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

| ⁄ S Edge 0.98000 C2 85.3 49.3 87.6 94.9 50–2.30 (2.38–2.30)b 111 339 15 803 96.7 (89.7)b 43.5 (11.5)b 18.7 (26.5)b 50–2.30 (2.38–2.30)b 111 287 15 798 96.5 (87.7)b 43.3 (11.3)b 17.8 (26.2)b Native PF-AR NW-12 1.0000 167.4 95.3 117.1 131.6 50–2.50 (2.66–2.50)b 181 528 47 691 100.0 (100.0)b 24.0 (8.6)b 6.9 (21.7)b 11 856 352 539 21.8 26.8 0.007 1.40 26.0 31.5 28.1 b The values for the highest resolution shells are given in parentheses and 283–330) and the C-domain (residues 131–279 and 334–397) (Fig 1B,C) Both domains have similar architectures; 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 differences 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, indicating 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 backbones 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 FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2111 Structure of cyclo ⁄ maltodextrin-binding protein T Tonozuka et al Fig 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 calculated from the coefficients of the (Fobs ) Fcalc) and the resultant phase angles after several cycles of refinement of the model excluding 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) C-domain The structure of c-CD shows a sliced conical 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 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 2112 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 functionally equivalent residues, but their positions are different (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 residue, Glc3¢, of maltotetraose bound to EcoMBP are superimposed well, and Glc2 and Glc4 also adopt similar 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 closer 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 mechanisms 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) FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS Structure of cyclo ⁄ maltodextrin-binding protein T Tonozuka et al Fig 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–maltotetraose (yellow; PDB ID, 4MBP) and TliTMBP–trehalose complex (magenta; PDB ID, 1EU8) (B) Comparison of the Ca backbones 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 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 residues produce a hydrophobic environment, which contributes 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 sugarbinding 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 bottom 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 residues are found to be few Between TvuCMBP and EcoMBP, many residues, including Leu and Trp, seem to be conserved, but the positions and the conformations 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 protrusions 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 Kd values of TvuCMBP for binding of sugars were determined by measuring changes in fluorescence (Table 2) TvuCMBP shows almost the same Kd values for CDs and maltooligosaccharides A similar experiment with a CD-binding protein from Klebsiella oxytoca, 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 Kd values of K oxytoca CymE is highly specific for CDs, while TvuCMBP FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2113 Structure of cyclo ⁄ maltodextrin-binding protein T Tonozuka et al Fig Schematic drawing of the residues located at C- (A) and N-domain (B) interacting 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 to 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 molecule; - - - -, hydrogen bond Several residues involved in hydrophobic interactions are also illustrated 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 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 2114 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 structure shows that the sugar-binding site of TvuCMBP differs structurally from those of EcoMBP and FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS Structure of cyclo ⁄ maltodextrin-binding protein T Tonozuka et al Fig 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 to (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 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 efficiently 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 FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2115 Structure of cyclo ⁄ maltodextrin-binding protein T Tonozuka et al Fig 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 sugarbinding proteins, TvuCMBP–c-CD (A), EcoMBP–maltotetraose (B) and TliTMBP– trehalose (C) are compared The numbering and the color representation of glucose residues of the ligands are as in Fig 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 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 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 ligand 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 2116 CDs In order to determine whether TvuCMBP complex 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-terminal cysteine codon for the mature TvuCMBP, and the pET expression system (Novagen, Darmstadt, Germany) FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS Structure of cyclo ⁄ maltodextrin-binding protein T Tonozuka et al pET21a, resulting in the plasmid pETCBP The sequence of the construct was verified by DNA sequencing Preparation of TvuCMBP Fig 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 Kd values of TvuCMBP for various sugars by measuring changes in fluorescence Ligand Kd (lM) Maltose Maltotriose Maltotetraose Maltopentaose a-CD b-CD c-CD 0.41 0.97 0.27 0.20 0.73 1.2 0.23 ± ± ± ± ± ± ± 0.11 0.06 0.06 0.02 0.02 0.1 0.06 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 To produce TvuCMBP, E coli BL21(DE3) harboring pETCBP was cultured in Luria-Bertani medium containing ampicillin (50 lgỈmL)1) to A600 ¼ 0.6–0.9, induced with isopropyl b-d-thiogalactopyranoside to a final concentration of 0.5 mm, and grown for another 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 previously [7] The protein was further purified using cationexchange 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 buffer at a flow rate of mLỈmin)1 As reported for a trehalose ⁄ 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 measurements SeMet-substituted TvuCMBP was prepared by overexpressing 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 lL of well solution (25% polyethylene glycol 6000, 0.1 m Mes pH 6.25, mm c-CD) and lL of protein solution (10 mgỈmL)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, 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 FEBS Journal 274 (2007) 2109–2120 ª 2007 The Authors Journal compilation ª 2007 FEBS 2117 Structure of cyclo ⁄ maltodextrin-binding protein T Tonozuka et al [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 dimensions are different (Table 1) Phasing and refinement Finding of the heavy-atom sites and determination of the initial phasing of the SeMet data set were carried out using the program solve [35] Since automatic model building programs 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 Rfree 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 (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 concentration of 0.1 mgỈmL)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 spectropolarimeter Fluorescence was measured and calculated based on the method of Hiromi et al [43] in a Shimadzu RF-5300PC spectrofluorophotometer at an excitation wavelength of 280 nm and an emission wavelength of 337 nm, and lL of 0.1 mm sugar solution in 20 mm sodium phosphate buffer (pH 6.0) was titrated into a cuvette containing mL of 0.47 lm (20 lgỈmL)1) TvuCMBP The fluorescence intensity was measured into the stirred cuvette at 37 °C, and the dissociation constants, Kd, were determined The two 2118 TvuCMBP solutions, derived from the major peak and the minor peak obtained from the step of cation-exchange chromatography in the purification procedure, gave almost identical Kd values The values of the major peak are listed in Table 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 performed with the approval of the Photon Factory Advisory Committee (2005G047 and 2006G149), the National 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