Maltose-binding protein is the periplasmic component of the ABC transporter

responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis

pv. citri maltose-binding protein MalE has been crystallized at 293 Kusing

the hanging-drop vapour-diffusion method. The crystal belonged to the

primitive hexagonal space group P6_122, with unit-cell parameters a = 123.59,

b = 123.59, c = 304.20 Å, and contained two molecules in the asymetric unit. It

diffracted to 2.24 Å resolution

Balan, A.

Barbosa, J. A. R. G.

Ferreira, L. C. S.

Souza, C. S.

Thomas, L.

English

PubMed

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 Kusing the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 angstrom, and contained two molecules in the asymetric unit. It diffracted to 2.24 angstrom resolution.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[01/07540-3]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[04/02716-4]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[00/10266-8]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Associacao Brasileira de Tecnologia de Luz Sincrotron (ABTLuS)US Department of Energy (DOE

SOUZA, C. S.

FERREIRA, L. C. S.

THOMAS, L.

BARBOSA, J. A. R. G.

BALAN, A.

Universidade de São Paulo

Acta Crystallographica Section F Structural Biology and Crystallization Communications

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

C. S. Souza

L. C. S. Ferreira

L. Thomas

J. A. R. G. Barbosa

A. Balan

Crossref

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogenXanthomonas axonopodispv.citri

Caltech Authors - Main

crystallization communications
Acta Cryst. (2009). F65, 105–107 doi:10.1107/S1744309108041833 105
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Crystallization, data collection and data processing
of maltose-binding protein (MalE) from the
phytopathogen Xanthomonas axonopodis pv. citri
C. S. Souza,a L. C. S. Ferreira,a
L. Thomas,b J. A. R. G. Barbosac*
and A. Balanc*
aDepartamento de Microbiologia, Instituto de
Cieˆncias Biome´dicas II, Universidade de Sa˜o
Paulo, Sa˜o Paulo, SP, Brazil, bHoward Hughes
Medical Institute, Division of Biology, California
Institute of Technology, 1200 East California
Boulevard, Pasadena, California, USA, and
cCenter for Structural Molecular Biology
(CeBiMe), Brazilian Synchrotron Light
Laboratory (LNLS), CP 6192, Campinas,
SP 13084-971, Brazil
Correspondence e-mail: joao@lnls.br,
abalan@usp.br
Received 27 October 2008
Accepted 9 December 2008
Maltose-binding protein is the periplasmic component of the ABC transporter
responsible for the uptake of maltose/maltodextrins. The Xanthomonas axono-
podis pv. citri maltose-binding protein MalE has been crystallized at 293 K using
the hanging-drop vapour-diffusion method. The crystal belonged to the
primitive hexagonal space group P6122, with unit-cell parameters a = 123.59,
b = 123.59, c = 304.20 A˚, and contained two molecules in the asymetric unit. It
diffracted to 2.24 A˚ resolution.
1. Introduction
Maltose- or maltodextrin-binding proteins (MBPs) are located in the
periplasm of Gram-negative bacterial species, where they interact
with substrates such as the disaccharide maltose, longer linear
maltodextrins and even larger and more complex cyclodextrin
molecules before uptake to the interior of the cell (Boos & Shuman,
1998; Tam& Saier, 1993). InEscherichia coliK12, the active transport
of maltose and maltodextrins requires MBP and an ATP-dependent
membrane-associated transport system made up of two integral
membrane proteins (MalF and MalG) and two copies of an ATP-
hydrolyzing subunit (MalK) (Hall, Ganesan et al., 1997; Hall, Gehring
et al., 1997; Hall, Thorgeirsson et al., 1997). Similar to other ABC-type
transport binding components, MBP or MalE typically consists of two
nearly symmetrical lobes separated by a hinge region in which the
substrate-binding site lies (Spurlino et al., 1991). After binding, the
protein undergoes a conformational change as a direct result of
bending and twisting movements of the lobes towards each other,
leading to substrate enclosure (Quiocho et al., 1997; Saul et al., 2003;
Millet et al., 2003; Telmer & Shilton, 2003). MBPs have also been
characterized as chemoreceptors for maltose taxis after interaction of
the bound complex with the periplasmic portion of the Tar protein
(Zhang et al., 1999).
Much of the existing knowledge about microbial ABC transporters
has been gleaned from studies of the high-affinity maltose-transport
system in E. coli. Indeed, E. coliMBP (Eco_MBP) has been subjected
to a battery of genetic, biochemical and biophysical studies that have
produced a wealth of information about the structure and ligand-
binding properties of Eco_MBP (Martineau et al., 1990; Spurlino et
al., 1991; Sharff et al., 1992). However, it is unclear to what degree
these observations can be generalized to other bacteria: that is, what
are the structural similarities and differences between Eco_MBP and
MBPs from distant relatives such as bacteria living in different
habitats or host organisms? The crystal structures of six bacterial
MBPs have been reported from Pyrococcus furiosus (PDB code 1elj;
Evdokimov et al., 2001), Thermococcus litoralis (1eu8; Diez et al.,
2001), Alicyclobacillus acidocaldarius (1urg; Schafer et al., 2004),
E. coli (1anf; Spurlino et al., 1991), Thermus thermophilus (2gh9; M. J.
Cuneo, A. Changela, L. S. Beese & H. W. Hellinga, unpublished
work) and Thermotoga maritima (2fnc; M. J. Cuneo & A. Changela,
unpublished work) and they all share a similar structural organization
irrespective of the relatively low amino-acid sequence conservation.
The crystal structures of MBPs in the closed and open states reveal
a cleft between two lobes in which the oligosaccharide substrate binds
# 2009 International Union of Crystallography
All rights reserved
and is completely engulfed by rotation of the lobes. While delivering
the substrate to the transmembrane subunits, the binding protein
seems to stimulate ATP hydrolysis. The observation that MBP is
trapped in a complex with MalFGK2 in the presence of a transition-
state analogue (Mg, ADP, vanadate) suggests that the binding of
MBP induces conformational changes in the transporter and thereby
triggers ATP hydrolysis (Oldham et al., 2007).
However, maltose/maltodextrin-binding proteins from plant-
associated bacteria have not been functionally or structurally
characterized. The genus Xanthomonas comprises a diverse and
economically important group of bacterial phytopathogens that
belong to the -subdivision of the proteobacteria. Xanthomonas
axonopodis pv. citri is the causative agent of citrus canker, a disease
that affects several citrus cultivars around the world and that results
in significant economic losses worldwide (Brunings & Gabriel, 2003).
Definition of the complete genome sequence of X. axonopodis pv.
citri revealed that approximately 4% of the genomic content
encoding structural genes is dedicated to the synthesis of ATP-
dependent transport systems, represented mainly by ABC transpor-
ters (da Silva et al., 2002). Most of the maltose-uptake system is
present in a single operon encompassing three cistrons (malElacFG)
that encode the periplasmic binding protein MalE and two trans-
membrane proteins LacF and LacG. The nucleotide-binding domain
corresponding to the MalK protein was identified in another region
of the genome (the ugpC gene, which shares 50% sequence identity
to E. coli malK). The periplasmic protein contains 456 amino acids,
including a putative 19-amino-acid signal peptide, and should drive
both the specificity and affinity of the maltose/maltodextrin-transport
system in this plant pathogen. Previous evidence has suggested that
recombinant X. axonopodis pv. citriMalE (Xac_MalE) binds maltose
(Balan et al., 2005), but the functionality of this operon in
X. axonopodis pv. citri is still unclear. In order to determinate the
three-dimensional structure of this protein in the presence of maltose,
we report the crystallization, data collection and initial structural
analysis of recombinant Xac_MalE expressed in E. coli. The X-ray
crystal structure of the present protein is expected to reveal structural
features that may be common to MBPs from other phytopathogenic
microorganisms, since the amino-acid sequences of MalE and of the
other components of the transporter share more than 80% identity.
2. Crystallization
The nucleotide sequence encoding mature MalE (without the initial
19-amino-acid signal peptide) was amplified by PCR from X. axono-
podis pv. citri var. 306 and cloned into a pET28a vector (Novagen) for
production of a cytoplasmic protein with a His6 tag genetically fused
at the N-terminal end. The recombinant protein was purified from
soluble extracts by immobilized nickel-affinity chromatography (1 ml
HisTrap HP fromAmersham Biosciences; Balan et al., 2005). Samples
dialyzed against 20 mM Tris–HCl pH 8.0, 50 mM NaCl were kept at
277 K at a final concentration of 40 mg ml1. Samples of recombinant
Xac_MalE protein (10 mg ml1) in the absence and the presence of
various sugars including maltose, trehalose, lactose, -cyclodextrin
and -cyclodextrin (the protein:sugar ratio was 1:2) were submitted
to crystallization trials using both the sitting-drop and hanging-drop
vapour-diffusion methods. Screenings were performed using Crystal
Screens 1 and 2, Index Screen, SaltRX and PEG/Ion Screen (all from
Hampton Research), JBScreen 1–5 (Jena Bioscience) and Wizard I
and II (Emerald Biostructure) according to the instructions of the
manufacturers. Drops were prepared by mixing equal volumes (2 ml)
of protein and reservoir solution and were equilibrated against 400 ml
reservoir solution at 294 K. Refinement of the crystallization condi-
tions was carried out by varying the precipitant and salt concentra-
tions as well as the pH and by the incorporation of additives.
3. Data collection and processing
The final crystallographic data were collected on protein crystallo-
graphy beamline 9-2 of the Stanford Synchrotron Radiation
Laboratory (Menlo Park, California, USA), a national user facility
operated by Stanford University on behalf of the US Department of
Energy, Office of Basic Energy Sciences and Biological and Envir-
onmental Research. The wavelength was 0.82 A˚ and a MarMosaic
crystallization communications
106 Souza et al.  MalE Acta Cryst. (2009). F65, 105–107
Table 1
Crystallization conditions and resolution obtained for the Xac_MalE crystals.
Nd, no diffraction.
Crystallization condition Additive Cryoprotectant Ligand Resolution (A˚)
4% PEG 8000 — 20% glycerol Maltose Nd
25% PEG MME 2000 — 15% glycerol Maltose Nd
0.1M Na MES pH 6.5, 0.6M sodium chloride, 20% PEG 4000 — 20% glycerol Maltose Nd
0.1M Na MES pH 6.5, 12% PEG 20 000 — — Maltose 10.0
0.1M Na HEPES pH 7.5, 12% PEG 20 000 — — Maltose 7.0
0.1M Na HEPES pH 7.5, 0.2M sodium acetate, 20% PEG 3000 30% dioxane 15% glycerol Maltose 3.7
0.1M Na HEPES pH 7.5, 0.2M sodium acetate, 20% PEG 3000 — — Maltose 3.4
0.1M Na HEPES pH 7.5, 0.2M sodium acetate, 20% PEG 3000 0.1M trimethylamine–HCl — Maltose 3.4
0.1M Tris–HCl pH 8.5, 0.2M lithium sulfate, 17% PEG 4000 — 20% glycerol Maltose 3.2
1.2M sodium tartrate, 0.1M bis-Tris propane pH 7.0 — 25% glycerol Maltose 2.8
3.5M sodium formate, 0.1M Tris–HCl pH 8.0 — — Maltose 2.24
Figure 1
X. axonopodis pv. citri MalE crystals obtained in 3.5M sodium formate and 0.1M
Tris pH 8.0; the crystals diffracted to 2.24 A˚ resolution.
325 CCD detector was used to record the oscillation data with
’ = 0.25. Crystals were cooled in a stream of nitrogen gas at 100 K
in order to minimize radiation damage. The data sets were integrated
usingMOSFLM v.6.2.6 (Leslie, 1999) and scaled with SCALA v.3.2.19
(Evans, 1993). The Matthews coefficient was calculated using the
CCP4 package (Collaborative Computational Project, Number 4,
1994).
4. Results and discussion
The single-step purification by affinity chromatography was sufficient
to produce crystallization-quality protein (Balan et al., 2005). Crystal
growth usually occurred in 6 d, leading to bipyramidal crystals with
dimensions ranging from 100 to 500 mm. Crystals were obtained in
more than 15 different crystallization conditions containing poly-
ethylene glycol (PEG), MES or HEPES in the pH range 6.5–7.5, but
were only obtained in the presence of maltose. However, none of
these crystals were capable of generating diffraction patterns with
resolution better than 7 A˚. Cryoprotection with 15–20% glycerol or
PEG 400 and annealing (1–15 s) were tested in order to increase the
resolution, with no success. Refinement of all crystallization condi-
tions revealed that crystals grown in 0.1M HEPES pH 7.5 or 0.1M
Tris pH 8.0 with lithium sulfate, sodium acetate or sodium formate
showed an increase in the resolution limit to around 3.2–3.4 A˚
(Table 1). All crystallization trials with sugars other than maltose
were unsuccessful.
In order to further improve the quality of the crystals, the salt
concentration in the samples was increased from 20 to 50 mM Tris–
HCl and the pH was increased to 8.0. Finally, good diffraction data
were obtained to a maximum resolution of 2.24 A˚ on increasing the
pH from 7.0 to 8.0 in 0.1M Tris–HCl and 3.5M sodium formate
(Fig. 1). Crystals showed the symmetry and systematic absences of the
primitive hexagonal space group P6122. The data-collection statistics
are summarized in Table 2. The Matthews coefficient (Matthews,
1968) was calculated to be 3.35 A˚3 Da1 and the solvent content was
63.4% assuming the presence of two molecules in the asymmetric
unit.
Ongoing experiments will elucidate the structural organization of
Xac_MalE, which will shed light on the mechanism of sugar transport
in Xanthomonas species as well as in other phytopathogenic bacteria.
Elucidation of the crystal structure of Xac_MalE will also reveal
possible conserved structural and functional features that are shared
with other substrate-binding proteins of maltose ABC transporters
reported from other bacterial and archaeal species.
This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do
Estado de Sa˜o Paulo (grants 01/07540-3, 04/02716-4 and 00/10266-8),
Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico
(CNPq), Associac¸a˜o Brasileira de Tecnologia de Luz Sı´ncrotron
(ABTLuS) and the US Department of Energy. We would like to
thank Andre´ia N. Meza and Camila Calderon for their technical
assistance and Dr Pamela Bjorkman for allowing part of this research
to be carried out in her laboratory.
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crystallization communications
Acta Cryst. (2009). F65, 105–107 Souza et al.  MalE 107
Table 2
Data-collection and processing statistics of X. axonopodis pv. citri MalE crystal.
Values in parentheses are for the last resolution shell.
Space group P6122
Unit-cell parameters (A˚, ) a = 123.59, b = 123.59, c = 304.20,
 = 90.00,  = 90.00,  = 120.00
Mosaicity () 0.24
Temperature (K) 100
Wavelength (A˚) 0.84
Oscillation () 0.25
Crystal-to-detector distance (mm) 325.0
No. of frames 360
Resolution limits (A˚) 50.00–2.24 (2.36–2.24)
I/(I) after merging 23.1 (4.5)
Completeness (%) 99.9 (99.8)
Multiplicity 10.9 (11.1)
Rmerge 0.07 (0.45)
No. of reflections 726568 (105326)
No. of unique reflections 66688 (9508)


Crystallization, data collection and data processing

of maltose-binding protein (MalE) from the

phytopathogen Xanthomonas axonopodis pv. citri

Biblioteca Digital da Produção Intelectual da Universidade de São Paulo (BDPI/USP)

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 Kusing the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 angstrom, and contained two molecules in the asymetric unit. It diffracted to 2.24 angstrom resolution

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Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

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