secondary structure prediction was made by using the amino acid sequence of EspB in conjunction with the program package cdpro, in which selcon3, cdsstr and continll programs are included [70,71] The reported values are the average of results from the above three programs
Fluorescence spectroscopy
The fluorescence spectra of intrinsic EspB tyrosines and ANS were measured by using a FP-777 fluorimeter (Jasco) For tyrosine fluorescence, the excitation wavelength was
280 nm and the fluorescence emission was 300–350 nm The protein concentration was 0.1 mgÆmL)1 For ANS fluores-cence, the excitation wavelength was 350 nm and the emis-sion was measured between 400 and 650 nm The protein concentration was 0.1 mgÆmL)1and the ANS concentration was 5 lm The temperature was maintained at 20C with a peltier-type thermostatically controlled cell holder
Fluorescence quenching of EspB tyrosines was measured
in the presence of various concentrations of acrylamide, with spectra acquired as described above
Urea-induced unfolding measurements
Urea-unfolding curves were plotted with [h] at 222 nm vs the urea concentration The data were analyzed assuming
a two-state unfolding mechanism and assuming that the change in free energy of unfolding (DG), is linearly depend-ent on urea concdepend-entration:
Here, DGwater corresponds to DG of unfolding in the absence of urea; m is a measure of the co-operativity
Trang 10of the unfolding transition; and [urea] is the urea
concen-tration
The fractions of unfolded (fU) and folded (fF) species at
various urea concentrations can be expressed as:
fU¼ 1=½1 þ expðDGR1T1Þ ð3Þ
and as:
where R is the gas constant and T is the temperature in
Kelvin
The theoretical value of [h] at 222 nm ([h]222), observed
in the presence of various concentrations of urea, can be
expressed as:
½h222¼ ½hFfFþ ½hUfU ð5Þ Here, [h]Fand [h]Uare the [h]222of the folded and unfolded
species, respectively
The values forDGwaterand m were obtained by nonlinear
curve fitting to the transition curves, according to
Eqns (2–5), by using the program igorpro (WaveMetrics
Inc., Lake Oswego, OR, USA) The linear dependences of
[h]F and [h]U on urea concentrations were also considered
in the fitting analysis The same baselines for folded and
unfolded species were used for the fitting of data obtained
at pH 2.0–7.0
Multiangle laser light scattering
Multiangle laser light scattering data were obtained by
using a dynapro Molecular Sizing Instrument (Protein
Solutions Inc., Milton Keynes, UK) at 20C Various
con-centrations of protein solution at pH 2.0, 4.0, 6.0 and 7.0
(400 lL) were passed through 0.22 lm of centrifugal filter
unit, ultrafree-MC from Millipore (Billerica, MA, USA),
and further centrifuged at 20 000 g for 10 min Only the
clear solution at the top of a tube (100 lL) was used for
the light scattering analysis
Ultracentrifugation
Sedimentation equilibrium experiments were performed by
using a Beckman Optima XL-I analytical ultracentrifuge
(Fullerton, CA, USA) at 11 300 g, 20C The protein
con-centration was 3 mgÆmL)1
NMR spectroscopy
2D15N-1H HSQC spectra were recorded at 15C on either
a 500 or an 800 MHz spectrometer (Brucker DRX500 or
DRX800, respectively, Brucker Biospin GmbH, Karlsruhe,
Germany), each equipped with a triple axis gradient and a
triple-resonance probe Protein concentrations were 1–2 mm
in buffered solution containing 10% 2H2O For DRX500
experiments, the number of complex points and spectral
widths were 1024, 12019 Hz (1H, F2) and 64, 1168 Hz (15N, F1), and for those using the DRX800 spectrometer, the parameters were 1024, 12821 Hz (1H, F2) and 64,
1866 Hz (15N, F1) The 1H carrier was set at 4.7 p.p.m., and the 15N carrier at 120 p.p.m The 15N-1H HSQC experiments included the WATERGATE and Water-flip-back techniques The data were processed by using nmrpipe [72] and visualized by using sparky (TD Goddard & DG Kneller, University of California, San Francisco, CA, USA; http://www.cgl.ucsf.edu/home/sparky/)
Acknowledgements
The authors acknowledge Prof Yuji Goto for access
to the CD spectropolarimeter, Prof Atsushi Nakagawa for access to light scattering, and Miyo Sakai for per-forming ultracentrifugation This work was supported,
in part, by grants-in-aid for scientific research from the Japan Ministry of Education, Science, Culture and Sports, and JSPS Research Fellowships for Young Sci-entists (to D.H.)
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