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

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

of a gibberellin mimic peptide as a peptidyl mimotope

for a hydrophobic ligand

Takashi Murata1,3, Hikaru Hemmi1, Shugo Nakamura2, Kentaro Shimizu2, Yoshihito Suzuki3and Isomaro Yamaguchi3

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 Peptnonpept-idyl 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 A4) 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 A4 (GA4) and peptide SD, we concluded that peptide SD

is bound to the antigen-binding site of mAb 4-B8(8)⁄ E9 as a GA4 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.

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), ACLPWSDGPC

(SD) and ACLPWGTGPC (GT), were screened as

peptidyl mimotopes of a hydrophobic ligand

gibberel-lin A4 (GA4) (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 thphag-ese peptidphag-es was replaced by antigen

GA4, but not by GA4methyl 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

1D1H-NMR spectra of two synthetic cyclized decapeptides

1D 1H-NMR spectra of the two peptides ACLPWSDGPC (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 1H 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 1D1H 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) or Ha(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

AC-LPWSDGPC (SD).

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 3JHaHb 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 structFig-ures 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 concentrasatura-tion 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 1H-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

1 H

a ,

3 3.19(HB3)

a b

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

rmsd from experimental restraints

rmsd from ideal covalent geometry

Whole molecule (residues 1–10)

Core region (residues 3–7)

analysis.

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

A

B

Fig 4 Reference NMR spectrum of mixture of peptide SD and

of the same sample (B) Prior to acquisition, a 30 ms spin-lock pulse was applied to remove residual protein resonance.

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 GA4, 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 GA4previously [11]

In the complex structure, NH of Ala33 and NH of Thr53 of the mAb formed hydrogen bonds with GA4 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.).

STD enhancement (%)

0.92–0.93

58 50

2.19–2.20

66 50

major conformer

minor conformer 1

2.81–2.86

22 22

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 GA4 mimics

We first measured 1D 1H-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

in complex with peptide SD Three models

of peptide SD are shown in the wire model (green) Surface electrostatic potentials of

[31], coloured by electrostatic potentials with positive regions in blue and negative regions in red Some residues of peptide SD

one-letter codes or three-letter codes, respectively, are also represented (B) Sche-matic drawing of the interaction between

docking simulation model The figure was

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 GA4 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-coccusantibody 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, peppep-tide

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

15N-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 GA4 [11] It shows that 3b-hydroxy and

6b-carb-oxyl groups of GA4 form hydrogen bonds with

Ala33H of the main chain, and with Thr53H of the

heavy chain, respectively Furthermore, C⁄ D rings of

GA4 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 GA4–mAb 4-B8(8)⁄ E9 interaction Previously [6], the binding of phages having peptides SD and GT was not inhibited

by excess GA4 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 GA4for 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 GA4mimic 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

hydrogen bond, the peptide solution was lyophilized and

(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,

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

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 specspec-tra width of 15 p.p.m in both

dimensions and 512 and 2048 complex points in both

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

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,

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

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

The on-resonance irradiation of the protein was performed

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

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 /

estimated from high digital resolution 2D DQF-COSY spectra, and sequential and short-range NOEs However,

we could not obtain / angle restraints from the

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

coupling constants qualitatively estimated from short-mix-ing TOCSY connectivities combined with NH-Ha and

g + (+ 60
), t (180
) or g– ()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

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

was performed by using GOLD 2.1 software [32–35] The

structure of the Fab was obtained from the crystal structure

pre-vious work (PDB ID: 1KFA) [11] The structure of the Fab

was fixed except for v angles of serine residues The search

area was set within 10 A˚ of the centroid of heavy atoms of

were set to the default values To validate these calculation

the Fab Ten simulation runs were executed and we found

that these calculation conditions could reproduce the

crys-tal structure (the average rmsd of all heavy atoms was

0.88 A˚) To consider the flexibility of peptide SD, 25 runs

were executed for each of the 50 structures of peptide SD

simulated annealing calculation above

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