Tài liệu Báo cáo khoa học: Structural basis for cyclodextrin recognition by Thermoactinomyces vulgaris cyclo⁄maltodextrin-binding protein ppt

Structural basis for cyclodextrin recognition by
Thermoactinomyces vulgaris cyclo⁄ maltodextrin-binding
protein
Takashi Tonozuka
1
, Akiko Sogawa
1
, Mitsugu Yamada
2
, Naoki Matsumoto
1
, Hiromi Yoshida
2,3
,
Shigehiro Kamitori
2,3
, Kazuhiro Ichikawa
1
, Masahiro Mizuno
1,
*, Atsushi Nishikawa
1
and
Yoshiyuki Sakano
1
1 Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Japan
2 Graduate School of Medicine, Kagawa University, Japan
3 Information Technology Center, Kagawa University, Japan
Cyclodextrins (CDs) are cyclic a-1,4-glucans, and the
central cavity of CDs can host a large number of che-
micals by hydrophobic interaction [1]. A thermophilic

actinomycete, Thermoactinomyces vulgaris R-47, pro-
duces two CD-hydrolyzing enzymes, TVA I [2] and
TVA II [3]. We have determined the crystal structures
Keywords
crystal structure; cyclodextrin; sugar-binding
protein; sugar transporter; Thermoactinomyces
vulgaris
Correspondence
T. Tonozuka, Department of Applied
Biological Science, Tokyo University of
Agriculture and Technology, 3-5-8 Saiwai-
cho, Fuchu, Tokyo 183–8509, Japan
Fax: +81 42 3675705
Tel: +81 42 3675702
E-mail:
*Present address
Department of Chemistry and Material
Engineering, Shinshu University, Nagano,
Japan
Database
The atomic coordinates and structural fac-
tors described in this paper have been
deposited in the Protein Data Bank (http://
www.rcsb.org/) with the accession code
2DFZ
(Received 11 December 2006, revised 13
February 2007, accepted 21 February 2007)
doi:10.1111/j.1742-4658.2007.05753.x
The crystal structure of a Thermoactinomyces vulgaris cyclo ⁄ maltodextrin-
binding protein (TvuCMBP) complexed with c-cyclodextrin has been deter-

mined. Like Escherichia coli maltodextrin-binding protein (EcoMBP) and
other bacterial sugar-binding proteins, TvuCMBP consists of two domains,
an N- and a C-domain, both of which are composed of a central b-sheet
surrounded by a-helices; the domains are joined by a hinge region contain-
ing three segments. c-Cyclodextrin is located at a cleft formed by the two
domains. A common functional conformational change has been reported
in this protein family, which involves switching from an open form
to a sugar-transporter bindable form, designated a closed form. The
TvuCMBP–c-cyclodextrin complex structurally resembles the closed form
of EcoMBP, indicating that TvuCMBP complexed with c-cyclodextrin
adopts the closed form. The fluorescence measurements also showed that
the affinities of TvuCMBP for cyclodextrins were almost equal to those for
maltooligosaccharides. Despite having similar folds, the sugar-binding site
of the N-domain part of TvuCMBP and other bacterial sugar-binding pro-
teins are strikingly different. In TvuCMBP, the side-chain of Leu59 pro-
trudes from the N-domain part into the sugar-binding cleft and orients
toward the central cavity of c-cyclodextrin, thus Leu59 appears to play the
key role in binding. The cleft of the sugar-binding site of TvuCMBP is also
wider than that of EcoMBP. These findings suggest that the sugar-binding
site of the N-domain part and the wide cleft are critical in determining the
specificity of TvuCMBP for c-cyclodextrin.
Abbreviations
CD, cyclodextrin; EcoMBP, Escherichia coli maltodextrin-binding protein; Mol, molecule; SeMet, selenomethionine; TliTMBP, Thermococcus
litoralis trehalose ⁄ maltose-binding protein; TvuCMBP, Thermoactinomyces vulgaris cyclo ⁄ maltodextrin-binding protein.
FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2109
of TVA I and TVA II [4,5] and TVA II complexed
with CDs [6]. To find the proteins physiologically rela-
ted to these enzymes, the flanking regions of the genes
were sequenced. A gene homologous to those of the
bacterial sugar-binding protein family was found to be

located upstream of the TVA II gene, and the affinities
of this protein for c-CD were higher than that for
maltose [7]. The results suggested that this protein par-
ticipates in binding to not only linear maltooligosac-
charides but also to CDs, and thus was designated
cyclo ⁄ maltodextrin-binding protein ( TvuCMBP).
The bacterial sugar-binding protein is a member of
the bacterial ATP-binding cassette transport system,
and the mechanism of the maltodextrin transport in
Escherichia coli has been well studied [8,9]. The malto-
dextrin-binding protein from E. coli (EcoMBP) is also
widely used as a tool for genetic engineering [10] and
as a biosensing platform [11,12]. The ATP-binding cas-
sette transporters of E. coli are composed of a mem-
brane-bound complex comprising the two permease
subunits, MalF and MalG, and two copies of the
ATPase subunit, MalK, and all together are named
the MalFGK
2
transporter. EcoMBP interacts with the
MalFGK
2
transporter complex, and thus is essential
for the transport activity [13]. Genetic analyses showed
that Klebsiella oxytoca [14] and T. vulgaris [7] have a
CD transport system similar to the maltodextrin trans-
port system in E. coli, and that the CD-binding protein
would participate in the CD transport.
Crystal structures of several sugar-binding proteins,
such as Eco MBP [15–19], Thermococcus litoralis treha-

lose ⁄ maltose-binding protein (TliTMBP) [20], Pyrococ-
cus furiosus maltodextrin-binding protein [21] and
Alicyclobacillus acidocaldarius maltose ⁄ maltodextrin-
binding protein [22], have been determined. These pro-
teins share a common structural motif that consists of
two domains, joined by a hinge region, which sur-
round a sugar-binding site [11,15]. A common drastic
conformational change is found in this protein family,
which participates in switching from an open form to
a closed form [16]. In EcoMBP, the closed form has
been observed in the complexes with linear maltooligo-
saccharides, such as maltose, maltotriose and malto-
tetraose [17], and this form is capable of interacting
with the MalFGK
2
sugar-transporter complex. In
contrast, the open form does not have the ability to
perform the proper interaction with the MalFGK
2
sugar-transporter complex. Interestingly, EcoMBP
adopts the open form in the unliganded protein but
also in the complex with b-CD [18].
Here we present the crystal structure of TvuCMBP
complexed with c-CD. Unlike EcoMBP complexed
with b-CD, the Tvu CMBP–c-CD complex was deter-
mined as the closed form. The structure provides
evidence that the architecture of TvuCMBP is well
optimized for interacting with the central hydrophobic
cavity of c-CD.
Results and Discussion

Determination of the structure of Tvu CMBP
complexed with c-CD
Crystals of native and selenomethionine (SeMet)-sub-
stituted TvuCMBP were obtained. Both crystals belong
to the C2 space group, but the unit cell parameters dif-
fer (Table 1). One molecule is found in an asymmetric
unit of the SeMet-substituted crystal, whereas four
molecules (Mol-A–D) are contained in an asymmetric
unit of the native crystal. A rough model of TvuCMBP
was built based on the data from the SeMet-substi-
tuted crystal, and the structure was further refined
using the native TvuCMBP data set at 2.5 A
˚
resolu-
tion. The refinement converged to R
cryst
¼ 21.8% and
R
free
¼ 26.8%. A Ramachandran plot was calculated
with the program procheck of CCP4 [23]. No residue
is present in the disallowed regions or the generously
allowed regions, and 91.7, 90.8, 89.8 and 89.2% of res-
idues in Mol-A–D, respectively, are in the most fav-
ored regions. Of all Mol-A–D, the first 16 N-terminal
amino acid residues are not visible. In the case of
A. acidocaldarius maltose ⁄ maltodextrin-binding protein,
no electron density corresponding to the N-terminal
segment was observed [22]. The N-terminal segment of
the sugar-binding protein family has been proposed to

be a flexible linker, which allows the proteins to inter-
act with carbohydrates as well as the membrane-bound
transport proteins [22,24].
An omit map shows that one c-CD binds to each
TvuCMBP molecule (Fig. 1A). Although noncrystallo-
graphic symmetry restraints were not applied in the
late stage of the refinement, c-CD was found to form
the same contacts with TvuCMBP in Mol-A–D. The
rmsd between Mol-A and Mol-B, Mol-A and Mol-C,
Mol-A and Mol-D are 0.77, 0.94, and 0.74 A
˚
, respect-
ively, for all atoms, and 0.42, 0.55, and 0.43 A
˚
,
respectively, for main-chain atoms, suggesting that the
four structures are almost identical. To facilitate des-
cription, the following depiction is based on Mol-A.
Overall structure of TvuCMBP
The bacterial sugar-binding proteins have been reported
to share a common structural motif [11,15]. Like other
bacterial sugar-binding proteins [15–22], TvuCMBP
consists of two domains, the N-domain (residues 17–127
Structure of cyclo ⁄ maltodextrin-binding protein T. Tonozuka et al.
2110 FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS
and 283–330) and the C-domain (residues 131–279 and
334–397) (Fig. 1B,C). Both domains have similar archi-
tectures; a b-sheet is located at the center, surrounded
by a-helices. The two domains are joined by a hinge
region, which contains three segments (residues 128–

130, 280–282 and 331–333). The sugar-binding site is
located at a cleft formed by the two domains. Structural
homology was searched for using the DALI server [25],
and many bacterial sugar-binding proteins and other
periplasmic binding proteins were listed. In this search,
TvuCMBP most resembled a closed form of EcoMBP
complexed with maltotetraose (4MBP; Z score, 44.0;
rmsd, 2.1 A
˚
; LALI (length of the alignment excluding
insertions and deletions), 363) [17] and TliTMBP
complexed with trehalose (1EU8, Z score, 36.5; rmsd,
2.7 A
˚
; LALI, 358) [20]. The Ca backbone of the
TvuCMBP–c-CD complex was superimposed with that
of EcoMBP–maltotetraose and TliTMBP–trehalose
complexes (Fig. 2A) using swiss-pdb viewer [26]. The
amino acid sequence of TvuCMBP is 27% identical to
that of EcoMBP and 22% identical to that of TliTMBP,
which are moderate values, and many structural differ-
ences were found among the three proteins, especially in
several regions composing the sugar-binding sites (as
will be discussed in detail below). The folds of these
three backbones are, however, almost identical, indica-
ting that TvuCMBP complexed with c-CD adopts the
closed form. The structure of EcoMBP complexed with
b-CD has been reported to adopt the open form, and
the superposition of C-domains of TvuCMBP–c-CD
and EcoMBP–b-CD complexes shows that the Ca back-

bones of their N-domains are completely different
(Fig. 2B).
The C-domain parts of the sugar-binding sites
of TvuCMBP and related proteins
As the sugar-binding site is formed by N- and
C-domains, residues involved in sugar binding are
grouped into two parts, the N-domain part and the
Table 1. Data collection and refinement statistics.
Derivative (SeMet)
Native
Peak Edge
Data collection
Beamline PF BL-5 A PF-AR NW-12
Wavelength 0.97932 0.98000 1.0000
Space group C2 C2
Cell dimensions
a (A
˚
) 85.3 167.4
b (A
˚
) 49.3 95.3
c (A
˚
) 87.6 117.1
b (°) 94.9 131.6
Resolution range (A
˚
) 50–2.30 (2.38–2.30)
b

50–2.30 (2.38–2.30)
b
50–2.50 (2.66–2.50)
b
Total reflections 111 339 111 287 181 528
Unique reflections 15 803 15 798 47 691
Completeness 96.7 (89.7)
b
96.5 (87.7)
b
100.0 (100.0)
b
I ⁄ r(I) 43.5 (11.5)
b
43.3 (11.3)
b
24.0 (8.6)
b
R
merge
(%)
a
18.7 (26.5)
b
17.8 (26.2)
b
6.9 (21.7)
b
Refinement statistics
Number of atoms

Protein 11 856
Ligand 352
Water 539
R
cryst
(%) 21.8
R
free
(%) 26.8
rmsd
Bond length (A
˚
) 0.007
Bond angles (°) 1.40
Average B
Protein 26.0
Ligand 31.5
Water 28.1
a
R
merge
¼ SS|Ii–<I>| ⁄S<I>0.
b
The values for the highest resolution shells are given in parentheses.
T. Tonozuka et al. Structure of cyclo ⁄ maltodextrin-binding protein
FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2111
C-domain. The structure of c-CD shows a sliced coni-
cal form; the OH-2 and OH-3 hydroxyl groups of all
glucose residues are positioned on one side, and the
OH-6 hydroxyl groups are located on the other side.

The glucose residues of c-CD are labeled from Glc1 to
Glc8 as shown in Fig. 3. The OH-2 and OH-3 groups
of c-CD face to the N-domain part, whereas the OH-6
groups interact with the C-domain part of the
sugar-binding site. The C-domain part consists of four
regions designated region C-I (residues 170–177),
region C-II (residues 227–232), region C-III (residues
248–251) and region C-IV (residues 359–365). Four
aromatic residues, Tyr175, Tyr176, Trp250 and Trp360
interact with c-CD. The three residues, Tyr175,
Trp250 and Trp360, make stacking interactions with
Glc4, Glc5 and Glc3, respectively, and appear to be
the most important for binding (Figs 3A and 4A).
There are also many hydrogen bonds, either direct or
via water, between residues from the C-domain and
Glc3–Glc5 (Fig. 3A).
Superimposition of TvuCMBP, EcoMBP and
TliTMBP shows that the positions of four aromatic
residues, Tyr155, Phe156, Trp230 and Trp340, of
EcoMBP are identical (Fig. 4B). In TliTMBP, Tyr177,
Trp257, Tyr259 and Tyr370, are identified as the func-
tionally equivalent residues, but their positions are dif-
ferent (Fig. 4C). The whole structure of maltotetraose
(labeled from Glc1¢ to Glc4¢ as shown in Fig. 4B)
bound to EcoMBP exhibits a curved form, which is
similar to the portion (Glc1–Glc4) of the round shape
of c-CD bound to TvuCMBP. The conformations of
Glc3 bound to TvuCMBP and the third glucose resi-
due, Glc3¢, of maltotetraose bound to EcoMBP are
superimposed well, and Glc2 and Glc4 also adopt sim-

ilar conformations to Glc2¢ and Glc4¢ of maltotetraose
bound to EcoMBP (Fig. 4D). In the case of trehalose
(labeled from Glc4¢¢ to Glc5¢¢, as shown in Fig. 4C)
bound to TliTMBP, although the conformations of
Glc4 of TvuCMBP and corresponding glucose residues
bound to EcoMBP (Glc4¢) and TliTMBP (Glc4¢¢) are
similar, those between EcoMBP and TliTMBP are clo-
ser than those between TvuCMBP and TliTMBP, and
neither the first (Glc4¢¢) nor the second residues
(Glc5¢¢) of trehalose bound to TliTMBP strictly fit to
the glucose residues of c-CD bound to TvuCMBP.
These findings indicate that the sugar-binding mecha-
nisms of the C-domains of TvuCMBP and EcoMBP are
relatively conserved, whereas the different architecture of
the C-domain of Tli TMBP may be more s uitable for the
specific b inding to small oligosaccharides lik e trehalose.
Comparison of the N-domain parts of the
sugar-binding sites of TvuCMBP, EcoMBP
and TliTMBP
The N-domain part of the sugar-binding site consists of
three loops, region N-I (residues 25–33), region N-II
(residues 56–61) and region N-III (residues 80–85)
(Figs 3B and 5A). Compared with TvuCMBP, EcoMBP
and TliTMBP, the positions and the conformations of
the three regions are strikingly different (Fig. 5A–C).
Fig. 1. Three-dimensional structure of TvuCMBP complexed with
c-CD. (A) Stereoview of the omit map electron density for c-CD
bound to Mol-A with 2.0 r contoured level. The omit map was cal-
culated from the coefficients of the (F
obs

) F
calc
) and the resultant
phase angles after several cycles of refinement of the model exclu-
ding c-CD. (B) Overall structure of TvuCMBP complexed with c-CD.
N- and C- domains and the hinge region are shown by different gray
scales. The N- and C-termini and the bound c-CD are indicated. The
figure was generated using
MOLSCRIPT [40]. (C) A side view of
TvuCMBP. The orientation is rotated through 90° from that of (B).
Structure of cyclo ⁄ maltodextrin-binding protein T. Tonozuka et al.
2112 FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS
In TvuCMBP, the side-chain of Leu59 orients toward
the central cavity of c-CD, and thus Leu59 appears to
play the key role in binding. Another leucine residue,
Leu58, is located close to Leu59, and these two resi-
dues produce a hydrophobic environment, which con-
tributes to interact with the hydrophobic central cavity
of c-CD. Although the number of hydrogen bonds
between the N-domain and c-CD are much fewer than
those between the C-domain and c-CD (Fig. 3B),
region N-II plays an important role to form the
TvuCMBP–c-CD complex (Fig. 5A). The positions
and the conformations of the three loops of EcoMBP
are different from those of TvuCMBP. Trp62, located
at region N-I, is found at the center of the curved
form of maltotetraose (Fig. 5B). In TliTMBP, which
binds to the smallest sugar among the three sugar-
binding proteins, the three loops seem to play only
auxiliary roles, and Tyr121 and Trp295, both of which

are from the hinge region (b-strands located at the bot-
tom of the sugar-binding cleft), appear to be most
responsible for the binding to trehalose (Fig. 5C). No
aromatic residues equivalent to Tyr121 and Trp295 of
TliTMBP are found in TvuCMBP and EcoMBP. The
primary structures of the sugar-binding sites of the
three proteins were aligned based on the structural
comparison (Fig. 6). In TliTMBP, the conserved resi-
dues are found to be few. Between TvuCMBP and
EcoMBP, many residues, including Leu and Trp, seem
to be conserved, but the positions and the conforma-
tions of the residues at regions N-I–III are different, as
described above.
The capacities of the sugar-binding sites of
TvuCMBP, EcoMBP and TliTMBP, where all of the
conformations are the closed form, were compared
(Fig. 7A–C). The cleft of the sugar-binding site of
TvuCMBP is the widest among the three sugar-binding
proteins (Fig. 7A). Although the side-chain of Leu59 is
located in the cleft, the sugar-binding site around
Leu59 is wide open. Lys229 and Glu361 form protru-
sions at the entrance of the cleft, and the distance
between Nf of Lys229 and Oe1 of Glu361 is 16.7 A
˚
.
On the other hand, the width of the sugar-binding cleft
of EcoMBP is apparently narrower than that of
TvuCMBP (Fig. 7B). Similar protrusions, which are
formed by Asp209 and Arg344, are observed at the
entrance of the cleft of EcoMBP, but the distance

between Od2 of Asp209 and Ng2 of Arg344 is only
10.2 A
˚
.InTliTMBP, the cleft is the smallest among
the three proteins (Fig. 7C). The ligand, trehalose,
is half-buried, and the form of the cleft is markedly
different from those of TvuCMBP and EcoMBP.
These observations suggest that the structure of the
N-domain part and the capacity of the sugar-binding
site are critical in determining the specificity of the
bacterial sugar-binding proteins.
Evaluation of the binding affinities by
fluorescence measurements
The K
d
values of TvuCMBP for binding of sugars were
determined by measuring changes in fluorescence
(Table 2). TvuCMBP shows almost the same K
d
values
for CDs and maltooligosaccharides. A similar experi-
ment with a CD-binding protein from Klebsiella oxyto-
ca, CymE, was carried out by Pajatsch et al. [27] and
the K
d
values for a-CD, b-CD and c-CD were 0.02,
0.14 and 0.30 lm, respectively, whereas the value for a
maltooligosaccharide, maltoheptaose, was 70 lm. The
results indicate that, while the K
d

values of K. oxytoca
CymE is highly specific for CDs, while TvuCMBP
Fig. 2. Superposition of the Ca backbones.
The figure was generated using
RASTOP. (A)
Stereoview of the Ca backbone of
TvuCMBP–c-CD complex (blue), which are
superimposed on those of EcoMBP–malto-
tetraose (yellow; PDB ID, 4MBP) and
TliTMBP–trehalose complex (magenta; PDB
ID, 1EU8). (B) Comparison of the Ca back-
bones of TvuCMBP–c-CD complex (blue)
and EcoMBP–b-CD (orange; PDB ID,
1DMB). C-domains of the two structures
were superimposed. CDs are represented
as stick models.
T. Tonozuka et al. Structure of cyclo ⁄ maltodextrin-binding protein
FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2113
shows the high affinities for not only CDs but also
higher maltooligosaccharides.
It is impossible to determine, however, whether
TvuCMBP adopts the open form or the closed form
with the sugars listed in Table 2 by this experiment.
Although the experimental conditions were different,
the K
d
values of EcoMBP for maltose, maltotriose,
maltotetraose, and b-CD are reportedly 1.0, 0.2, 1.6
and 1.0 lm, respectively [13], indicating that the K
d

values of EcoMBP for linear maltooligosaccharides
and CDs are not markedly different [13,28]. A series
of studies of the crystal structures of EcoMBP show
that EcoMBP complexed with maltose, maltotriose or
maltotetraose adopts the closed form [17], while that
complexed with b-CD adopts the open form [18].
Because only the closed form is capable of interacting
with the membrane-bound sugar-transporter complex,
the specificity of the bacterial sugar-binding proteins
should be defined in terms not of the affinities for
sugars but of whether the protein adopts the open
form or the closed form. TvuCMBP–c-CD complex is
seen as the closed form (Fig. 2A,B), and this struc-
ture shows that the sugar-binding site of TvuCMBP
differs structurally from those of EcoMBP and
Fig. 3. Schematic drawing of the residues
located at C- (A) and N-domain (B) interact-
ing with c-CD. The figures were based on a
cartoon generated by the program
LIGPLOT
[41]. The number of glucose residues of
c-CD is labeled from 1 to 8. The C-domain
and N-domain parts are categorized into four
(C-I–C-IV) and three (N-I–N-III) regions,
respectively. Residues from hinge region
are also shown. s, oxygen atom; d, carbon
atom;
, nitrogen atom; Wat, water mole-
cule; ,hydrogen bond. Several residues
involved in hydrophobic interactions are also

illustrated.
Structure of cyclo ⁄ maltodextrin-binding protein T. Tonozuka et al.
2114 FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS
TliTMBP, both of which engage in binding to linear
maltooligosaccharides. The most remarkable feature is
that Leu59 protrudes into the sugar binding cleft
(Fig. 5A), which enables TvuCMBP to interact effi-
ciently with the hydrophobic cavity of CDs. The
hydrophobic environment provided by Leu58 and
Leu59 could also promote to incorporate CDs into
the sugar-binding cleft of TvuCMBP. In addition, the
wide cleft of TvuCMBP (Fig. 7A) is large enough to
accommodate CDs. These findings indicate that the
architecture of TvuCMBP is suitable for binding to
c-CD.
Fig. 4. Stereoview of four regions (regions C-I–C-IV) located at C-domain involving the sugar binding. Regions C-I, C-II, C-III and C-IV are blue,
yellow, magenta and red, respectively. The ligands shown in (A–C) are in gray. The figures were generated using
MOLSCRIPT [40] and
RASTER3D [42]. (A) TvuCMBP–c-CD complex. The glucose residues of c-CD are labeled from 1 to 8. (B) EcoMBP–maltotetraose complex. The
glucose residues of maltotetraose are labeled from 1¢ to 4¢. (C) TliTMBP–trehalose complex. The glucose residues of trehalose are labeled
from 4¢¢ to 5¢¢. (D) A superimposition of c-CD bound to TvuCMBP (blue) and maltotetraose bound to EcoMBP (orange). The two structures
(A) and (B), are superimposed and the portions of c-CD and maltotetraose are illustrated.
T. Tonozuka et al. Structure of cyclo ⁄ maltodextrin-binding protein
FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2115
The positions of the major aromatic residues located
at the C-domain part are conserved between
TvuCMBP and EcoMBP, but b-CD is not a proper lig-
and for EcoMBP, and in fact, glucose residues of
b-CD undergo stacking with aromatic residues derived
from C-domain, Tyr155, Trp230, and Trp340, whereas

poor interactions with the N-domain are observed in
the structure of the EcoMBP–b-CD complex [18].
Compared with the cyclodextrin glycosyltransferases
(CGTases) [29,30] and the CD-hydrolyzing enzymes
[31,32], their entire structure is completely different
from that of TvuCMBP, and also unlike TvuCMBP,
aromatic residues Tyr and Phe of the enzymes are
important for the interaction with the central cavity of
CDs. In order to determine whether TvuCMBP com-
plex with linear maltooligosaccharides adopts the open
form or the closed form, analyses of the structures of
TvuCMBP complexed with various sugars are now in
progress.
Experimental procedures
Construction of the expression plasmid
To construct the efficient expression plasmid of Tvu CMBP,
the initiation methionine codon was linked to the N-ter-
minal cysteine codon for the mature TvuCMBP, and the
pET expression system (Novagen, Darmstadt, Germany)
Fig. 5. Stereoview of three loops (regions
N-I–N-III) located at N-domain involving the
sugar binding in TvuCMBP, EcoMBP and
TliTMBP. Complex forms of three sugar-
binding proteins, TvuCMBP–c-CD (A),
EcoMBP–maltotetraose (B) and TliTMBP–
trehalose (C) are compared. The numbering
and the color representation of glucose resi-
dues of the ligands are as in Fig. 4. Regions
N-I, N-II and N-III are green, orange and
cyan, respectively. Other residues, which

are from the hinge region, are shown in
pink. The figures were generated using
MOLSCRIPT [40] and RASTER3D [42].
Fig. 6. Alignment of the primary structures
of regions N-I–III and C-I–IV. Identical amino
acid residues are shown in white on black.
The numbering of the amino acid sequences
is given.
Structure of cyclo ⁄ maltodextrin-binding protein T. Tonozuka et al.
2116 FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS
was also employed. A DNA fragment encoding the mature
TvuCMBP was prepared by polymerase chain reaction
using a plasmid, pTP-TVE [7], and oligonucleotides, 5¢-
GGG AAT TCC ATA TGT GCG GGC CAA AGC GGG
ATC CC-3¢ and 5¢-GTT TTC CCA GTC ACG ACG TTG
T-3¢, which have restriction sites of NdeI and EcoRI sites,
respectively, to facilitate cloning of the fragment. The
amplified fragment was digested with the enzymes NdeI and
EcoRI, and inserted into the NdeI and EcoRI sites of
pET21a, resulting in the plasmid pETCBP. The sequence of
the construct was verified by DNA sequencing.
Preparation of TvuCMBP
To produce TvuCMBP, E. coli BL21(DE3) harboring
pETCBP was cultured in Luria-Bertani medium containing
ampicillin (50 lgÆmL
)1
)toA
600
¼ 0.6–0.9, induced with iso-
propyl b-d-thiogalactopyranoside to a final concentration

of 0.5 mm, and grown for another 4 h at 37 °C. Cells were
centrifuged at 10 000 g using a Himac CR21G centrifuge
with R10A3 rotor (Hitachi, Tokyo, Japan), resuspended in
a buffer containing 50 mm sodium phosphate buffer
pH 6.0, and disrupted by sonication. The supernatant
obtained by centrifugation at 10 000 g using a Himac
CR21G centrifuge with R12A2 rotor (Hitachi) was pooled,
and batch-purified with amylose resin (New England
Biolabs, Ipswich, MA, USA) and c-CD as described previ-
ously [7]. The protein was further purified using cation-
exchange chromatography. The sample was applied onto a
HiLoad SP-Sepharose HR 16 ⁄ 10 column (1.6 · 10 cm, GE
Healthcare, Chalfont St Giles, UK) equilibrated with
50 mm sodium phosphate buffer pH 6.0, and eluted with a
linear gradient of 0–0.5 m sodium chloride in the same buf-
fer at a flow rate of 3 mLÆmin
)1
. As reported for a treha-
lose ⁄ maltose-binding protein from TliTMBP [20], two (one
major and one minor) peaks for TvuCMBP were obtained.
The N-terminal amino acid sequences of the two peaks
were analyzed using an ABI 476 A Protein Sequencer, and
both peaks were determined to be identical (CGPKRD-).
The protein from the major peak was crystallized. Protein
concentrations were determined by the measurement of
absorbance at 280 nm using the formula of Gill and von
Hippel [33] for the crystallization and the binding measure-
ments. SeMet-substituted TvuCMBP was prepared by over-
expressing the construct in E. coli strain B834(DE3), grown
in minimal medium supplemented with SeMet and purified

using a protocol similar to that of the native protein.
Crystallization and data collection
Crystals were grown by the hanging drop vapor diffusion
method at 20 °C. Crystals of TvuCMBP complexed with
c-CD were obtained by mixing 1 lL of well solution (25%
polyethylene glycol 6000, 0.1 m Mes pH 6.25, 5 mm c-CD)
and 1 lL of protein solution (10 mgÆmL
)1
TvuCMBP).
Crystals of SeMet-substituted TvuCMBP were obtained
with the same procedure. The crystals were transferred to a
solution consisting of 30% polyethylene glycol 6000, 0.1 m
Mes pH 6.25, 5 mm c-CD, and frozen in a 100 K nitrogen
stream. A native diffraction data set was collected at the
PF-AR NW-12 beamline (Tsukuba, Japan). Data were
processed with the program hkl2000 [34]. An attempt to
solve the structure by molecular replacement, using various
sugar-binding proteins, such as TliTMBP [20], EcoMBP
Fig. 7. Surface models of the sugar-binding sites of TvuCMBP,
EcoMBP and TliTMBP. Sugars are drawn in red sticks. The figures
were generated using
PYMOL. (A) TvuCMBP–c-CD complex. Leu59,
Lys229 and Glu361 are indicated in orange or magenta. (B)
EcoMBP–maltotetraose complex. Trp62, Asp209 and Arg344 are
indicated in cyan or yellow. (C) TliTMBP–trehalose complex.
Table 2. K
d
values of TvuCMBP for various sugars by measuring
changes in fluorescence.
Ligand K

d
(lM)
Maltose 0.41 ± 0.11
Maltotriose 0.97 ± 0.06
Maltotetraose 0.27 ± 0.06
Maltopentaose 0.20 ± 0.02
a-CD 0.73 ± 0.02
b-CD 1.2 ± 0.1
c-CD 0.23 ± 0.06
T. Tonozuka et al. Structure of cyclo ⁄ maltodextrin-binding protein
FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2117
[15–19], P. furiosus maltodextrin-binding protein [21]
and A. acidocaldarius maltose ⁄ maltodextrin-binding protein
[22], was unsuccessful. Therefore, a MAD data collection
of the SeMet derivative was also carried out at the PF
BL-5 A beamline (Tsukuba, Japan) at the wavelengths of
peak (0.97932 A
˚
) and edge (0.98000 A
˚
). The remote data
set could not be obtained probably because of the X-ray
damage during the data collection. Although the SeMet
crystal belongs to the same space group, its unit cell dimen-
sions are different (Table 1).
Phasing and refinement
Finding of the heavy-atom sites and determination of the ini-
tial phasing of the SeMet data set were carried out using the
program solve [35]. Since automatic model building pro-
grams such as resolve [36] and arp ⁄ warp [37] did not give

adequate structures, the model was manually built with the
program xfit in the xtalview package [38]. The refinement
was performed with the program cns [39]. Although the
model of the SeMet-substituted TvuCMBP was initially built,
a high R
free
value (32%) was yielded after placing all of the
residues, water molecules and c-CD, probably because of the
high mosaicity of the MAD data set. Thus, the native data
set was used for further refinement. Molecular replacement
was carried out using the program molrep of CCP4 [23]
with the rough model of the SeMet derivative as a probe
model. Four TvuCMBP molecules were in an asymmetric
unit and further refined with CNS. Figures were prepared
using xtalview, pymol ( />rastop (einfinity.org/rastop/), swiss-pdb
viewer [26], molscript [40], ligplot [41] and raster3d [42].
The atomic coordinates and structural factors (code 2DFZ)
have been deposited in the Protein Data Bank (http://
www.rcsb.org/).
Fluorescence measurements
To remove c-CD, which was derived from the purification
procedure, the purified TvuCMBP was denatured at a con-
centration of 0.1 mgÆmL
)1
in 2.5 m guanidine hydrochloride,
20 mm sodium phosphate buffer (pH 6.0) at 37 °C. The
denatured TvuCMBP was then dialyzed against 20 mm
sodium phosphate buffer (pH 6.0). To confirm that the
renaturation was completed, the circular dichroism spectra
of each step were monitored using a Jasco J-720WI spectro-

polarimeter. Fluorescence was measured and calculated
based on the method of Hiromi et al. [43] in a Shimadzu
RF-5300PC spectrofluorophotometer at an excitation wave-
length of 280 nm and an emission wavelength of 337 nm,
and 1 lL of 0.1 mm sugar solution in 20 mm sodium phos-
phate buffer (pH 6.0) was titrated into a cuvette containing
2 mL of 0.47 lm (20 lgÆmL
)1
) TvuCMBP. The fluorescence
intensity was measured into the stirred cuvette at 37 °C, and
the dissociation constants, K
d
, were determined. The two
TvuCMBP solutions, derived from the major peak and the
minor peak obtained from the step of cation-exchange chro-
matography in the purification procedure, gave almost iden-
tical K
d
values. The values of the major peak are listed in
Table 2.
Acknowledgements
This work was supported by the National Project on
Protein Structural and Functional Analyses and a
grant-in-aid for Scientific Research (16370048) from
the Ministry of Education, Culture, Sports, Science
and Technology of Japan. This research was per-
formed with the approval of the Photon Factory
Advisory Committee (2005G047 and 2006G149),
the National Laboratory for High Energy Physics,
Tsukuba, Japan.

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