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

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

Thermoactinomyces vulgaris cyclo⁄maltodextrin-binding protein

Takashi Tonozuka1, Akiko Sogawa1, Mitsugu Yamada2, Naoki Matsumoto1, Hiromi Yoshida2,3, Shigehiro Kamitori2,3, Kazuhiro Ichikawa1, Masahiro Mizuno1,*, Atsushi Nishikawa1and

Yoshiyuki Sakano1

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: tonozuka@cc.tuat.ac.jp

*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 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.

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 MalFGK2 transporter EcoMBP interacts with the

MalFGK2 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 EcoMBP [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 MalFGK2 sugar-transporter complex In

contrast, the open form does not have the ability to

perform the proper interaction with the MalFGK2

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 TvuCMBP–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 TvuCMBP 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 Rcryst¼ 21.8% and

Rfree¼ 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

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

Data collection

Cell dimensions

Refinement statistics

Number of atoms

rmsd

Average B

a Rmerge¼ SS|Ii – <I>| ⁄ S<I> 0 b The values for the highest resolution shells are given in parentheses.

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 TliTMBP may be more suitable for the specific binding to small oligosaccharides like 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).

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˚ In TliTMBP, 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 Kdvalues of TvuCMBP for binding of sugars were determined by measuring changes in fluorescence (Table 2) TvuCMBP shows almost the same Kdvalues 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 Kd 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 Kdvalues 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.

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 Kd 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 Kd

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.

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

RASTER 3 D [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.

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, lig-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 TvuCMBP, 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 RASTER 3 D [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.

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 5¢-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) to A600¼ 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 37C 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 20C 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.

[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 Rfreevalue (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 (http://pymol.sourceforge.net/),

rastop (http://www.geneinfinity.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)1in 2.5 m guanidine hydrochloride,

20 mm sodium phosphate buffer (pH 6.0) at 37C 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 wavewave-length 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 37C, and

the dissociation constants, Kd, 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 Kd 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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