Báo cáo khoa học: Structure, epitope mapping, and docking simulation of a gibberellin mimic peptide as a peptidyl mimotope for a hydrophobic ligand pot

Structure, epitope mapping, and docking simulation of a gibberellin mimic peptide as a peptidyl mimotope for a hydrophobic ligand Takashi Murata 1,3 , Hikaru Hemmi 1 , Shugo Nakamura 2 , Kentaro Shimizu 2 , Yoshihito Suzuki 3 and Isomaro Yamaguchi 3 1 National Food Research Institute, Kannondai, Tsukuba, Japan 2 Department of Biotechnology, Division of Agriculture and Agricultural Life Sciences, The University of Tokyo, Japan 3 Department of Applied Biological Chemistry, Division of Agriculture and Agricultural Life Sciences, The University of Tokyo, Japan The mimotope is a structure that acts as a mimic of an epitope recognized by an antibody. Because a com- pound with the similar tertiary structure to the epitope could work as a mimotope, peptidyl mimotopes could be prepared even to an epitope composed of nonpept- idyl molecules. Peptidyl mimics for carbohydrates and double-stranded DNA have been reported [1–3]. It is difficult to obtain sufficiently high titre antibodies when using nonpeptidyl molecules such as carbohydrates, because they elicit only a T-cell independent immune response, while peptidyl molecules can raise high titre antibodies in a T-cell dependent manner. These peptidyl mimics of carbohydrates could thus potentially serve as surrogate antigens in discovering vaccines to overcome the T-cell independent immune response and to obtain anticarbohydrate antibodies with high binding activity [1]. Few reports have been made on peptidyl mimics for other ligands, especially hydrophobic ones, with the Keywords solution structure; STD-NMR; docking simulation; hydrophobic ligand; mimic peptide Correspondence H. Hemmi, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan Fax: +81 29 8387996 Tel: +81 29 8388033 E-mail: hemmi@affrc.go.jp Note The atomic coordinates for the 50 conform- ers of peptide SD described in this paper have been deposited with the Protein Data Bank (PDB ID 1YT6). Chemical shifts for peptide SD have been deposited in the BioMagRes Bank as entry 6511. (Received 9 June 2005, revised 29 July 2005, accepted 4 August 2005) doi:10.1111/j.1742-4658.2005.04902.x Using NMR spectroscopy and simulated annealing calculations, we deter- mined the solution structure of the disulfide-linked cyclized decapeptide ACLPWSDGPC (SD), which is bound to an anti-(gibberellin A 4 ) mAb 4-B8(8) ⁄ E9 and was found to be the first peptidyl mimotope for a hydro- phobic ligand. The resulting structure of the peptide showed a b-turn-like conformation in residues three to seven and the region converges well (average rmsd 0.54 A ˚ ). The binding activity and the epitopes of the peptide to the antibody were assessed using saturation transfer difference (STD)- NMR experiments. We also conducted docking simulations between the peptide and the mAb to determine how the peptide is bound to the mAb. Resonances around the b-turn-like conformation of peptide SD (residues 3–5) showed strong STD enhancement, which agreed well with results from docking simulation between peptide SD and the mAb. Together with the commonality of amino acid residues of the mAb involved in interactions with gibberellin A 4 (GA 4 ) and peptide SD, we concluded that peptide SD is bound to the antigen-binding site of mAb 4-B8(8) ⁄ E9 as a GA 4 mimic, confirming evidence for the existence of peptide mimics even for hydropho- bic ligands. Abbreviations GAs, gibberellins; mAb, monoclonal antibody; STD, saturation transfer difference; DQF, double-quantum-filtered; Fab, antigen binding fragment. 4938 FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS exception of the water-soluble ligands biotin [4] and deoxynivalenol (DON) [5]. If peptidyl mimics for hydro- phobic ligands become generally available, they could work as ideal immunogens to create antibodies that pos- sess high binding activities to various organic com- pounds such as plant hormones. In our previous paper [6], two types of homologous peptides with two different successive amino acids in the middle of peptides (underlined), ACLPW SDGPC (SD) and ACLPW GTGPC (GT), were screened as peptidyl mimotopes of a hydrophobic ligand gibberel- lin A 4 (GA 4 ) (Fig. 1) against mAb 4-B8(8) ⁄ E9 by a phage display method using a disulfide constrained phage display peptide library (Ph.D C7CTM phage display peptide library kit), because disulfide-con- strained peptide libraries have proved to be useful in identification of structural epitopes. As far as we are aware this is the first report on peptidyl mimics for hydrophobic ligands. Both peptides are composed mostly of hydrophobic amino acid residues. These are cyclized, forming a disulfide cross-link between Cys2 and Cys10. Gibberellins (GAs), a class of plant hor- mones, play important roles in various plant growth phenomena, including seed germination, stem elonga- tion, and flower development [7]. We assumed that the peptides interacted with the mAb at the GA binding site based on the observation that the binding of phag- es displaying these peptides was replaced by antigen GA 4 , but not by GA 4 methyl ester which is not recog- nized by the mAb. To confirm that peptides are bound to the antigen binding site of the mAb and to discuss interactions between peptides and the mAb in detail, it is essential to determine the conformation of the pep- tides and then investigate their interactions with atomic resolution. It is thus worthwhile to determine the conformations of these peptides and to analyse the interaction between peptides and the mAb to obtain clear evidence that the peptides are real mimotopes of GAs and to confirm the existence of peptidyl mimics even for hydrophobic ligands. In this paper, we first report the solution structure of the GA-mimic peptide, peptide SD, by 2D NMR methods. Second, we report epitope mapping of the peptide against the mAb by saturation transfer differ- ence (STD)-NMR methods. Finally, we report compu- tational docking simulation between peptide SD and the mAb, and discuss the interaction between the GA-mimic peptide and the mAb. Results 1D 1 H-NMR spectra of two synthetic cyclized decapeptides 1D 1 H-NMR spectra of the two peptides ACLPW SDGPC (SD) and ACLPWGTGPC (GT) clearly showed that both had three conformations, based on the number of resonance signals (data not shown). We could not separate the three conformers of these peptides by reversed-phase HPLC in this study. Ratios of the three conformers for peptide SD or pep- tide GT were estimated to be 3 : 1.4 : 1 or 2 : 2 : 1, based on the differential signal intensity of the resolved side-chain NH resonance of tryptophane residue in each conformer. We assigned the 1 H chemical shifts only for peptide SD using 2D NMR experiments because: (a) peptide SD and peptide GT have high binding activity for the antibody [6]; and (b) resonance signals in the 1D proton spectrum of the peptide GT are more complicated for resonance assignment than those in the 1D proton spectrum of peptide SD, a result of the ratio of the three conformers of peptide GT where the larger two are almost equal. Resonance assignments of peptide SD The sequence-specific assignments of the proton reson- ance from the residue in the three conformers (denoted the three conformers in order of the signal intensity in 1D 1 H spectrum as major conformer, minor conformer 1, and minor conformer 2) of peptide SD were made using standard procedures [8] from 2D NMR spectra collected at 20, 25, 30, and 35 °C. For assignments of Pro residues Ha(i)–Hd(i +1 : Pro) (dad)orHa(i)– Ha(i + 1 : Pro) (daa) NOEs were used instead of daN. Both proline residues, Pro4 and Pro9, of the major con- former showed strong dad NOEs, indicating that all proline residues in the major conformer of peptide SD have a trans configuration. Pro4 of minor conformer 1 and Pro9 of minor conformer 2, however, showed dad NOEs, but Pro4 of minor conformer 2 and Pro9 of minor conformer 1 did not. daa NOEs between Leu3 Ha and Pro4 Ha in minor conformer 2 or between Gly8 Fig. 1. Structures of gibberellin A 4 and cyclized decapeptide AC- LPWSDGPC (SD). T. Murata et al. Gibberellin mimics peptide-antibody recognition FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS 4939 Ha and Pro9 Ha in minor conformer 1 were observed, indicating that peptide linkages of Leu3–Pro4 in minor conformer 2 and Gly8–Pro9 in minor conformer 1 exhi- bit a cis configuration. Peptide SD thus exists in three isoforms due to cis–trans isomerization about the pep- tide linkages of Leu3–Pro4 and Gly8–Pro9. In STD- NMR experiments, binding between all conformers of peptide SD and mAb 4-B8(8) ⁄ E9 were observed as detailed later. We therefore propose that the cis ⁄ trans configuration of proline residues is basically not critical to binding to the mAb. Proton peaks of all conformers in the SD peptide were completely assigned (see Table 1). Resonance assignments were extended by determining stereospecific assignments of some methy- lene protons to obtain high-precision NMR structures. b-Methylene protons were stereospecifically assigned for three of 10 residues in the major conformer of peptide SD using information on 3 J HaHb coupling constants qualitatively estimated from the short-mixing time TOCSY spectrum combined with intraresidue NH-Hb and Ha-Hb NOEs. Sequential- and medium-range NOE connectivities and slowly exchanging amide protons in the major conformer of peptide SD is summarized in Fig. 2. Unfortunately, the two minor conformers of peptide SD had concentrations too low to detect med- ium-range NOEs. Therefore, we could not determine the tertiary structures of the two minor conformers of pep- tide SD. Conformation of the major conformer of peptide SD The 3D structure of the major conformer of peptide SD was determined by simulated annealing calcula- tions using 49 NOE-derived distance restraints (inclu- ding 11 intraresidue, 28 sequential-residue, and 10 medium range), four hydrogen bond restraints, and eight dihedral angle restraints. Fifty conformations that give low conformation energy and that give no distance and dihedral angle violations greater than 0.5 A ˚ and 5 A ˚ , respectively, were obtained. Statistical data for the 50 structures of the major conformer of peptide SD are given in Table 2. The structures thus obtained had good covalent geometry and stereochem- istry, as evidenced by the low rmsd values for bond, angle and improper from idealized geometry. The Ramachandran plot confirmed the high quality of these structures, which showed that 100% of / and w angles are found within core and allowed regions. Fig- ure 3A shows the resulting solution structures of the major conformer of peptide SD, where these structures are superimposed to give the best fit in space. The rmsd value from the mean structure is 1.60 A ˚ for all backbone atoms in the whole molecule, while the cor- responding value is 0.54 A ˚ for all backbone atoms in the region of residues 3–7. This data indicates that the region from Leu3 to Asp7 converges very well in cal- culated structures. Figure 3B shows the schematic drawing of the lowest energy structure of the major conformer of peptide SD among the 50 calculated structures, which is well characterized by a b-turn-like conformation in the sequence Leu3-Pro4-Trp5-Ser6. Interactions of peptide SD with mAb 4-B8(8)/E9 by STD-NMR experiments To investigate the interaction between peptide SD and mAb 4-B8(8) ⁄ E9, we performed STD-NMR experi- ments. The STD-NMR technique is a method of epi- tope mapping by NMR spectroscopy. During the experiment, resonances of the protein are selectively saturated and the signals of a ligand that is specifically bound to a target protein show changes in resonance intensity and are observed in the difference NMR spectrum, while those of nonassociating ligands are cancelled out and not observed in the difference spec- trum. The time course of saturation was determined by plotting the STD amplification factor against satura- tion time in the fixed concentration of peptide SD in the presence of mAb 4-B8(8) ⁄ E9, since the absolute magnitude of the STD effect depends on the concen- tration of a ligand and saturation time [9]. Saturation profiles of peptide SD showed that a 3-s saturation time was sufficient for efficient saturation transfer from a proton in the protein to that in peptide SD, and we carried out STD-NMR experiments with a 3-s satura- tion time for the epitope mapping of mAb 4-B8(8) ⁄ E9 (data not shown). Figure 4 shows (A) the 1D 1 H-NMR spectrum of peptide SD incubated with mAb 4-B8(8) ⁄ E9 at a ratio of 100 : 1; and (B) the corresponding 1D STD spec- trum. 1D STD-NMR signals of peptide SD were assigned and some signals of the three conformers overlapped. We confirmed STD-NMR signal assign- ment by 2D STD-TOCSY spectra, and overlapping signals were treated as a group to calculate their STD intensity (Table 3). The integral value of the signal of one of the b protons of Leu3 of minor conformer 1, the largest STD intensity of peptide SD, was much lar- ger than those of other STD signals, and thus this was set to 200%. Table 3 shows the relative degree of sat- uration of individual protons normalized to that of one of the b protons of Leu3 of minor conformer 1. STD enhancement was observed for all three conform- ers, indicating that they all interact with the mAb. We also found that the pattern of STD enhancement for Gibberellin mimics peptide-antibody recognition T. Murata et al. 4940 FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS Table 1. 1 H chemical shifts of three conformers in peptide SD (p.p.m., from 2,2-dimethyl-2-silapentane-5-sulfonate). Residue Major conformer Minor conformer 1 Minor conformer 2 NH HA HB Others NH HA HB Others NH HA HB Others Ala1 – 4.10 1.50 – 4.10 1.48 – 4.10 1.57 Cys2 8.76 4.68 2.95(HB2) a , 3.12(HB3) 8.82 4.63 2.95, 3.12 8.61 4.75 2.98, 3.33 Leu3 8.65 4.52 0.72(HB3), HG 1.56 8.52 4.48 0.58, 1.16 HG 1.57 8.05 4.28 1.59 HG 1.43 1.23(HB2) HD 0.85, 0.92 HD 0.84, 0.93 HD 0.89 Pro4 – 4.24 1.80, 2.20 HG 1.95, 2.00 – 4.11 1.80, 2.19 HG 1.95, 2.01 – 4.44 1.68, 2.19 HD 1.93 HD 3.21, 3.69 HD 3.19, 3.69 HD 3.15, 3.30 Trp5 7.04 4.74 3.33, 3.49 HD1 7.22 HE1 10.31 HE3 7.67 HH2 7.30 HZ2 7.55 HZ3 7.23 6.88 4.72 3.34, 3.51 HD1 7.21 HE1 10.33 HE3 7.66 HH2 7.30 HZ2 7.56 HZ3 7.22 8.13 4.53 3.35 HD1 7.33 HE1 10.18 HE3 7.66 HH2 7.26 HZ2 7.51 HZ3 7.17 Ser6 7.62 4.42 3.77 7.44 4.58 3.67 7.74 4.39 3.62, 3.81 Asp7 8.48 4.77 2.74, 2.81 8.61 4.77 2.74, 2.86 8.20 4.60 2.70, 2.75 Gly8 7.92 4.05, 4.12 8.06 3.80, 4.02 8.00 3.97, 4.20 Pro9 – 4.43 1.93, 2.25 HG 2.01 – 4.58 1.93, 2.39 HG 2.15 – 4.43 1.94, 2.24 HG 2.00 HD 3.60 HD 3.57 HD 3.59 Cys10 8.16 4.44 3.06(HB2), 3.19(HB3) 8.25 4.48 3.02, 3.21 8.53 4.53 2.98, 3.26 a b-Protons in parentheses were stereospecifically assigned. T. Murata et al. Gibberellin mimics peptide-antibody recognition FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS 4941 some residues among the three conformers such as HE1 of Trp5 differed from one another (Table 3). These results indicate that the conformational change among the three conformers due to cis–trans isomeri- zation at the position of Pro4 or Pro9 may affect the difference in the pattern of STD enhancement. Strong STD enhancement (> 60%) of all three conformers was observed, however, only for residues, Leu3-Trp5, constituting a b-turn-like structure, while C-terminal residues Ser6-Cys10, have lower STD enhancement (12–28%) except for NH of Ser6 in minor conformer 1 (Table 3), suggesting that the region from Leu3 to Trp5 has more and tighter contacts to the surface of the mAb. Docking simulation between the conformation of peptide SD and the mAb Docking simulation of the conformation of peptide SD obtained in this study to the crystal structure of mAb 4-B8(8) ⁄ E9 antigen binding fragment (Fab) [Protein Data Bank (PDB) ID 1KFA] was performed by using gold 2.1 software. Default parameters for the energy function were used, including hydrogen bond energy between the protein and ligand, van der Waals energy between the protein and ligand and within the ligand, and internal torsion energy for the ligand. To consider Fig. 2. Summary of sequential and medium-range NOE connectivi- ties observed for the major conformer of peptide SD. Bars, the size of which indicates the NOE intensity (strong, medium, and weak), represent sequential NOEs. Slow exchanging amide protons are also represented as closed circles. Table 2. Statistics for 50 NMR structures of peptide SD. Number of restraints Total distance restraints 53 Intraresidue 11 Sequential 28 Medium (1 < | i–j | < 5) 10 Long (| i–j | ‡ 5) 0 Hydrogen bond (2 per bond) 4 Total dihedral angle restraints 8 / 5 w 0 v 1 3 rmsd from experimental restraints NOE distance restraints (A ˚ ) 0.0305 ± 0.0123 Dihedral angle restraints (degree) 0.3631 ± 0.1474 rmsd from ideal covalent geometry Bonds (A ˚ ) 0.0023 ± 0.00071 Angles (degree) 0.5644 ± 0.0294 Impropers 0.1668 ± 0.0529 / and w in core and allowed regions (%) a 100 rmsd relative to the mean structure (A ˚ ) Backbone (N, Ca and C¢ atoms) All non-H Whole molecule (residues 1–10) 1.60 ± 0.46 2.22 ± 0.58 Core region (residues 3–7) 0.54 ± 0.19 1.47 ± 0.31 a The program PROCHECK-NMR [30] was used for Ramachandran plot analysis. AB N C C N A1 C2 L3 W5 S6 D7 P9 C10 P4 G8 Fig. 3. Superimposition of 50 structures (A) and ribbon diagram of the lowest energy structure of the major conformer of peptide SD (B). One disulfide bridge (Cys2–Cys10) and side chains of all resi- dues are ball-and-stick representations. This figure was generated using MOLMOL [31]. A B Fig. 4. Reference NMR spectrum of mixture of peptide SD and mAb 4-B8(8) ⁄ E9 in the ratio of 100 : 1 (A) and STD-NMR spectrum of the same sample (B). Prior to acquisition, a 30 ms spin-lock pulse was applied to remove residual protein resonance. Gibberellin mimics peptide-antibody recognition T. Murata et al. 4942 FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS the flexibility of peptide SD, 25 runs were executed for each of the 50 NMR structures while fixing the main- chain atoms of peptide SD and optimizing its side- chain atoms. Among 1250 peptide SD-mAb 4-B8(8) ⁄ E9 Fab complex structures predicted in this study, 904 complex structures with positive fitness scores were selected, and clustered using the method of Baker et al. [10]. During clustering, the rmsd of Ca atoms was used as the measure of distance between structures. Three large clusters were found among 904 predicted complex structures by the clustering analysis, and three complex structures that were closest to each centre of the three large clusters were obtained. We then com- pared the fitness values of the three complex structures to find the best model of the peptide SD-mAb 4-B8(8) ⁄ E9 complex structure. The fitness values of the three complex structures were 51.26, 49.49, and 1.97. The complex structure with the highest fitness value, 51.26, was obtained from the largest cluster, indicating that this complex structure is the best model of all pre- dicted complex structures in this study. Firstly, the three complex structures obtained by clustering analy- sis showed that peptide SD interacted with the antigen binding site in mAb 4-B8(8) ⁄ E9 (Fig. 5A). Residues of Pro4 and Trp5 of peptide SD in each of three complex structures are located at almost the same positions in the three complex structures, and these residues of peptide SD showed hydrophobic interaction with mAb 4-B8(8) ⁄ E9 in complex structures. The three complex structures also showed that amide proton of Ala33, which is very important for binding with GA 4 , in mAb 4-B8(8) ⁄ E9 was located at a position to possibly form hydrogen bonding with peptide SD. We speculated from the three complex structures, however, that pep- tide SD is mainly bound to mAb 4-B8(8) ⁄ E9 by hydro- phobic interaction. Next, we analysed the interaction between peptide SD and mAb 4-B8(8) ⁄ E9 in detail using the best model pre- dicted (Fig. 5B). In this model of peptide SD-mAb 4-B8(8) ⁄ E9 complex, two hydrogen bonds exist between the antibody and the peptide: Ala33a NH–CO Pro4p (where a denotes an antibody residue and p denotes a peptide residue); and Thr53a OHsc–CO Trp5p (where sc denotes side-chain) (Fig. 5B). We reported the crystal structure of mAb 4-B8(8) ⁄ E9 with GA 4 previously [11]. In the complex structure, NH of Ala33 and NH of Thr53 of the mAb formed hydrogen bonds with GA 4 . The results indicate that peptide SD in the best complex model interacts with very important residues, Ala33 and Thr53, of the mAb for antigen recognition. This com- plex model obtained from docking simulations in this study thus appears to be extremely suitable. As described, we found from STD-NMR experiments in this study that the region Leu3-Trp5 of peptide SD is an important epitope for interaction with the mAb. The corresponding region of peptide SD in the three com- plex models, also shown to interact with the mAb from docking simulations in this study, is in good agreement with the results obtained from STD-NMR experiments. In complex models, the region from Ser6 to Cys10, which showed the lower STD enhancement, had no interactions with the mAb. We thus conclude that Table 3. STD enhancement of peptide SD in the presence of monoclonal antibody 4-B8(8) ⁄ E9. Resonance signals overlapping between major conformer and minor conformer 1 are shown in ital- ics. STD enhancement was normalized to the strongest enhance- ment, Leu3 HB of minor conformer 1 (0.58 p.p.m.). Resonances d (p.p.m) STD enhancement (%) Ala-1 HA 4.10 28 Ala-1 HB 1.48–1.50 36 Cys-2 NH of minor conformer 1 8.82 6 Cys-2 HB 2.95, 3.12 46, 26 Leu-3 NH of minor conformer 1 8.52 46 Leu-3 HB2 of major conformer 1.23 50 Leu-3 HB3 of major conformer 0.72 68 Leu-3 HB of minor conformer 1 1.16 58 Leu-3 HB of minor conformer 1 0.58 200 Leu-3 HG 1.56–1.57 42 Leu-3 HD 0.84–0.85, 0.92–0.93 58 50 Pro-4 HB 1.80, 2.19–2.20 66 50 Pro-4 HB of minor conformer 2 1.68 44 Pro-4 HG ⁄ Pro9 HB ⁄ Pro9 HG of major conformer 1.93–2.01 50 Pro-4 HD ⁄ Ser6 HB of minor conformer 1 3.69–3.77 14 Pro-4 HD ⁄ Cys10 HB 3.19–3.21 24 Trp-5 NH of major conformer 7.04 108 Trp-5 NH of minor conformer 1 6.88 102 Trp-5 HB 3.33–3.51 34 Trp-5 HD1 ⁄ HZ3 7.21–7.23 80 Trp-5 HE1 of major conformer 10.31 20 Trp-5 HE1 of minor conformer 1 10.33 60 Trp-5 HE3 7.66–7.67 116 Trp-5 HH2 7.30 80 Trp-5 HZ2 7.55–7.56 76 Trp-5 HZ2 of minor conformer 2 7.51 64 Ser-6 NH of major conformer 7.62 20 Ser-6 NH of minor conformer 1 7.44 68 Ser-6 HB of major conformer 3.77 12 Asp-7 NH of minor conformer 1 8.61 24 Asp-7 HB 2.74, 2.81–2.86 22 22 Gly-8 NH of minor conformer 1 8.06 28 Pro-9 HB 2.25–2.39 24 Cys-10 HB 3.02–3.06 22 T. Murata et al. Gibberellin mimics peptide-antibody recognition FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS 4943 complex models between peptide SD and the mAb obtained from docking simulation in this study are suit- able since epitopes of peptide SD to the mAb obtained from STD-NMR are in good agreement with those obtained from docking simulation. Discussion We previously identified two disulfide linked cyclized decapeptides, SD and GT, which have affinity with mAb 4-B8(8) ⁄ E9, an antibioactive GA antibody, by screening a phage display peptide library [6]. In this study, we performed NMR spectroscopic analysis of the peptides to determine the conformation and an epi- tope for mAb 4-B8(8) ⁄ E9 in order to obtain structural information showing that the peptides are bound to the antigen-binding site of the mAb as GA 4 mimics. We first measured 1D 1 H-NMR spectra of peptides SD and GT. 1D proton spectra of the peptides showed that each of the two peptides has three cis ⁄ trans iso- mers due to two proline residues; resonance signals in 1D proton spectra are complicated by resonance sig- nals overlapping among the three isomers. Fortunately, in the 1D proton spectrum of peptide SD, the intensity of the resonance signals of one isomer (all trans-confi- guration) is much stronger than those of the other two isomers (one cis-configuration and one trans-configur- ation). For the large isomer (major conformer) of pep- tide SD, we therefore assigned resonance signals and determined the solution structure by 2D NMR Phe100BH Leu97H Leu98H Tyr100AH A B Fig. 5. Docking simulation models of pep- tide SD and mAb 4-B8(8) ⁄ E9. (A) Electro- static potential surface of mAb 4-B8(8) ⁄ E9 in complex with peptide SD. Three models of peptide SD are shown in the wire model (green). Surface electrostatic potentials of mAb 4-B8(8) ⁄ E9 were calculated in MOLMOL [31], coloured by electrostatic potentials with positive regions in blue and negative regions in red. Some residues of peptide SD or mAb 4-B8(8) ⁄ E9 described in text using one-letter codes or three-letter codes, respectively, are also represented. (B) Sche- matic drawing of the interaction between peptide SD and mAb 4-B8(8) ⁄ E9 in the docking simulation model. The figure was generated by LIGPLOT [36]. Gibberellin mimics peptide-antibody recognition T. Murata et al. 4944 FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS spectroscopy. We measured the STD-NMR spectrum for the mixture of peptide SD and mAb 4-B8(8) ⁄ E9 to investigate the interaction of peptide SD with the mAb. We also performed the docking simulation using the NMR structures of peptide SD and the crystal structure of the mAb Fab in the complex with GA 4 to analyse interactions between them in more detail. The solution structure we determined for peptide SD showed a b-turn-like conformation in residues 3–7 and the region converges well (average rmsd 0.54 A ˚ ). This conformation would be stabilized by two intramolecular Leu3 NH–Asp7 CO and Ser6 NH–Leu3 CO hydrogen bonds. The b-turn motif has been observed in other antigenic peptides free in solution [12–15] and bound to antibodies [16–19]. The 12-residue carbohydrate- mimetic peptide recognized by an antigroup B Strepto- coccus antibody was recently reported to have a type I b-turn both free and bound to the antibody [20]. The turns present in the bound and free peptide are very sim- ilar and residues forming this turn are recognized by the mAb as demonstrated by STD-NMR experiments, which indicates that the b-turn conformation may be an important reason for the effective immunogenicity of the peptide. In our study, bound conformation of pep- tide SD has not been determined yet. However, peptide SD has the b-turn-like conformation stabilized by two hydrogen bonds when free and residues (Leu3-Trp5) forming this turn are recognized by the mAb, as demon- strated by STD-NMR experiments. We propose that the b-turn-like conformation of peptide SD is important for binding to the mAb, this being supported by the reason- ably good simulated docking between peptide SD and the mAb when fixing the b-turn-like conformation of peptide SD. If we can monitor the changes in chemical shifts of peptide SD on the addition of mAb using 15 N-labelled peptide, expected results will make it clearer that the b-turn-like conformation of peptide SD is important for binding to the mAb. We previously determined the crystal structure of the complex formed with the mAb 4-B8(8)⁄ E9 Fab and GA 4 [11]. It shows that 3b-hydroxy and 6b-carb- oxyl groups of GA 4 form hydrogen bonds with Ala33H of the main chain, and with Thr53H of the heavy chain, respectively. Furthermore, C ⁄ D rings of GA 4 were in van der Waals’ contact mainly with the aromatic side chain of Tyr100AH and Phe100BH of the third complementarity-determining region of the heavy chain in mAb 4-B8(8) ⁄ E9. Our complex model between mAb 4-B8(8) ⁄ E9 and peptide SD in this study shows that Pro4 CO and Trp5 CO of peptide SD are hydrogen-bonded to Ala33H NH and to Thr53H side- chain OH of the mAb, respectively. Furthermore, the region composed of hydrophobic amino acid residues, Leu3-Pro4-Trp5, of peptide SD form hydrophobic interactiona with the hydrophobic surface of the mAb including Tyr100AH and Phe100BH, as also demon- strated by STD-NMR experiments (Figure 5; Table 3). These results indicate that the peptide SD–mAb 4-B8(8) ⁄ E9 interaction is very similar to the GA 4 –mAb 4-B8(8) ⁄ E9 interaction. Previously [6], the binding of phages having peptides SD and GT was not inhibited by excess GA 4 methylester, which is not reactive with mAb 4-B8(8) ⁄ E9, suggesting that the binding of the peptides is tightly related to the binding property of the mAb to its antigen. We therefore conclude that peptide SD is a real mimotope of GA 4 for the mAb. So far, quite a number of peptidyl mimics for carbo- hydrates or double-stranded DNA have been prepared [1–3]. Mimicry peptides for water-soluble ligands, bio- tin and DON, have also been reported [4,5]. No reports exist, however, on peptidyl mimics for hydro- phobic ligands, such as GAs, except for our previous study [6]. In this study, we confirmed that peptide SD formed hydrophobic interactions with mAb 4-B8(8) ⁄ E9 as a GA 4 mimic. This is thus the first proof that peptidyl mimics can be prepared even though the ligands are hydrophobic, such as GAs. This finding would provide further availability of peptidyl mimics for other hydrophobic ligands as ideal immunogens to create antibodies that possess high binding activities to the organic compounds. Experimental procedures Sample preparation Two synthetic cyclized decapeptides, ACLPWSDGPC (SD) and ACLPWGTGPC (GT), made by the Fmoc method were purchased from Bex (Tokyo, Japan). A disulfide bond in these peptides was formed under oxidized conditions. Synthetic peptides were dissolved in 50 mm phosphate buf- fer (pH 5.0, 90% H 2 O ⁄ 10% D 2 O, v,v) to give a final con- centration of  5mm for NMR experiments. To detect the hydrogen bond, the peptide solution was lyophilized and redissolved in an equal volume of 100% D 2 O. mAb 4-B8(8) ⁄ E9 was prepared as reported elsewhere [6]. The mAb was dissolved in NaCl ⁄ P i (10% D 2 O), and concentra- ted to  50 lm by centrifugation using Centricon-10 (Milli- pore, Billerica, MA). The peptide was dissolved into the mAb solution and the molar ratio of the peptide to mAb was adjusted to 100 : 1 for STD-NMR experiments. NMR spectroscopy All NMR spectra were obtained on Bruker Avance 600 MHz and 800 MHz spectrometers at 293 K, 298 K, T. Murata et al. Gibberellin mimics peptide-antibody recognition FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS 4945 and 303 K. Standard Bruker software (xwinnmr 2.6) was used to acquire and process NMR data. Water suppression was performed using watergate sequence [21,22]. Chem- ical shifts were referenced to internal 2,2-dimethyl-2-silapen- tane-5-sulfonate at 25 °C. All 1D 1 H-NMR spectra were recorded with 128 scans and 60 000 data points, and proc- essed by zero-filling to 60 k points and multiplication by an exponential function, followed by Fourier transformation. Resonance signals were assigned based on 2D double-quan- tum-filtered (DQF)-COSY, TOCSY, ROESY and NOESY spectra. The 2D spectra were recorded with quadrature detection in the phase-sensitive mode by time proportional phase increment (TPPI) [23] and States-TPPI [24]. 2D spec- tra were acquired with a spectra width of 15 p.p.m. in both dimensions and 512 and 2048 complex points in both dimensions. TOCSY spectra with a DIPSI-2 mixing sequence were recorded with mixing times of 35, 60 or 80 ms. NOESY spectra were obtained with mixing times of 60, 100, 200, and 400 ms, and ROESY with a mixing time of 100 ms. The high digital resolution DQF-COSY spec- trum was recorded using 400 and 4096 complex points in both dimensions. Slowly exchanging amide protons were identified by lyophilizing peptide from a H 2 O solution, dis- solving the peptide in D 2 O, and collecting sequential 2-h 2D TOCSY spectra. Before Fourier transformation, the shifted sine-bell window function was applied to the t1 and t2 dimensions. Peak picking and assignment were per- formed with sparky (T. D. Goddard and D. G. Kneller, sparky 3, University of California, San Francisco, CA). 1D and 2D STD-NMR spectra were performed as des- cribed by Mayer and Meyer [9]. The time dependence of the saturation transfer was determined by recording 1D STD spectra with 1 k scans and saturation times from 0.25 s to 6.0 s. The irradiation power in all STD-NMR experiments was set to  0.15 W. Relative STD values were calculated by dividing STD signal intensities by the intensi- ties of the corresponding signals in a reference spectrum of the same sample recorded with 64 scans. All STD-NMR spectra for epitope mapping were acquired using a series of equally spaced 50 ms Gaussian-shaped pulses for saturation with 1 ms intervals and the total saturation time of  3s. The on-resonance irradiation of the protein was performed at the chemical shift of )2.0 p.p.m., and the off-resonance at 40 p.p.m. where no protein signal was present. Free induction decay values with on- and off-resonance protein saturation were recorded in alternative fashion. Subtraction of the 1D STD spectra was achieved via phase cycling. Pro- tein resonance was suppressed by application of a 30 ms spin-lock pulse prior to acquisition. 2D STD-TOCSY spec- tra with on- and off-resonance protein saturation were recorded with 128 scans per t 1 increment in alternative fash- ion. The 2D spectra were acquired with a spectra width of 15 p.p.m. in both dimensions, and 256 and 2048 complex points in both dimensions. A MLEV-17 mixing time of 100 ms was applied in STD-TOCSY spectra. Structure calculations NOE-derived distance restraints were classified into three ranges, 1.8–3.0 A ˚ , 1.8–4.0 A ˚ and 1.8–5.0 A ˚ , according to the relative NOE intensities. Upper distance limits for NOEs involving methyl protons and nonstereospecifically assigned methylene protons were corrected appropriately for centre averaging [25]. In addition, a distance of 0.5 A ˚ was added to the upper distance limits only for NOEs involving methyl protons [26] after correction for centre averaging. Torsion angle constraints on the backbone / angle are usually derived from 3 J HNHa coupling constants estimated from high digital resolution 2D DQF-COSY spectra, and sequential and short-range NOEs. However, we could not obtain / angle restraints from the DQF- COSY spectra, because all 3 J HNHa coupling constants observed were between 6 Hz and 8 Hz. The additional / angle restraint of 100° ±80° was applied to residues for which the intraresidue Ha-HN NOE was clearly weaker than the NOE between HN and the Ha of the preceding residue [27]. Five / angle restraints were obtained for the peptide. Side-chain v 1 angles were determined by 3 J HaHb coupling constants qualitatively estimated from short-mix- ing TOCSY connectivities combined with NH-Ha and Ha-Hb NOEs [28]. Three v 1 angle restraints for the peptide were obtained. v 1 angle restraints were normally restricted to a ± 60° range from staggered conformations, g + (+ 60°), t (180°)org–()60°). Hydrogen-deuterium exchange experiments identified four hydrogen bond donors for the peptide. Corresponding hydrogen bond acceptors were determined based on NOE patterns observed for regu- lar secondary structural regions and preliminary calculated structures without restraints regarding hydrogen bonds. Hydrogen bond constraints were applied to N–H and C¼O groups: 1.7–2.4 A ˚ for the H-O distance and 2.7–3.4 A ˚ for the N-O distance. The peptide structures were calculated by simulated annealing using torsion angle dynamics with the program cns [29]. The structure calculation proceeded in two stages. In the first stage, a low-resolution structure was preliminar- ily determined using only NOE-derived distance restraints. In the second stage, the same protocol was applied by add- ing hydrogen bond restraints and dihedral angle restraints. Additional NOE constraints were added in each round of calculations, and restraints that were consistently violated were removed. Additional NOE constraints were then added and used in the final structure calculation. All subse- quent numerical analyses were performed using procheck- NMR [30] and molmol [31]. Structure figures were gener- ated using molmol. Docking simulation Docking simulation of peptide SD to mAb 4-B8(8) ⁄ E9 Fab was performed by using GOLD 2.1 software [32–35]. The Gibberellin mimics peptide-antibody recognition T. Murata et al. 4946 FEBS Journal 272 (2005) 4938–4948 ª 2005 FEBS structure of the Fab was obtained from the crystal structure of the complex of the Fab and GA 4 determined in our pre- vious work (PDB ID: 1KFA) [11]. The structure of the Fab was fixed except for v angles of serine residues. 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