Near-atomic resolution analysis of BipD, a component of the type III secretion system of Burkholderia pseudomallei.

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, possesses a type III protein secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to inject virulence-associated proteins into target cells of the host organism. The bipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and is most likely to be functionally analogous to IpaD from Shigella and SipD from Salmonella. Proteins in this family are thought to act as extracellular chaperones at the tip of the secretion needle to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and may also link the translocon pore with the secretion needle. BipD has been crystallized in a monoclinic crystal form that diffracted X-rays to 1.5 A resolution and the structure was refined to an R factor of 16.1% and an Rfree of 19.8% at this resolution. The putative dimer interface that was observed in previous crystal structures was retained and a larger surface area was buried in the new crystal form.

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structural communications
990
doi:10.1107/S1744309110026333
Acta Cryst. (2010). F66, 990–993
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Near-atomic resolution analysis of BipD, a
component of the type III secretion system of
Burkholderia pseudomallei
M. Pal,a,bP. T. Erskine,b
R. S. Gill,bS. P. Woodband
J. B. Cooperb*
aSchool of Biological Sciences, University of
Southampton, Bassett Crescent East,
Southampton SO16 7PX, England, and
bLaboratory for Protein Crystallography, Centre
for Amyloidosis and Acute Phase Proteins, UCL
Department of Medicine (Royal Free Campus),
Rowland Hill Street, London NW3 2PF, England
Correspondence e-mail:
j.b.cooper@medsch.ucl.ac.uk
Received 10 June 2010
Accepted 3 July 2010
PDB Reference: BipD, 3nft.
Burkholderia pseudomallei, the causative agent of melioidosis, possesses a type
III protein secretion apparatus that is similar to those found in Salmonella and
Shigella. A major function of these secretion systems is to inject virulence-
associated proteins into target cells of the host organism. The bipD gene of
B. pseudomallei encodes a secreted virulence factor that is similar in sequence
and is most likely to be functionally analogous to IpaD from Shigella and SipD
from Salmonella. Proteins in this family are thought to act as extracellular
chaperones at the tip of the secretion needle to help the hydrophobic
translocator proteins enter the target cell membrane, where they form a pore
and may also link the translocon pore with the secretion needle. BipD has been
crystallized in a monoclinic crystal form that diffracted X-rays to 1.5 A˚
resolution and the structure was refined to an R factor of 16.1% and an Rfreeof
19.8% at this resolution. The putative dimer interface that was observed in
previous crystal structures was retained and a larger surface area was buried in
the new crystal form.
1. Introduction
Burkholderia pseudomallei causes the disease melioidosis, which is
endemic to tropical and subtropical regions, particularly southeast
Asia and northern Australia (Dance, 2002; Gan, 2005). Most
commonly, the disease manifests itself clinically as abscesses, pneu-
monia and, at worst, a fatal septicaemia (Aldhous, 2005; Gan, 2005).
The genome of B. pseudomallei comprises two chromosomes of 4.07
and 3.17 megabase pairs, which show significant functional parti-
tioning of genes between them (Holden et al., 2004). The large
chromosome encodes many of the core functions associated with
central metabolism and cell growth, whereas the small chromosome
carries more accessory functions associated with adaptation and
survival in different environments.
Type III secretion systems (T3SSs) are large assemblies of proteins
that span the inner bacterial membrane, the periplasmic space, the
peptidoglycan layer, the outer bacterial membrane, the extracellular
space and the target cell membrane (Yip & Strynadka, 2006). The
function of bacterial protein secretion systems is to transport
‘effector’ and other proteins across the bacterial inner membrane and
the outer envelope in an ATP-dependent manner (Mecsas & Strauss,
1996). Each secretion system involves a hollow tube or needle (the
injectisome) through which the secreted proteins travel (Mota et al.,
2005). The injectisome varies between 45 and 80 nm in length
depending on thebacterial species, is made by the polymerization of a
major subunit and has a hollow interior of approximately 25 A˚in
diameter. The ring-like assembly that spans the membrane of the host
cell is referred to as the translocon. This is formed by the initial
secretion of a small number of proteins into the extracellular envir-
onment as a result of contact between the bacterium and the target
cell (Pettersson et al., 1996). The translocator proteins act to transport
bacterial proteins across the plasma membrane into the host cell,
where they essentially subvert the cell’s normal processes to aid
replication of the bacterium. It appears that the translocator proteins
# 2010 International Union of Crystallography
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form a pore in the lipid membrane of the target cell through which
the effector proteins are able to pass (Blocker et al., 2000). Inter-
estingly, the T3SS and the flagellar motor have several common
protein components, which together with the ability of flagella to
secrete certain proteins suggests a common evolutionary origin (Yip
& Strynadka, 2006).
It has been found that B. pseudomallei contains at least three loci
encoding putative type III protein secretion systems (Rainbow et al.,
2002), one of which shares homology with one of the T3SSs of
Salmonella typhimurium (Attree & Attree, 2001) and Shigella flexneri
(Stevens et al., 2002) and has been designated as the Burkholderia
secretion apparatus or BSA (Hueck, 1998). The BSA effector
proteins are termed Bop proteins and the translocators are termed
Bip proteins (short for Burkholderia invasion proteins). In Salmo-
nella the homologous proteins SipB, SipC and SipD are required for
the injection of effector molecules and the invasion of epithelial cells
in vitro (Kaniga et al., 1995) and likewise for IpaB, IpaC and IpaD
from Shigella (Me ´nard et al., 1994). Hence, it is thought that the
B. pseudomallei homologues BipB, BipC and BipD perform a similar
function. Disruption of the bipD gene reduces the ability of
B. pseudomallei to invade eukaryotic cells and reduces virulence in
mice (Stevens et al., 2002, 2004), indicating that the BipD protein is
an important secreted virulence factor. A BipD mutant exhibited
impaired invasion of HeLa cells, reduced intracellular survival in
murine macrophage-like cells and a marked reduction in actin-tail
formation (Stevens et al., 2002, 2004). Hence, it has been suggested
that BipD is involved in the actin polymerization that facilitates the
escape of B. pseudomallei from endocytic vesicles during the initial
infection and the subsequent escape of progeny bacteria into
surrounding host cells. It has also been suggested that proteins in
the same class as BipD (e.g. IpaD and SipD) act as extracellular
chaperones to help the hydrophobic translocators (equivalent to
BipB and BipC) enter the target cell membrane and may even link
the translocon pore with the secretion needle (Mecsas & Strauss,
1996).
The BipD protein from B. pseudomallei consists of 310 amino acids
and has a molecular weight of 33 kDa. Recently, the biophysical
properties of BipD and its homologues have been extensively
analysed by Espina et al. (2007) and their role in the type III secretion
system has been reviewed in Moraes et al. (2008). Previously, we have
crystallized BipD in a monoclinic crystal form that diffracted X-rays
to 2.1 A˚resolution and determined its structure by selenomethionine
MAD (Knight et al., 2006; Erskine et al., 2006) in parallel with similar
studies elsewhere (Roversi et al., 2006; Johnson et al., 2007). In this
paper, we report that the crystallization of the BipD protein in the
presence of the lipid head-group phosphocholine gave a crystal form
that diffracted to 1.5 A˚resolution, thus allowing the structure to be
refined at near-atomic resolution.
2. Crystallization of BipD
The expression of BipD in Escherichia coli as a GST-fusion protein
and affinity purification have been described elsewhere (Knight et al.,
2006) together with the crystallization of the native protein. In the
current study, the expressed BipD protein was concentrated to
6 mg ml?1(as determined using a Nanodrop ND1000 spectrophoto-
meter at 280 nm) in 10 mM Tris–HCl, 140 mM NaCl pH 7.5. Crystals
were obtained by inclusion of phosphocholine in the mother liquor
since preliminary isothermal titration calorimetric studies had
suggested that this compound interacted with the protein, although
the result was difficult to reproduce. Use of Molecular Dimensions
Structure Screens I and II and subsequent optimization of hits
established that the best crystals could be obtained in 35%(w/v) PEG
4000, 100 mM glycine, 20 mM EDTA, 60 mM cacodylate buffer pH
6.0. Using the hanging-drop method, 2 ml protein solution was mixed
with 2 ml 10 mM phosphocholine (in 10 mM Tris–HCl pH 7.5) and
4 ml well solution on siliconized glass cover slips. The crystals, which
appeared in 2–3 weeks (Fig. 1), were cryoprotected with 30% glycerol
(added stepwise) and mounted in loops for flash-freezing with an
Oxford Cryosystems cryostat and subsequent storage under liquid
nitrogen.
3. X-ray data collection and processing
Data were collected at Diamond Light Source (UK) on beamline I02
(? = 0.976 A˚) with an ADSC Q315 CCD detector and with the sample
maintained at a temperature of 100 K. The crystal diffracted to a
resolution of at least 1.5 A˚and 360 images were collected with an
oscillation angle of 0.5?. The data were processed in space group C2
using iMOSFLM (Leslie, 2006), SCALA (Evans, 2006), TRUNCATE
(French & Wilson, 1978) and other utilities in the CCP4 program
suite (Collaborative Computational Project, Number 4, 1994). The
corresponding unit-cell parameters were a = 51.15, b = 60.42,
c = 90.42 A˚, ? = 96.01?. The Matthews coefficient (Kantardjieff &
structural communications
Acta Cryst. (2010). F66, 990–993Pal et al.
? BipD
991
Figure 1
The crystal of BipD which diffracted to beyond 1.5 A˚resolution. The crystal is
approximately 1.0 mm in its largest dimension.
Table 1
Data-collection and refinement statistics for BipD.
Values in parentheses are for the outer resolution shell.
Data processing
Space group
Unit-cell parameters
a (A˚)
b (A˚)
c (A˚)
? (?)
Resolution range (A˚)
Rmerge† (%)
No. of reflections
No. of unique reflections
Mean I/?(I)
Completeness (%)
Multiplicity
Refinement
No. of atoms in protein
No. of solvent atoms
Resolution range (A˚)
No. of reflections in working set
No. of reflections in free set
R factor (%)
Rfree(%)
R.m.s. bond-length deviation (A˚)
R.m.s. bond-angle deviation (?)
C2
51.15
60.42
90.42
96.01
38.9–1.5 (1.6–1.5)
6.7 (55.6)
218736 (21656)
42898 (6210)
13.7 (2.0)
98.3 (99.4)
5.1 (3.5)
2089
259
34.7–1.5
40761
2038
16.1
19.8
0.012
1.26
† Rmerge= 100 ?P
hkl
P
ijIiðhklÞ ? hIðhklÞij=P
hkl
P
iIiðhklÞ, where hI(hkl)i is the mean
intensity of the scaled observations Ii(hkl).

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Rupp, 2003; Matthews, 1968) was calculated using the program
MATTHEWS_COEF (Collaborative
Number 4, 1994), which suggested the presence of only one BipD
molecule in the asymmetric unit, with a solvent content of 42%. The
data set had overall and outer shell Rmergevalues of 6.7% and 55.6%,
respectively (Table 1).
ComputationalProject,
4. Structure determination
One monomer of the previously solved structure of BipD (Erskine et
al., 2006; PDB code 2izp) was used as the search model in molecular
replacement using Phaser (Read, 2001). Of the possible alternative
space groups, only one (C2) yielded a solution (rotational Z score
16.7, translational Z score 13.6, log-likelihood gain 1788) which gave
good crystal packing. The model was then subjected to several rounds
of refinement with REFMAC (Murshudov et al., 1997) and rebuilding
using Coot (Emsley & Cowtan, 2004) and 259 water molecules were
added. After anisotropic temperature-factor refinement, the final R
factor and Rfreewere 16.1% and 19.8%, respectively (Table 1). The
coordinates and structure factors have been deposited in the Protein
Data Bank (http://www.wwpdb.org) with accession code 3nft. The
significance of putative oligomeric states arising from symmetry
operations of the new crystal form were analysed by use of the PISA
server provided by the European Bioinformatics Institute (Krissinel
& Henrick, 2007).
Continuous electron density was visible for most of the protein
except for the first 33 N-terminal residues, the loop formed by resi-
dues 113–121 and six residues at the C-terminal end. A sample of the
electron density showing its appreciable atomicity is shown in Fig. 2.
The current structure superimposes on the previous structure with an
r.m.s. deviation of 0.65 A˚for the 253 equivalent C?atoms that are
closer than 3.5 A˚following least-squares fitting (Fig. 3). Residues
104–112, which were rather poorly defined in the previous electron-
density map at 2.1 A˚resolution (Erskine et al., 2006), are extremely
well defined in the new map, thus improving the definition of the final
2–3 turns of helix ?3. The same improvement in map quality is seen
for residues at the other end of this loop in the range 125–128 which
lead into helix ?4 and are involved in putative dimer interactions.
Interestingly, the putative dimer interface relating the two mono-
mers in the asymmetric unit of the earlier crystal form (shown in
Fig. 7 of Erskine et al., 2006) is conserved in the new crystal form, with
the only differences being that in the new form the two monomers are
re-oriented slightly and are related by crystallographic rather than
noncrystallographic symmetry. These interactions involve helices ?4
and ?8 pairing with their counterparts in the adjacent monomer in an
antiparallel manner owing to an intervening (pseudo)twofold axis.
The C-terminal end of ?8 is the most highly conserved region of the
molecule. The total surface-accessible area of each monomer that is
buried in this putative dimer interface (1271 A˚2) is larger than that
reported for the previous crystal form (970 A˚2; Erskine et al., 2006).
The apparent strengthening of the dimer interface may stem from the
improved definition of the residues in the range 125–128 which form
more extensive contacts in the new crystal form. It is interesting that
the same putative dimer interface is observed in yet further crystal
forms of BipD that have been reported by others (Johnson et al.,
2007; PDB codes 2ixr and 2j9t). Intriguingly, the putative ligand
(phosphocholine) was not visible in the electron-density map,
although it appears to have acted as a crystallization additive in
producing the significantly improved crystal form which we report
here.
We thank the Gerald Kerkut Charitable Trust for a studentship to
MP. We are indebted to Diamond Light Source for beam time and
travel support.
References
Aldhous, P. (2005). Nature (London), 434, 692–693.
Attree, O. & Attree, I. (2001). Microbiology, 147, 3197–3199.
Blocker, A., Holden, D. & Cornelis, G. (2000). Cell. Microbiol. 2, 387–390.
Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50,
760–763.
Dance, D. A. (2002). Curr. Opin. Infect. Dis. 15, 127–132.
Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–2132.
Erskine, P. T., Knight, M. J., Ruaux, A., Mikolajek, H., Wong Fat Sang, N.,
Withers, J., Gill, R., Wood, S. P., Wood, M., Fox, G. C. & Cooper, J. B. (2006).
J. Mol. Biol. 363, 125–136.
Espina, M., Ausar, S. F., Middaugh, C. R., Baxter, M. A., Picking, W. D. &
Picking, W. L. (2007). Protein Sci. 16, 704–714.
Evans, P. (2006). Acta Cryst. D62, 72–82.
French, S. & Wilson, K. (1978). Acta Cryst. A34, 517–525.
Gan, Y.-H. (2005). J. Infect. Dis. 192, 1845–1850.
Holden, M. T. G. et al. (2004). Proc. Natl Acad. Sci. USA, 101, 14240–14245.
Hueck, C. J. (1998). Microbiol. Mol. Biol. Rev. 62, 379–433.
Johnson, S., Roversi, P., Espina, M., Olive, A., Deane, J. E., Birket, S., Field, T.,
Picking, W. D., Blocker, A. J., Galyov, E. E., Picking, W. L. & Lea, S. M.
(2007). J. Biol. Chem. 282, 4035–4044.
Kaniga, K., Trollinger, D. & Galan, J. E. (1995). J. Bacteriol. 177, 7078–7085.
Kantardjieff, K. A. & Rupp, B. (2003). Protein Sci. 12, 1865–1871.
Knight, M. J., Ruaux, A., Mikolajek, H., Erskine, P. T., Gill, R., Wood, S. P.,
Wood, M. & Cooper, J. B. (2006). Acta Cryst. F62, 761–764.
Krissinel, E. & Henrick, K. (2007). J. Mol. Biol. 372, 774–797.
Leslie, A. G. W. (2006). Acta Cryst. D62, 48–57.
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.
Mecsas, J. & Strauss, E. J. (1996). Emerg. Infect. Dis. 2, 271–288.
structural communications
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Pal et al.
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Acta Cryst. (2010). F66, 990–993
Figure 2
A sample of the 2Fo? Fcelectron-density map at 1.5 A˚resolution contoured at
2 r.m.s. (blue contours).
Figure 3
A superposition of the 1.5 A˚resolution structure of BipD (dark yellow) with the
previous model solved at 2.1 A˚resolution (cyan) in a different space group. The
only regions where the structures differ appreciably were very poorly defined in the
earlier analysis.

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Me ´nard, R., Sansonetti, P. & Parsot, C. (1994). EMBO J. 13, 5293–5302.
Mota, L. J., Sorg, I. & Cornelis, G. R. (2005). FEMS Microbiol. Lett. 252, 1–10.
Moraes, T. F., Spreter, T. & Strynadka, N. C. J. (2008). Curr. Opin. Struct. Biol.
18, 258–266.
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Acta Cryst. D53,
240–255.
Pettersson, J., Nordfelth, R., Dubinina, E., Bergman, T., Gustafsson, M.,
Magnusson, K. E. & Wolf-Watz, H. (1996). Science, 273, 1231–1233.
Rainbow, L., Hart, C. A. & Winstanley, C. (2002). J. Med. Microbiol. 51,
374–384.
Read, R. J. (2001). Acta Cryst. D57, 1373–1382.
Roversi, P., Johnson, S., Field, T., Deane, J. E., Galyov, E. E. & Lea, S. M.
(2006). Acta Cryst. F62, 861–864.
Stevens, M. P., Haque, A., Atkins, T., Hill, J., Wood, M. W., Easton, A., Nelson,
M., Underwood-Fowler, C., Titball, R. W., Bancroft, G. J. & Galyov, E. E.
(2004). Microbiology, 150, 2669–2676.
Stevens, M. P., Wood, M. W., Taylor, L. A., Monaghan, P., Hawes, P., Jones,
P. W., Wallis, T. S. & Galyov, E. E. (2002). Mol. Microbiol. 46, 649–659.
Yip, C. K. & Strynadka, N. C. J. (2006). Trends Biochem. Sci. 31, 223–
230.
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