An inter-action between YopD278300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the pept
Trang 1Conformational analysis by CD and NMR spectroscopy of a peptide
Tobias Tengel1, Ingmar Sethson1and Matthew S Francis2
1
Departments of Organic Chemistry and2Molecular Biology, Umea˚ University, Umea˚, Sweden
To establish an infection, Yersinia pseudotuberculosis utilizes
a plasmid-encoded type III secretion machine that permits
the translocation of several anti-host factors into the cytosol
of target eukaryotic cells Secreted YopDis essential for this
process Pre-secretory stabilization of YopDis mediated by
an interaction with its cognate chaperone, LcrH YopD
possesses LcrH binding domains located in the N-terminus
and in a predicted amphipathic domain located near the
C-terminus This latter domain is also critical for Yersinia
virulence In this study, we designed synthetic peptides
encompassing the C-terminal amphipathic domain of YopD
A solution structure of YopD278)300, a peptide that strongly
interacted with LcrH, was obtained by NMR methods The
structure is composed of a well-defined amphipathic a helix
ranging from Phe280 to Tyr291, followed by a type I b turn between residues Val292 and His295 The C-terminal trun-cated peptides, YopD278)292and YopD271)292, lacked helical structure, implicating the b turn in helix stability An inter-action between YopD278)300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide–LcrH complex These effects were not observed for the unstructured peptide, YopD278)292, which confirms that the a helical structure of the YopDamphi-pathic domain is a critical binding region of LcrH
Keywords: YopD; amphipathic helix; LcrH; NMR solution structure; 2,2,2-trifluoroethanol
Injection of anti-host factors into eukaryotic cells by
numerous economically important animal- and
plant-inter-acting Gram-negative bacteria is achieved by functionally
homologous type III secretion systems (TTSS) [1,2] This
TTSS-dependent process is essential to establish bacterial
infections The enteropathogen Yersinia pseudotuberculosis
is a model system used to study the basic molecular
mechanisms of type III secretion All pathogenic Yersinia
spp harbor a 70-kb virulence plasmid that encodes
numerous Yop (Yersinia outer protein) and Lcr (low
calcium response) virulence determinants that are secreted
by the Ysc (Yersinia secretion) type III apparatus [3,4] Two
protein classes are secreted by the Ysc apparatus; antihost
Yop-effector proteins and those required for their efficient
injection into target cells Collectively, these determinants
co-operate to allow Yersinia to resist uptake by both professional and nonprofessional cells [5–7] and subvert host cell signalling that would normally lead to effective bacterial clearance [8]
YopDis a crucial TTSS component during a Yersinia infection being essential for the injection of antihost Yop-effectors into target cells, possibly through stabilization
of a YopB–LcrV pore complex in the plasma membrane through which Yop-effectors are injected into host cells [4,9] However, involvement of YopDin pore formation is only transitory, because a portion of YopDis also localized to the host cell cytosol [10] In addition, we and others observed that a yopD null mutant is constitutively induced for synthesis of Yops in vitro, while Yop synthesis in wild type bacteria remained tightly regulated in response to temper-ature and Ca2+[10,11] This highlights important dual roles for YopDin both negative regulation of Yop synthesis and injection of Yop-effectors into target cells
While the mechanism of YopDfunction is unknown, it is dependent on an interaction with the nonsecreted TTSS chaperone LcrH [12,13] This interaction is responsible for the presecretory stabilization and efficient secretion of YopD[12,13], and is important for control of yop regulation [14,15] It follows that protein interactions involving several TTSS chaperones and their cognate secreted partner are now recognized as having pivotal roles in temporal and spatial control of virulence [16,17] Therefore, to better understand this relationship, we have chosen to analyze the YopD–LcrH complex because functional homologues exist
in other systems [18,19] and their interactive domains have already been mapped in vitro [13] In fact, we have previously identified several hydrophobic residues within a putative C-terminal amphipathic domain of YopDthat are necessary for binding LcrH [13] This finding was significant
as it coincides with the additional requirements for this
Correspondence to M S Francis, Department of Molecular
Biology Umea˚ University, SE-901 87 Umea˚, Sweden.
Fax: + 46 90 77 14 20, Tel.: + 46 90 785 25 36,
E-mail: matthew.francis@molbiol.umu.se
or I Sethson, Department of Organic Chemistry,
Umea˚ University, SE-901 87 Umea˚, Sweden.
Fax: + 46 90 13 88 85, Tel.: + 46 90 786 99 76,
E-mail: ingmar.sethson@chem.umu.se
Abbreviations: CSI, chemical shift index; SA, simulated annealing;
TTSS, type III secretion system(s).
Note: Web pages are available at http://www.cmb.umu.se
and http://www.chem.umu.se/Department/orgchem
Note: Individual amino acids are indicated by the three-letter
abbreviation followed by a number indicating sequence position
relevant to the full length YopDprotein Complete peptide
sequences are presented in one-letter amino acid code.
(Received 18 March 2002, revised 6 June 2002,
accepted 17 June 2002)
Trang 2domain in Yersinia pathogenesis, being essential for both
regulation of Yop production and injection of antihost
effectors into host cells [10]
Thus, in this initial structural study of YopD, we focused
on the putative amphipathic domain This strategy was
advantageous because full length YopDis susceptible to
aggregation [20] and the amphipathic domain is clearly
biologically relevant [10,13] The utilization of small
peptides to evaluate smaller domains to build up the tertiary
structure of large polypeptides has made a substantial
contribution to the understanding of protein structures and
initial protein folding events [21,22] In this study, we
therefore designed synthetic peptides that encompassed the
C-terminal amphipathic domain of YopD The peptide
structures were examined using CDspectroscopy and 2D
homonuclear/heteronuclear NMR spectroscopy Using
these peptides, the interaction between the amphipathic
domain of YopDand its cognate chaperone LcrH was
investigated by NMR
E X P E R I M E N T A L P R O C E D U R E S
Materials
Peptides spanning the C-terminal amphipathic domain of
YopDwere purchased from Chemical R & DLaboratory
(Copenhagen, Denmark) Peptide purity was confirmed by
HPLC The peptide sequences were as follows, YopD278)292
(DNFMKDVLRLIEQYV); YopD271)292(EEAMNYND
NFMKDVLRLIEQYV); YopD278)300(DNFMKDVLRL
IEQYVSSHTHAMK) and YopD271)300 (EEAMNYN
DNFMKDVLRLIEQYVSSHTHAMK)
2,2,2-trifluoro-ethanol-d3(99%) was purchased from Larodan Chemicals
(Malmo¨, Sweden) and nondeuterated trifluoroethanol used
in CDexperiments was obtained from Sigma-Aldrich All
other chemicals were analytical grade and obtained from
various manufacturers
Cloning, expression and purification of LcrH
The lcrH gene was amplified by PCR on a 540-bp NdeI/BglII
DNA fragment using the primer combination of plcrH10:
5¢-CGAGGTACATATGCAACAAGAGACG-3¢ and
plcrH11: 5¢-ACGTACAGATCTCCTTGTCGTCGTCGT
CTGGGTTATCAACGCACTC-3¢ This fragment was
then cloned into the expression vector pET30a (Novagen,
Wisconsin, USA) giving rise to pMF322, encoding LcrH
containing a C-terminal enterokinase cleavage site upstream
of a His6-tag To express this recombinant protein, an
overnight culture of Escherichia coli BL21(DE3)/pMF322
grown at 26C in Luria–Bertani broth (1% (w/v) NaCl,
0.5% (w/v) yeast extract, 1% (w/v) tryptone) was
subcul-tured (0.1 volume) into 500 mL fresh medium After 1.5 h
incubation at 26C, protein expression from pMF322 was
induced by the addition of isopropyl thio-b-D-galactoside to
1 mMfor a further 3.5 h Cells were pelleted by
centrifuga-tion at 9820 g and stored overnight at)80 C, from which
10 mL of cleared lysate was prepared under native
condi-tions using the QIAexpressionist protocol (Qiagen, CA,
USA) To the cleared lysate, 1.5 mL of nickel-nitrilotriacetic
acid slurry (Qiagen) was added, followed by a 1-h incubation
at 4C on a rotary shaker The sample was then loaded on a
Poly Prep chromatography column (Bio-Rad, CA, USA)
and each subsequent flow-through collected The column was washed twice with wash buffer (50 mMsodium phos-phate, pH 8, 300 mMNaCl, 0.8 mMimidazole) containing complete protease inhibitor cocktail (Roche Molecular Biochemicals, Basel, Switzerland) and once with wash buffer without inhibitors The LcrH::His protein was eluted from the column in 50 mM sodium phosphate, pH 8, 300 mM
NaCl, 8 mMImidazole SDS/PAGE analysis and Coomassie Brilliant Blue staining was used to assess the purity of LcrH::His contained in each column flow-through fraction Pure fractions were combined and dialyzed for several days
in large volumes of 50 mMsodium phosphate buffer, pH 8 The concentration of LcrH::His was determined with the Bradford Reagent (Sigma) using known concentrations of bovine serum albumin (New England Biolabs, Massachu-setts, USA) as the standard
Size-exclusion chromatography Size-exclusion chromatography was performed on Super-dex 75 HR 10/30 columns using an FPLC-system (Amer-sham Pharmacia Biotech, New Jersey, USA) The mobile phase for the SEC experiments was 50 mM sodium phosphate buffer, pH 8, 150 mM NaCl with a flow rate
of 0.75 mLÆmin)1 Circular dichroism Samples for CDwere either 60 lM peptide in 5 mM
sodium phosphate buffer at pH 4.5 and 6 or 60 lMLcrH
in 10 mMbuffer at pH 8 CDexperiments were conducted
on YopD278)292, YopD271)292 and YopD278)300 using different concentrations of 2,2,2-trifluoroethanol, 0–40%
In addition, a temperature study between 25 and 60C was performed on YopD278)300in 40% 2,2,2-trifluoroeth-anol No CDdata was collected for YopD271)300because this peptide was difficult to solubilize in phosphate buffer CDspectra were recorded on a CD6 spectrodichrograph (Jobin-Yvon Instruments SA, Longjumeau, France) Spectra were collected between 185 and 260 nm at
25C using a 0.5-mm quartz cell Data were collected
at 0.5-nm intervals with an integration time of 2 s Three spectra per sample were acquired and averaged, followed
by subtraction of the CDsignal of the solvent Ellipticity
is expressed in terms of mean residue molar ellipticity [h] (degÆcm2Ædmol)1)
Nuclear magnetic resonance Peptide samples for NMR were 2–4 mMin 20 mMsodium phosphate buffer and 1 mMNaN3, pH 4.5 However, the YopD278)300and YopD271)300peptides were also examined
in 2,2,2-trifluoroethanol/water mixtures YopD278)300was studied in 40% 2,2,2-trifluoroethanol-d3/H2O/D2O solution (4 : 5 : 1, v/v/v) at pH 4.5 and 6.3, whereas experiments involving YopD271)300 were carried out in a 40% 2,2,2-trifluoroethanol/water mixture at pH 3.8 When analyzing the peptide–LcrH interaction, 0.25 mM samples of YopD278)300 and YopD278)292 were prepared in 10% 2,2,2-trifluoroethanol at pH 6.3 and purified LcrH was added in sequential steps to a final peptide/protein molar ratio of 2 : 1 The appropriate pH was corrected by the addition of small aliquots of HCl and NaOH NMR
Trang 3experiments were also conducted between 10 and 50C to
elucidate the appropriate temperature for further NMR
analysis A temperature of 40C was chosen in order to
minimize peptide aggregation and obtain a better resolved
spectrum
All NMR spectra were recorded on a Bruker DRX and a
Bruker AMX2 spectrometer operating at a proton frequency
of 600.13 MHz and 500.13 MHz, respectively Both were
equipped with a triple resonance gradient probe The spectra
used for resonance assignments and structure elucidation
included phase sensitive DQF-COSY [23], TOCSY [24],
NOESY [25] and gradient enhanced HSQC [26]
In TOCSY and NOESY experiments the solvent signal
was suppressed just before the FIDacquisition using the
WATERGATE pulse sequence [27] The DIPSI pulse
sequence with a spin lock time of 85 ms was used in the
TOCSY experiments and the NOESY spectra was recorded
with a mixing time of 150 ms Data were processed on a
Silicon Graphic workstation using theXWINNMRsoftware
(Bruker) Prior to Fourier transformation, the data were
multiplied by appropriate window functions Zero-filling
was applied in both dimensions and linear prediction in the
indirect dimension The chemical shift of the water signal
was used as a reference and calibrated to 4.60 p.p.m at
40C The HSQC spectra were calibrated using the ratio
13C/1H¼ 0.25144953 for carbon and 15N/1H¼
0.101329118 for nitrogen [28]
Derivation of distance and dihedral restraints
Distance restraints for YopD278)300were obtained from the
NOESY spectrum recorded at 40C, pH 4.5 and 40%
2,2,2-trifluoroethanol, using a mixing time of 150 ms
Assigned NOE cross peaks were volume integrated and
converted to distance restraints usingMARDIGRAS[29] An
extended structure of YopD278)300was subjected to
unre-strained molecular dynamics calculations at 1000 K to
generate 10 different structures These 10 divergent
struc-tures served as a representation of the conformational space,
and each of them was used in theMARDIGRAScalculations
The extreme values were used as upper and lower bonds in
the structure calculation As no specific assignment could be
made for the methyl and methylene protons, appropriate
pseudoatom correction was applied [30] The rotational
correlation time, sc, used in the MARDIGRAS calculations,
was calculated from experimental spin-lattice (T1) and
spin-spin (T2) relaxation time measurements of well resolved
peaks in YopD278)300 T1and T2values were obtained for
residues 5–8, 11–16, 20 and 22 The rotational correlation
time was calculated for each residue using the equation
sc¼ 2x)1(3T2/T1))1/2[31] resulting in scvalues between 6
and 10 ns The average value, 8 ns, was used in the
following MARDIGRAS calculations Backbone / dihedral
angle restraints were obtained using the programTALOS[32]
Structure calculations
Structure calculations were carried out usingX-PLOR3.851
[33] This involved simulated annealing (SA) [34] and SA
refinement The starting structures for the SA calculations
were varied to ensure that the resulting structure represented
a global energy minimum in the conformational space
From three structures with a pair-wise rmsd of 2 A˚ or more
for the backbone heavy atoms, 150 structures were calcu-lated using the SA and SA refinement protocols
To describe the quality of the solution structure of YopD278)300, rmsd values between all the accepted struc-tures and the average structure were studied The strucstruc-tures were analyzed using INSIGHT II (Accelrys Inc., California, USA),MOLMOL[35] andVMD[36] In order to verify that no residues were in disallowed regions, Ramachandran plot analysis was conducted using the programPROCHECK-NMR
[37]
R E S U L T S A N D D I S C U S S I O N
CD and NMR studies Computer analysis of YopDprimary sequence predicts a central hydrophobic membrane spanning domain and a C-terminal amphipathic domain (Fig 1A) [38] This latter region can be presented on a helical wheel projection to reveal an amino-acid sidedness (Fig 1B) [13] While the
Fig 1 Overview and helical wheel projection of biologically significant domains in YopD (306 amino acids) (A) Computer prediction [38] was used to define the central hydrophobic and the C-terminal amphipathic domains of YopD (B) A helical wheel projection of the amphipathic domain of YopDincorporates residues 278–292 [13] Amino acids are presented in one-letter amino-acid code with hydrophobic residues boxed.
Trang 4spatial distribution of these amino acids appeared crucial
for binding the LcrH chaperone [13], we wished to extend
these findings using a chemical approach In particular, this
initial study aimed at obtaining the secondary structure of
the predicted C-terminal amphipathic domain of YopD To
overcome the risk of YopDaggregation [20] we designed
small YopD-specific peptides that encompassed the
pre-dicted C-terminal amphipathic domain As an efficient
means to confirm the presence of a helical structure of these
peptides, CDexperiments were conducted on YopD278)292,
YopD271)292 and YopD278)300 The CDspectrum of
YopD278)300, in aqueous buffer, showed two minima at
208 and 222 nm and an isodichroic point at 200 nm, which
are characteristics of a a helical conformation (Fig 2) We
were unable to detect any secondary structure for the
peptides, YopD278)292and YopD271)292, even in the
pres-ence of 2,2,2-trifluoroethanol (data not shown) The fact
that neither peptide displayed any helical structure indicates
that the amino acids downstream of residue 292 may be
essential for helical stability
Because no secondary structure was detected for the
YopD278)292and YopD271)292peptides, NMR structural
characterization was conducted on YopD278)300 However,
in the first attempts to determine the structure in aqueous
buffer at pH 4.5, the peptide severely aggregated
Accord-ingly, under these conditions the spectrum of YopD278)300
showed extensive line broadening (Fig 3A) Several studies
have reported that the addition of organic solvents can
reduce the incidence of peptide aggregation [39,40] In view of
this, 2,2,2-trifluoroethanol was added to this sample to give
different final concentrations in the range of 0–40% When
NMR experiments were recorded to monitor the effects of
adding 2,2,2-trifluoroethanol, a resolved NMR spectrum
indicative of the disruption of large aggregates was observed
even at low concentrations of 2,2,2-trifluoroethanol
(Fig 3B) However, as 2,2,2-trifluoroethanol is known to
stabilize helical structure [41], additional CDexperiments
were conducted to investigate whether this solvent induced
structural changes in the YopDpeptide Furthermore, the
helical structure may be pH-dependent due to variations in charge distribution of the histidine side chains This fact was taken into account by conducting CDexperiments at both
pH 4.5 (data not shown) and pH 6 (Fig 2) as well as NMR experiments at pH 4.5 and 6.3 Collectively, no significant change in peptide helical content was observed, indicating that, at the pH conditions used in this study, the addition
of 2,2,2-trifluoroethanol did not significantly alter the secondary structure of YopD278)300
As NMR spectra of YopD278)300were recorded at 40C,
we used CDspectroscopy to verify that only minimal variations in helical content of the peptide occurred when the temperature was varied between 25 and 60C (data not shown) Thus, in this range, temperature had no significant impact on the secondary structure
1
H resonance assignment and secondary structure All NMR spectra were assigned according to classical procedures including spin system identification and sequen-tial assignment [42] Inisequen-tial spin system assignments of YopD278)300 were obtained using COSY and TOCSY spectra and a NOESY spectrum was used to identify sequential backbone connectivities A comparison of the
H and C chemical shift deviation from random coil values
Fig 2 Plot of the residual molecular ellipticity from 185 to 260 nm of
YopD278)300peptide samples at different 2,2,2-trifluoroethanol
concen-trations From below at 222 nm, the spectra are of peptide in 0, 30, 20,
40 and 10% 2,2,2-trifluoroethanol, respectively All spectra were
obtained with 60 l M of the peptide in 5 m M sodium phosphate buffer,
pH 6 and conducted at 25 C.
Fig 3 1D1H NMR spectrum of YopD 278)300 (A) Spectrum of a 3 m M
peptide sample prepared in 50 m M sodium phosphate buffer pH 4.5, obtained with a probe temperature of 40 C (amide region is shown) (B) Spectra of a 3 m M peptide sample containing 2,2,2-trifluoroethanol
at a concentration of 0, 10, 20, 25, 30, 35 or 40% (percent 2,2,2-trifluoroethanol shown for each individual spectrum) All experiments were conducted at pH 4.5 in 20 m M sodium phosphate buffer at 40 C (amide region is shown).
Trang 5according to the chemical shift index (CSI) [43], highlighted
a region of the peptide incorporating residues 280–295
where an a helical structure was predicted (Fig 4) These
observations support the presence of an a helix as suggested
from the CDanalysis and define the location of the helical
region
Structural restraints
Several medium range NOEs, daN(i,i + 3); daN(i,i + 4)
and dab(i,i + 3), and strong sequential NOEs between
amide protons also support a helical structured peptide
(Fig 4) NOEs assigned from the NOESY spectrum were
converted to distance restraints usingMARDIGRAS[29] and
used as input for the structure calculations The final
number of restraints, after removal of those that according
to the relaxation matrix originated from spin diffusion, was
242, which consisted of 134 intraresidue, 53 sequential and
55 medium range restraints The proton, carbon and
nitrogen chemical shifts of each residue were used to extract
the / dihedral restraints using the programTALOS[32] The
/ dihedral restraints obtained byTALOSwere then used for
residues in whichTALOSindicated a good prediction relative
to a known structure These values were collected for
residues 279 through to 295 and used as dihedral restraints
in the structure calculations
NOEs were found that were not compatible with a
monomeric structure Accordingly, these NOEs were
ascribed to intermolecular interactions and have been
excluded in the structure calculations conducted in this
study However, it is still possible that the intermolecular
interactions do not only appear as resolved peaks in the
NOESY spectrum They may also have the same
frequen-cies as NOEs reflecting intramolecular interactions and
thereby affecting the NOE intensities Such influences will
obviously affect the calculated monomeric structure
For-tunately, however, these influences appear minor for two reasons Firstly, structure calculations with the used NOEs proceed without violations Secondly, the dihedral restraints, which only represent intramolecular interactions, are completely compatible with the NOEs Taken together, this implies that the structure of YopD278)300presented in this work does represent the monomeric structure, even though intermolecular interactions are present
Description and quality of the calculated structures The peptide YopD278)300 adopts a well-defined helical structure with a more flexible C-terminal region (Fig 5), with the hydrophobic and hydrophilic residues mainly located at opposite sides of the helix (Fig 6) The a helix incorporates residues Phe280 to Tyr291 with the following four residues, Val292 to His295, forming a type I b turn The exclusion of the TALOS dihedral restraints from the structure calculations generates an almost identical structure containing an a helix with a C-terminal turn
The presence of the b turn is also supported by the lowfield shift of 9.2 p.p.m for the Val292 amide proton, as such shifts are rarely found in helical regions of
Fig 5 Superposition of the backbone atoms for the 25 lowest energy structures of YopD 278)300 The structures were aligned for the best overlap of the backbone of residues 280–295 and superimposed on the lowest energy structure This image was constructed with the VMD
software [36].
Fig 6 NMR structure of the amphipathic domain of YopD illustrating the hydrophobic and hydrophilic sidedness of the peptide The side chains are displayed for residues 279–295 with the hydrophobic residues colored in red, the hydrophilic in blue and the tyrosine in grey This image was constructed using the software [35].
Fig 4 Overview of NOE connectivities and chemical shift data of
YopD 278)300 in 40% 2,2,2-trifluoroethanol at pH 4.5 The relative
intensities of each filled bar indicate the strength of the NOE restraints.
Distance restraints were derived from a NOESY spectrum with a
mixing time of 150 ms The chemical shift indices are indicated by an
index with values of )1, 0 and +1, which corresponds to upfield,
random coil and downfield, respectively This image was constructed
using the VINCE software (The Rowland Institute for Science, MA,
USA).
Trang 6polypeptides Significantly, the amino acids involved in this
turn appear to be essential for stabilizing the a helical
structure because the two synthesized peptides lacking this
motif (YopD278)292and YopD271)292) did not contain any
helical structure (data not shown) Therefore, the formation
of the b turn may act as a stabilizer, capping the C-terminal
end of the a helix This phenomenon has been previously
described by Forood and colleagues [44] However, we have
not been able to identify any specific hydrogen bonds or
other favourable interactions within the calculated
struc-tures that would support this conclusion Rather, the
stabilizing effect may well occur via intermolecular
interac-tions within the observed aggregates of YopD278)300 The presence of these putative intermolecular interactions would
be consistent with the fact that the most significant chemical shift changes upon aggregation state variation occurred for the amide protons in the b turn (Fig 3B) However, further detailed structural descriptions of the aggregate are needed to better understand the stabilizing function of the b turn
To examine whether the helix extended upstream of the N-terminus, the properties of the longer YopD271)300 peptide in a 2,2,2-trifluoroethanol/water preparation were analyzed The chemical shifts and NOE patterns of this peptide, compared to those of YopD278)300, confirmed that the a helix begins at residue Phe280 (data not shown)
Of the 150 calculated peptide structures, 145 were accepted The criteria for acceptance were as follows: rmsd for bonds < 0.01 A˚; rmsd for angles < 2; no NOE violation > 0.3 A˚ and no constraint dihedral violation
> 5 To verify the quality of the YopD278)300solution structure, rmsd values between all 145 accepted structures and the average structure as well as pair-wise rmsd were studied When superposition was performed using residues 280–295, this region displayed a well-defined structure with
a rmsd value of 0.18 A˚ for the backbone atoms An illustration of the rmsd on a per residue basis compared to the number of NOE restraints per residue is presented in Fig 7 Ramachandran plot analysis using the program
PROCHECK-NMR[37] was used to verify that no residues were located in disallowed regions From the minimized average structure, 81% of the residues were in most favoured regions with the remaining 19% in additional allowed regions Structural restraints, rmsd values and the results from Ramachandran plot analysis are summarized in Table 1
Fig 7 Distribution of distance restraints and rmsd values for
YopD278)300 (A) Distribution of NOE restraints in YopD 278 )300 on a
per residue basis Three types of restraints are specified: black,
intra-residue; light grey, sequential; dark grey, medium range All
interres-idue NOEs are plotted twice NOE data was obtained from a 150 ms
NOESY spectrum conducted in 40% 2,2,2-trifluoroethanol at pH 4.5
with a sample temperature of 40 C (B) Distribution of rmsd values
on a per amino acid basis The structures were superpositioned
according to the best fit of the backbone of residues 280–295 and the
rmsd value was calculated for all of the accepted 145 structures (see
Table 1).
Table 1 Summary of the structural statistics and rmsd differences Unless stated, all 145 accepted structures have been used to calculate structural statistics.
NOE statisticsa
Dihedral angle restraints a
Ramachandran plot analysisb Residues in most favorable regions 81% Residues in additional allowed regions 19% rmsd from average structure (A˚)c
rmsd pair-wise (A˚) c
a No structure exhibited distance violations greater than 0.3 A˚ or dihedral angle violations greater than 3 b The minimized average structure was used to perform Ramachandran plot analysis.
c Backbone/heavy atoms.
Trang 7Aggregation of YopD
The NMR experiments conducted on YopD278)300revealed
that the peptide severely aggregated in aqueous buffer,
pH 4.5 The 1DNMR spectrum of YopD278)300was poorly
resolved with extensive line broadening (Fig 3A) This is
indicative of an aggregate having a size well above the limit
allowing high resolution NMR By adding
2,2,2-trifluoro-ethanol it was possible to disrupt this large aggregate without
any significant changes in peptide secondary structure (see
above) However, even though well resolved spectra were
recorded, several observations indicate that the aggregate
was not completely disrupted but forms smaller aggregates
We observed long range NOEs from the aromatic protons of
Phe280 to the side chains of Ile288 and Val292 This indicates
that the peptide forms an aggregate with the a helices
oriented in an antiparallel direction with their hydrophobic
sides facing each other In addition, the formation of small
aggregates is also supported by the rotational correlation
time (sc) In our case, scwas determined to be 8 ns, which is
too long to be explained by the viscosity of the
2,2,2-trifluoroethanol/water mixture [45] Hence, this value
sug-gests that the peptide is not monomeric but rather forms a
smaller aggregate that reflects this scvalue
It is also noteworthy that even in 40%
2,2,2-trifluoro-ethanol, the aggregation state can be affected by changing
the temperature When the temperature was lowered,
extensive line broadening and an upfield shift of the
a-protons occurred for all the residues We interpret these
findings to further indicate the formation of a larger
aggregate that stabilizes the helical structure of the peptide
In addition, analysis of the 2,2,2-trifluoroethanol titration
of YopD278)300 identified two different amide proton
behavioural patterns, residues that experienced a downfield
shift upon the addition of 2,2,2-trifluoroethanol and those
which displayed an upfield shift Interestingly, most residues
on the hydrophobic side of the helix experienced a large
downfield shift whereas those on the hydrophilic side of
the helix experienced a minor downfield shift or in some cases
an upfield shift This supports the hypothesis that
hydro-phobic residues form the aggregate, as these residues are
likely to be most affected when the aggregate is destabilized
It follows that the aggregation of full length secreted
YopDobserved by Michiels and colleagues [20], may
involve the same interaction between the amphipathic
C-terminal helices Because the nonsecreted LcrH
chaper-one prevents premature aggregation of presecretory YopD
[12,13] and binds to a region incorporating the amphipathic
a helix [13], it would be very interesting to determine if
LcrH-binding modulates the extent of peptide aggregation
In fact, such a biophysical study would provide valuable
information towards understanding the biological relevance
of YopDmultimerization and even the general role of TTSS
chaperone function, because functional homologues of
YopDand LcrH exist in other bacterial pathogens [18,19]
Information on the latter would be an important
develop-ment because dual roles for these specialized molecules have
been recently proposed [16,17]
Structure and aggregation of LcrH
Several observations support the fact that LcrH forms higher
order structures in aqueous solution These multimers may
explain why high quality NMR spectra, even at LcrH concentrations above 1 mM, were difficult to obtain Nev-ertheless, the fact that the chemical shifts of the amide protons and a-protons were found in a relatively restrained area does indicate that LcrH is a a helical protein (Fig 8) This is consistent with the CDspectra of 60 lMLcrH, which also displayed characteristics of a helical conformation (Fig 9) Interestingly, size-exclusion chromatography of
Fig 8 NOESY spectrum of LcrH A 0.5 m M sample of LcrH in
50 m M phosphate buffer, pH 8, was used for the experiment The spectrum was recorded with a mixing time of 150 ms at 25 C (amide region is shown).
Fig 9 Plot of the residual molecular ellipticity from 195 to 240 nm of LcrH The spectrum was obtained with a protein concentration of
60 l M in 10 m M sodium phosphate buffer, pH 8 and conducted at
25 C.
Trang 8LcrH suggested that the protein forms aggregates in aqueous
solution because two main fractions were detected
corres-ponding to 2.5 and 3.7 times the expected monomeric mass
(data not shown) This implied the presence of multimers
ranging from dimers (most abundant) to tetramers
Homod-imer formation by LcrH is consistent to that observed for
other TTSS chaperones [46–51] Significantly, this feature is
apparently required to necessitate substrate secretion in
diverse TTSS associated with both pathogenesis and flagellar
biogenesis [49,50]
Interaction of the YopD peptide with LcrH
In the yeast two hybrid assay, the C-terminal
amphi-pathic domain of YopDwas required for LcrH binding
[13] Because the YopD–LcrH complex is important for
regulatory control of virulence gene expression in
Yersinia infections [14,15], a detailed structural analysis
of this complex is required As part of this initial
structural study we wanted to confirm the involvement
of the C-terminal YopDdomain in LcrH binding For
interaction studies with the YopD278)300 peptide, LcrH
was purified as a His-tag recombinant fusion by Nickel
exchange chromatography The peptide was prepared in
a concentration of 0.25 mM in buffer supplemented with
10% 2,2,2-trifluoroethanol These conditions minimized
peptide aggregation and provided a better resolved
spectrum (Fig 3B) A 10% 2,2,2-trifluoroethanol
con-centration was specifically chosen because LcrH
precip-itated at higher concentrations In addition, interaction
studies were performed at pH 6.3 to avoid precipitation
of LcrH under acidic conditions Importantly, although
the conditions chosen to examine the peptide–LcrH
interaction are different from those used to describe the
YopD278)300 peptide solution structure, we clearly
con-firmed that they did not influence the peptide structure
(see above)
LcrH was added in a stepwise manner to the peptide
sample to give a final peptide:protein molar ratio of 2 : 1
An interaction between YopD278)300 and LcrH was
observed from line-broadening and chemical shift
differ-ences within the amide region from a 1DNMR spectrum of
the free peptide and the peptide-protein solution (Fig 10)
The amide-proton resonances of Tyr291 and Val292 are
considerably broadened in the presence of LcrH, consistent
with their induced chemical shift differences upon addition
of LcrH In addition, we observed a decreased relaxation
time of the peptide in the presence of LcrH, which supports
peptide/LcrH binding (data not shown) Moreover, when
we examined the unstructured YopD278)292peptide for the
ability to bind LcrH, no such interaction was observed (data
not shown) We interpret this finding to indicate the
absolute requirement of the YopDhelical structure for the
YopD–LcrH interaction It follows that line broadening
and chemical shift differences between the bound and the
unbound YopD278)300peptide indicate that at least Tyr291
and Val292 are directly involved in the peptide–LcrH
complex This is consistent with the view that hydrophobic
residues within the amphipathic domain of YopDare
required for LcrH binding [13] The fact that amino acid
replacements of Tyr291 and Val292 did reduce LcrH
binding to YopDin the yeast two-hybrid assay also
corroborates with this study [13]
Moreover, the chemical shift behaviour of YopD278)300
in the presence of LcrH is similar to the behaviour when peptide aggregates increase in size In particular, assigned amide protons in the YopD278)300peptide experienced an upfield frequency shift upon LcrH addition and this same trend is also observed when the 2,2,2-trifluoroethanol concentration is lowered These findings imply that the peptide aggregate mimics the interaction between the peptide and LcrH Therefore, analysis of LcrH binding on the dynamics of peptide aggregation warrants further investigation
C O N C L U S I O N S
In this study, we have initialized a means to understand the role of the YopD–LcrH complex in Yersinia pathogenesis
by determining the a helical structure of the biologically relevant C-terminal amphipathic domain of YopD Impor-tantly, this domain precedes a type I b turn that is essential for stability of the helical structure An interesting feature of the peptide encompassing this domain was its tendency to form small aggregates that were likely composed of a helices layered in an antiparallel manner In addition, we confirmed that this domain interacts with LcrH through hydrophobic interactions that include at least two residues, Tyr291 and
Fig 10 1D1H NMR experiment at 20 °C of a 0.25 m M YopD 278)300
sample in 10% 2,2,2-trifluoroethanol and 50 m M phosphate buffer at
pH 6.3 (A) In the absence of purified LcrH, and (B) In the presence of purified LcrH to give a peptide/protein molar ratio of 2 : 1 The peptide residues Tyr291 and Val292 identified to bind LcrH are indi-cated.
Trang 9Val292 Although our laboratory and others have recently
proposed new roles for TTSS chaperones, it is clear that
chaperone-substrate complexes are fundamental to the
process of functional type III secretion and ultimately for
successful infection by the bacterium Based on the recent
crystal structure determination of a TTSS chaperone/
effector protein complex from Salmonella spp., it is likely
that at least one function of chaperones is to maintain their
cognate partner in an elongated unfolded state, presumably
as a prerequisite for efficient secretion [50] We have begun
to reveal the secrets of a biologically relevant YopD–LcrH
complex in Yersinia infections However, a detailed
struc-tural study of this intriguing TTSS complex is ongoing
A C K N O W L E D G E M E N T S
This work was supported by grants from the Swedish Medical Research
Council, Swedish Natural Science Research Council and Swedish
Foundation for Strategic Research We are indebted to Hans
Wolf-Watz for insightful discussions, financial assistance and critical reading
of this manuscript We also thank Peter Stenlund and Gull-Britt Trogen
for excellent technical assistance.
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S U P P L E M E N T A R Y M A T E R I A L
The following material is available from http://www.black-well-science.com/products/journals/suppmat/EJB/EJB3051/ EJB3051sm.htm
Table S2 Phi dihedral angles for YopD (278 )300) Table S1 Chemical shifts (p.p.m.) of YopD(278)300).