Tài liệu Báo cáo khoa học: Design, structure and biological activity of b-turn peptides of CD2 protein for inhibition of T-cell adhesion ppt

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Design, structure and biological activity of b-turn peptides
of CD2 protein for inhibition of T-cell adhesion
Liu Jining
1
, Irwan Makagiansar
3
, Helena Yusuf-Makagiansar
3
, Vincent T. K. Chow
2
, Teruna J. Siahaan
3
and Seetharama D. S. Jois
1
1
Department of Pharmacy and
2
Department of Microbiology, National University of Singapore, Singapore;
3
Department of
Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS, USA
The interaction between cell-adhesion molecules CD2 and
CD58 is critical for an immune response. Modulation or
inhibition of these i nteractions has been shown t o be thera-
peutically useful. Synthetic 12-mer linear and cyclic pe ptides,
and cyclic hexapeptides based on rat CD2 protein, were
designed to modulate CD2–CD58 interaction. The synthetic
peptides effectively blocked the interaction between CD2–
CD58 proteins as demonstrated by antibody binding,
E-rosetting and heterotypic adhesion assays. NMR and
molecular modeling studies indicated that the synthetic
cyclic peptides exhibit b-turn structure in solution and clo-
sely mimic the b-turn s tructure of the surface epitopes of the
CD2 protein. Docking studies of CD2 peptides and CD58
protein revealed the possible binding sites of the cyclic
peptides on CD58 protein. The designed cyclic peptides
with b-turn structure have the ability to modulate the
CD2–CD58 interaction.
Keywords:CD2,b-turn, cyclic peptide, E-rosetting, LFA-3
(CD58).
Accessory m olecules, C D2–CD58 receptor-ligand pair [1 –4]
are important for adhesion and costimulation in the normal
immune response. The CD2 molecule is a transmembrane
glycoprotein expressed on all subsets of T-cells, NK cells
and lymphokine-activated killer cells, all known to b e
effectors of autoimmune disease and allograft rejection. Its
ligand, CD58 or leukocyte function associated antigen-3
(LFA-3), is also a transmembrane glycoprotein, distributed
widely on T and B lymphocytes, e rythrocytes, endothelium,
platelets, and g ranulocytes. It has been found that this
heterophilic adhesion facilitates initial cell–cell contact
before specific antigen recognition, and also enhances
T-cell receptor (TcR) trigge ring by fostering interaction
with peptide-class II m ajor histocompatability complex
(pMHC). The affinity of CD2–CD58 interaction is relat-
ively low (K
d
% 1 l
M
), with very rapid k
off
and k
on
that
supports dynamic binding with rapid counter-receptor
exchange. This creates an optimal intercellular membrane
distance (% 135 A
˚
) on opposing cell surfaces suitable for
TcR-pMHC or NK receptor–MHC interactions to foster
immune recognition. Hence, in the presence of human
CD2–CD58 interaction, T-cells recognize the correct
pMHC with a 50- to 100-fold greater efficiency than its
absence [4]. In addition, endothelial cells (EC) in rheuma-
toid arthritis (RA) have been shown to express elevated
levels of CD58, and RA lymphocytes in synovial fluid
(SF) express increased levels of CD2 and CD58 relative
to RA or normal peripheral blood lymphocytes [5,6].
Thus, the inhibition of CD2–CD58 interaction can
potentially be used for t he treatment o f autoimmune
diseases.
It has been shown that b lockade of t he CD2–CD58
interaction [7,8] and/or modulation of the CD2 costim-
ulatory pathway [9–12] can result in prolonged tolerance
towards allografts. The soluble CD58–Ig fusion protein
Amevive (LFA3TIP) has been used to treat psoriasis
[13]. However, antibodies are huge protein molecules and
therapeutic antibodies are nonhuman in origin, these
often elicit significant side-effects attributed to th eir
immunogenicity. The humanized versions of antibodies
BTI-322 [14] and MEDI-507 [15] have been tested for the
treatment of acute organ rejection and graft-vs host-
disease. Furthermore, MEDI-507 is also investigated for
autoimmune and other inflammatory diseases. Antibodies
are susceptible to enzymatic degradation and hence pose
a challenge for formulation and delivery. To circumvent
this problem, one approach is to design short peptides
or small molecular mimics that will bind to critical areas
in target proteins (CD58) and, like antibodies, i nterfere
with their activity. Currently, no peptide or small
molecules targeting CD2 or CD58 have been yet
reported.
Therefore, this study was undertaken to design small
peptides based on CD2 p rotein e pitopes t o m odulate
CD2–CD58 interaction . We designed linear a nd cyclic pep-
tides (Table 1) from the b-turn regions of rat CD2 protein
(Fig. 1), and evaluated their ability to inhibit cell a dhesion
using antibody, E-rosetting and heterotypic-adhesion
Correspondence to S. D. S. Jois, Department of Pharmacy, 18 Science
drive 4, National University of Singapore, Singapore 117543.
Fax: + 65 6 779 1554, Tel.: + 65 6 874 2653,
E-mail: phasdsj@nus.edu.sg
Abbreviations: AET, 2-aminoethylisothiouronium hydrobromide;
BCECF-AM, bis-carboxyethyl-carboxyfluorescein, acetoxymethyl;
FITC, fluorecein isothiocynate; hCD2, human CD2; hCD58, human
CD58; MEM-a, m inimum essential medium-a; MTT,
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide];
PAL-resin, 5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeryl-resin.
(Received 5 January 2004, revised 22 April 2004,
accepted 30 April 2004)
Eur. J. Biochem. 271, 2873–2886 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04198.x
inhibition assays. In order to understand structure–func-
tion relationship of peptides, we also carried out detailed
NMR, molecular modeling and docking studies of pep-
tide-protein complexes. Our results indicate that the
designed peptides are useful for inhibition of the T-cell
adhesion mechanism.
Materials and methods
Peptides
The linear and cyclic peptides lER, lVY, cER, cVY, cEL
and cYT (Table 1) were designed and purchased from
Multiple Peptide Systems (San Diego, CA, USA). The pure
product was analyzed by HPLC and fast atom bombard-
ment mass spectrometry (FABMS). The HPLC chromato-
gram showed that the purities of peptides were more than
90%, and FABMS showed the correct molecular ion for the
peptides. The control peptide was synthesized u sing auto-
matic solid-phase peptide s ynthesizer (Pioneer, Perspective
Biosystem, Foster, CA, USA) using Fmoc chemistry with
PAL resin. T he Fmoc-protected amino acids were obtained
from Novabiochem. All the solvents used in the Pioneer
peptide synthesizer were obtained from Applied Biosystems.
Peptide was purified by preparative HPLC (Waters 600
HPLC system), on a reversed-phase C18 column (Inertsil,
10 · 250 mm, 5 lm, 300 A
˚
) w ith a linear gradient o f
solvent A (0.1% (v/v) trifluoroacetic acid/H
2
O) and solvent
B (0.1% (v/v) trifluoroacetic acid/acetonitrile). The peptides
were detected by UV absorbance at k ¼ 215 and 280 nm.
The purity of each peptide was verified by an analytical
HPLC (Shimadzu LC-10AT VP) using a reverse-phase C18
column (Lichrosorb RP18, 4.6 · 200 mm, 10 lm) with the
same solvent system a s in t he preparative HPLC. The
molecular mass of the peptide was determined by using
electro-spray ionization mass spectrometry (ESI-MS,
Finnigon MAT).
Antibodies
Fluorescence-conjugated monoclonal antibody to human
CD58 (FITC-anti-CD58) and C D2 (FITC-anti-CD2) were
purchased from Biodesign Internation al (Saco, ME, USA)
and Immunotech, respectively.
Cell lines
The Jurkat, MOLT-3 T-leukemia and the human colon
adenocarcinoma (Caco-2) cell lines were obtained from the
American Type Culture Collection (Rockville, MD, USA).
Jurkat and M OLT-3 cells were maintained in suspension in
RPMI1640 medium supplemented with 10% (w/v) heat-
inactivated fetal bovine serum and 100 mgÆL
)1
of penicillin/
streptomycin. Caco-2 cells were maintained in minimum
essential m edium-a containing 10% (w/v) f etal bovine
serum, 1% (v/v) nonessential amino acids, 1 m
M
Na-pyruvate, 1% (v/v)
L
-glutamine and 100 mgÆL
)1
of
penicillin/streptomycin. Caco-2 cells were used between
passages 5 0 and 60. Sheep blood in Alsever’s solution was
purchased from TCS Biosciences Ltd., Singapore.
CD2 detection and flow-cytometry assay
To detect CD2 expression, 10
6
Jurkat cells were washed
with 0.5% (w/v) BSA/NaCl/P
i
, a nd incubated with FITC-
CD2 monoclonal antibody (mAb) for 1 h at 37 °C. After
washing three times with 0.5% (w/v) BSA/10 m
M
Hepes/
NaCl/P
i
, the cells were fixed using 1% (v/v) paraformalde-
hyde/NaCl/P
i
andanalyzedwithaflowcytometer(FAC-
Scan apparatus, Becton Dickinson) equipped with the
CELL
QUEST
software. Ten thousand c ells were counted for every
sample during acquisition.
Inhibition of antibody binding
MOLT-3 cells were grown and activated with 0.2 l
M
of
phorbol 12-myristate-13-acetate (PMA) (Sigma) in 75 cm
2
tissue culture flasks at 37 °C in a saturating humidified
atmosphere of 95% air and 5% CO
2
. Cells were pelleted
at %100 g for 5 min, and re-suspended i n serum-free
medium to reach a cell count of 2.5 · 10
6
per mL.
Peptide stock solution was prepared in phosphate
buffered saline (NaCl/P
i
) and 0.25% (v/v) dimethylsulf-
oxide. Cell suspensions and peptide solutions (80, 200
and 500 l
M
) were aliqu oted into a 48-well cell culture
cluster a nd incubated at 37 °C for 1 h. At the end of
incubation, unbound peptide was removed by washing
with 10 m
M
Hepes/NaCl/P
i
. FITC-anti-CD58 was added
to the cell p ellets, f ollowed by incubation on ice f or
45 min. After washing three t imes with 10 m
M
Hepes/
NaCl/P
i
, the cells were fixed using 2% (v/v) paraformal-
dehyde/NaCl/P
i
and analyzed with a flow cytometer
(FACScan, Becton Dickinson) equipped with
CELL QUEST
software. Binding of FITC-anti-CD58, following incuba-
tion with F
c
blocker (Biodesign International) was used
as a positive control. Median values of fluorescence
intensity were taken as the binding intensities. As many
as 10 000 cells were counted for e very sample during
acquisition, and each experiment was performed in
triplicate. The control histogram (cells without peptide
treatment) was placed within 100–101 on the log scale of
FL1-Height by adjusting the FL1 detector. The data
were represented as their relative inhibition or enhance-
ment to the positive control.
Table 1. Peptides used in this study that are derived from rat CD2
protein. The sequence number refers to the residues from th e second
position in the peptide to eleventh position. Pen1 an d Cys12 were
introduced for cyclization p urpose.
Code Name
Sequence number
in the native protein
lER PenERGSTLVAEFC 36–45
cER Cyclo(1,12) PenERGSTLVAEFC 36–45
lVY PenVYSTNGTRILC 85–94
cVY Cyclo (1,12) PenVYSTNGTRILC 85–94
cEL Cyclo (1, 6) ERGSTL 36–41
cYT Cyclo (1, 6) YSTNGT 85–90
Control
peptide
KGKTDAISVKAI 91–80
a
a
Sequence from human CD2. The sequence was reversed, Tyr81
and Ty86 were replaced by Ala.
2874 L. Jining et al. (Eur. J. Biochem. 271) Ó FEBS 2004
E-rosetting
Sheep red blood cells (SRBCs) were isolated by centrifu-
ging sheep blood in Alsever’s solution at 200 g for 5 min.
SRBCs were washed three times with NaCl/P
i
and
incubated with four volumes of 2-aminoethylisothiouro-
nium hydrobromide (AET) solution (Sigma) at 37 °Cfor
15 min. The cells were washed three times in NaCl/P
i
,and
resuspended in RPMI-1640 containing 20% fetal bovine
serum to give a 10% suspension. For use, the cell
suspension was further diluted 1 : 20 ( 0.5%) with
medium. Serial dilution s of peptides i n NaCl/P
i
were
added to 0.2 mL of 0.5% (w/v) AET-treated SRBCs, and
incubated at 37 °C for 30 min. After that, 0.2 mL of
Jurkat cell suspension (2· 10
6
per mL) was added to the
mixture, and incubated for another 15 min. The cells were
pelleted by centrifugation (200 g,5min,4°C) and then
incubated a t 4 °C f or 1 h. The cell pellet w as gently
resuspended, and the E-rosettes counted with a haemo-
cytometer [16]. Cells with five or more SRBCs bound
were counted as rosettes. At least 200 cells were counted
to determine t he percentage of E-rosette cells. The
inhibitory activity was calculated by the following Eqn (1):
inhibition ð%Þ
¼½ðnegative E- rosette %
peptid e
À negative E-rosette %
blank
Þ=
E-rosette %
blank
Â100 ð1Þ
where, negative E-rosette %
peptide
¼ (Jurkat cells without
formation of E-rosettes/total Jurkat cells) · 100%.
Lymphocyte-epithelial adhesion assay
Caco-2 cells were used between passages 50 and 60 and w ere
plated onto 48-well plates at % 2 · 10
4
cellsÆwell
)1
.When
the cells reached confluency, the monolayers were washed
once with MEM-a. J urkat cells were labeled the same day as
the adhesion assay by loading with 2 l
M
fluorescent d ye bis-
carboxyethyl-carboxyfluorescein (BCECF-AM) at 3 7 °C
for 1 h. Peptide dissolved in MEM-a was added at various
concentrations to Caco-2 cell monolayers. After incubation
at 37 °C for 30 min, the l abeled Jurkat ce lls (1 · 10
6
cellsÆwell
)1
) were added onto the monolayers. After incu-
bation at 37 °C for 45 min, nonadherent Jurkat cells
were removed by washing three times with NaCl/P
i
,and
the monolayer-associated Jurkat cells were lysed with 2%
(v/v) Triton X-100 in 0.2
M
NaOH. Soluble lysates were
Fig. 1. Sequence alignment of rat CD2 and
human CD2 (hCD 2;
CLUSTALW
alignment).
Residues of domain 1 and 2 are shown.
#, Interface contact residues in the hCD 2 –
hCD 58 s tructure; *, residues in the interface;
b-turn regions are in b old letters; designed 12
amino acid residue peptide sequences are
underlined.
Ó FEBS 2004 Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2875
transferred to 96-well plates for reading with a microplate
fluorescence analyzer.
Cell viability assay
Peptides which exhibited effects on Jurkat/Caco-2 adher-
ence were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyl-tetrazolium bromide (MTT) assay [17] to determine if
their effects were d ue to frank toxicity. A final concentration
of 180 l
M
peptide was added to Caco-2 or Jurkat cells for 1
or 2 h, which is the time of exposure of Caco-2/Jurkat cells
during the adherence assay. The cell viabilities were
validated by incubating with 5 mgÆmL
)1
MTT at 37 °C
for 3 h. The MTT-labeled cells were lysed by d imethylsulf-
oxide and the absorbance was measured with a microplate
reader at a wavelength of 570 nm.
NMR spectroscopy
The samples for the NMR spectra of the peptide were
prepared by dissolving 3 mg of the peptides in 0.5 mL of
90% H
2
O/10% D
2
O. For pH titration experim ents, the pH
of the solution was varied by the a ddition of DCl or NaOD
(pH was not corrected for isotopic effects). The temperature
dependence of the amide proton chemical shift was
measured by collecting data from 283–303K in steps of
5K using a variable temperature probe. The one- and
two-dimensional NMR experiments were performed and
processed on 300 MHz and 500 MHz Bruker DRX spec-
trometers equipped with a 5-mm broad-band inverse probe,
at a proton frequency of 300.3414 MHz and 500.134 MHz,
respectively, using
XWINNMR
version 1.0 software. Spectra
were acquired at 298K unless otherwise specified. TOCSY
[18], DQF-COSY [19] and rotating frame nuclear Overha-
user spectroscopy (ROESY) [20] and NOESY [21] experi-
ments were performed by presaturation of water during
relaxation delay. Data were collected by the TPPI method
[22] with a sweep width of 5000 Hz. ROE cross-peak
volumes were measured using ROESY spectra with 300 ms
spin-lock times and NOESY cross-peak volumes for
hexapeptides were measured at 200 ms mixing time. Coup-
ling c onstants (
3
J
HNa
) were measured from the DQF-COSY
spectrum. Intensities were assigned as strong, medium and
weak with upper and lower boundaries of distance for d
Na
(i, i ), d
aN
(i,i+1)andd
NN
(i, i +1), 1.9–3.0, 2.2–3.6 and
3.0–5.0 A
˚
, respectively [23]. Side chain protons were not
stereospecifically assigned; hence, ROE/NOE restraints for
the side chain protons were calculated by considering
pseudoatoms [23].
Determination of peptide structures
Conformational space was searched for the peptides using
the
DISCOVER
program version 2000 (Accelrys Inc., San
Diego, CA, USA) to identify conformations consistent
with the experimental ROE and coupling constant data
[24,25]. Briefly, the linear peptide was subjected to MD
simulations in vacuo at 300K with ROE and disulfide bond
constraints [26]. The resulting structure w as cyclized by
forming disulfide bo nds. The cyclic structure obtained was
slowly heated t o 900 K in steps of 100 K d ynamics for 5 ps
duration at each step. At 900 K, MD simulations were
performed for 20 ps to explore several possible conforma-
tions that the peptide can acquire. The trajectory from high
temperature dynamics was analyzed for similarities be-
tween the structures by comparing the root mean square
deviations (rmsd) between each possible pair of structures,
and was divided into several conformational families. The
average structure was taken from each family and chosen
as starting structures for the calculation of corrected
interproton distances from ROESY intensities using Mat-
rix Analysis of Relaxation for Discerning the G eometry of
an Aqueous Structure (
MARDIGRAS
) [27], which takes into
account TOCSY contributions for the calculated intensities
in the ROESY spectrum.
MARDIGRAS
runs with correlation
time (s
c
) of 0.25, 0.35, 0.45, 0.55 and 0.65 ns were
performed with coupling constants c alculated from s tarting
model and observed
3
J
HNa.
The correlation tim e was
expected to be in the range 0.25–0.65 ns as the observed
intensities in 2D NOESY spectrum of this peptide were
almost zero. The interproton distances were calculated
based on the distance of 1.78 A
˚
between the two GlyH
a
protons in peptide cVY and PheH
b
protons in peptide
cER, respectively. For cyclic hexapeptides NOESY cross-
peaks were observed at 200 ms mixing time and GlyH
a
protons were used for calibration. After high temperature
dynamics with NOE constraints the folded peptide was
cyclized by p eptide bond to arrive at th e starting s tructures
for cyclic hexapeptides. In the case of ROESY spectrum
for 12 amino acid residue cyclic peptides (12-mers), the
corrected interproton distances were used for subsequent
calculation of the structure. Each structure obtained during
high temperature dynamics was then slowly cooled down
to 400 K. Each structure was then soaked with water
molecules, followed by MD simulations at 300 K with all
the ROE/NOE constraints. These structures were further
energy minimized w ith solvent molecule s using the steepest
descents and conjugate gradient methods until the rms
derivative was 0.3 kcalÆmol
)1
ÆA
˚
)1
. The resulting structures
were analyzed again by
MARDIGRAS
, and the final struc-
tures chosen wh en two criteria w ere fulfilled: (a) t he
conformation of backbone had an interproton error of less
than 0.2 A
˚
compared to upper and lower boundaries of
distances from ROE/NOE data and (b) the conformation
had / angles within 30° of the calculated /-values from
3
J
HNa
[28]. The final structures which satisfy most of the
NMR distance constraints were clustered together based
on the rms deviation of the backbone atoms and the
structures which had similar NOE/ROE violations were
clustered together as one family. Each family/cluster had
10–12 structures. An average structure was also chosen
from this family as representative structure.
Modeling of the peptide-CD58 complexes
Complexes of CD58–CD2 peptide were generated by
docking studies of CD2 peptide to CD58 protein crystal
structure. All docking studies were performed with the
AUTODOCK
program [29] (version 3.0). The coordinates of
peptides were retrieved from the NMR determined structure
(studies presented in this paper) and the coordinates of
ligated hCD58 were retrieved from the Protein Data Bank
(accession code 1qa9; the monomer of hCD58 was
unmerged from the complex of hCD2–hCD58) [30].
2876 L. Jining et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Hydrogen atoms were added to the protein using
INSIGHTII
(Accelrys Inc., San Diego, CA, USA). The appropriate
partial atomic charges were assigned by consistent valence
force field (Cvff). To eliminate the steric hindran ce between
peptide and protein, and to relax the hydrogen added to the
protein, the peptide and protein were merged and minimized
before docking. A tomic solvation parameters and fragmen-
tal volumes were assigned to the protein atoms by the
auxiliary program,
ADDSOL
. Affinity grid files were gener-
ated using the other auxiliary program,
AUTOGRID
.The
dimension of the grid box was chosen to cover the whole
protein with grid-point spacing of 0.375 A
˚
and centered at
the positions describe below. As there are two major cavities
in the top and bottom of hCD58 besides the binding sites in
the hCD2–hCD58 complex, the starting positions of
peptides were generated at three sites on CD58 protein
surface (Fig. 2A). The parameters were set as the default
values of the
AUTODOCK
Lamarckian genetic algorithm.
First, a randomized rigid docking (blind docking) was
performed and the conformers with lowest energy or in
significant clusters were chosen to perform further docking
studies with flexible docking.
During flexible docking, the dihedrals of backbone of the
ligand were kept rigid, whereas the dihedrals of side chain
were allowed to rotate. After docking, all structures
generated were assigned to clusters based on a tolerance
of 1 A
˚
all-atom rmsd from the lowest energy structure. The
energies were listed in the increasing order o f energy. If the
rmsd of a structure is less than 1 A
˚
compared to the lowest
energy structure in that starting position, that was grouped
together with the lowest energy structure forming a cluster
of structure. The clusters were ranked by the lowest energy
representative of each cluster. Only low energy structures
with more number of conformers in each cluster were used
for final analysis.
Results
Biological activity of the peptides
12-mer linear and cyclic peptides. The inhibitory activities
of the peptides designed from rat CD2 were assayed by three
methods. In the first method, the inhibition of anti-CD58
binding to CD58 expressed on t he surface o f MOLT-3 cells
was evaluated. Figure 3 shows that the peptide lVY
enhanced the binding of FITC-anti-CD58 in a concentra-
tion-dependent manner, while the peptides lER, cVY and
cER inhibited antibody binding. Compared with the two
cyclic peptides, line ar peptide lER d isplayed less inhibitory
activity, inhibiting only 6% at 500 l
M
. The peptide cER
showed better inhibitory activity (18% at 200 l
M
)com-
paredtopeptidecVY(7%).
A second method, E-rosetting, was carried out to test
the biological activity of peptides [16]. E-rosetting is the
most widely used method to identify T-cells by CD2–
CD58 interaction. SRBCs express CD58 protein, while
Jurkat leukemic T -cells express CD2 protein o n t heir
surface. The ability of Jurkat cells to express CD2 was
Fig. 2. Ribbon diagram of crystal structure o f CD2–CD58 complex and crystal structure of rat CD2. (A) Ribbon diagram of crystal structure of
CD2–CD58 (LFA-3) complex. Starting positions of peptides for docking s tudies are shown in the figure. The residues of hCD 2 t hat are in b-turn
region are shown as red sticks. Tyr86 from CD2 is shown in green. Residues from C D58 that are important in the interaction of CD2–CD58 are
shown in t he following colors: Lys32, Glu25 (purple); Asp33, Lys29, Glu37 (blue); Lys30 (magenta). Residues, Asp33 and Lys29 were shown to be
important in binding to peptides from CD 2 in docking stud ies. (B) Crystal structure of ratCD2. Residues in the b-turn re gion are shown as sticks
and labeled.
Ó FEBS 2004 Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2877
measured by flow-cytometry assay. Binding of Jurkat cells
to SRBCs due to CD2 and CD58 interaction results in the
formation of E-rosettes. The ability of each of the
designed CD2 peptides to inhibit CD2–CD58 interaction
was evaluated by inhibition of E-rosette formation
between Jurkat cells and SRBCs. As depicted in Fig. 4,
the CD2 peptides showed 30–40% inhibitory activity at
200 l
M
. When the concentration of the peptide was
decreased, the inhibitory activity of the peptide was
correspondingly decreased. Even at 50 l
M
, peptide cVY
displayed nearly 30% activity. Among the four peptides
(12-mers) studied, cVY showed the highest inhibitory
activity of 40% at 100 l
M
concentration. Both linear and
cyclic ER peptides (lER and cER) showed similar
inhibitory activities, whereas in the case of VY peptides,
the cyclic cVY peptide show ed increased activity comp ared
to its linear counterpart lVY. Correspondingly, a control
peptide showed less than 5% inhibitory activity by the
E-rosetting assay.
As a third me thod, inhibition of adh esion between
Caco-2 cells and Jurkat cells was used to evaluate the
biological activity of peptid es designed. Caco-2 cells express
CD58 while Jurkat cells express CD2 protein. The inhib-
itory activity observed between Caco-2 cells and Jurkat cells
provides evidence that the peptides designed from CD2 c an
inhibit the adhesion between the heterotypic cells. The
inhibitory activities of designed CD2 peptides were meas-
ured by using fluorescently labeled Jurkat-cells by fluores-
cence spectrometer. The activities of the peptides from CD2
in the heterotypic cell adhesion assay are shown in the Fig. 5
along with a control peptide. Among the 12-mers, cER,
lVY, cVY showed 30–50% inhibitory activity at 90 l
M
concentration. The c yclic peptide cVY showed % 50%
inhibition at 90 l
M
concentration. However, as the peptide
concentration was decreased to 10 l
M
, cVY showed less
than 15% activity whereas lER and cER peptides retained
20% inhibitory activity. Compared to linear peptides, cyclic
peptides showed a slight increase in activity. A control
peptide showed less than 5% inhibitory activity at three
different concentrations studied. These peptides were also
tested for their toxicity using the MTT assay [17]. All the
four peptides tested in the study resulted in 90–100%
viability indicating that these peptides were not toxic to cells
and the inhibition data observed were not due to changes in
the cells arising from peptide toxicity.
Cyclic hexapeptides. In order to understand the amino
acid residues i nvolved in the biological activity and to study
the effect of reducing the chain length of peptides on
Fig. 3. Inhibition or enhancement of FITC-labeled CD58-antibody
binding to MOLT-3 cells by synthetic peptides derived from CD2
examined by FACS. MOLT-3 cells were activated by 0.2 l
M
PMA to
induce CD58 expression. FITC-anti-CD58 w as added to t he peptide-
treated c ells, followed b y a further incubation. B inding of FITC-anti-
CD58, following incubation with F
c
blocker was used as a positive
control. Median values of fluorescence intensity were taken as the
binding intensities. As many as 10
4
cells were counted for every sample
during acquisition. The control histogram (cells without peptide
treatment) was placed within 100–101 on the log scale of FL1-height.
The data were r epresented as their relative inhibition or enhancement
to the p ositive control. Each data point repre sents the mean of tripli-
cate assay at different p eptide concen tration (lM).
Fig. 4. In hibition of E-rosette form atio n by syn thet ic peptides derived
from CD2 protein. Peptides were added t o A ET-treated Sheep Red
BloodCells(expressingCD58protein)firstandthenanequalamount
of Jurkat cells (expressing CD2 protein) were added later. The cells
were pelleted by centrifugation and incubated at 4 °C. The cell pellet
was resuspended gently before coun ting the E-ro settes. Cells with five
or more SRBCs bound were counted as rosettes. At least 200 cells were
counted to determine the percentage of E-rosette cells. Values are
percentage inhibition of peptide-treated cells and expressed as the
mean of three independent experiments.
Fig. 5. Inhibition of lymphocyte-epithelial adhesion by synthetic peptides
derived from CD2 protein. CD58 and CD2 expressing on Caco-2 cells
and Jurkat cells, respectively, were pre-examined. Peptides were added
to the confluent Caco-2 monolayer and then the BCECF labeled
Jurkat cells were added to the mixture. After the incubation for 45 min
at 37 °C, nonadherent Jurkat cells were removed b y washing with
NaCl/P
i
and the monolayers associated Jurkat cells were lysed with
Triton X-100 solution. Soluble l ysates are transferred to 9 6-well plates
for reading in a microplate fluorescence analyzer. Values are showed in
the percentage i nhibition of peptide-treated cells and expressed as the
mean of three independent experiments.
2878 L. Jining et al. (Eur. J. Biochem. 271) Ó FEBS 2004
biological activity, cyclic hexapeptides w ere designed. These
hexapeptides were truncated forms of the 12-mers described
above, and were cyclized by peptide bonds. The inhibitory
activities of the cyclic hexapeptides are shown in Figs 4
and 5. In the E-rosetting assay ( Fig. 4), peptide cEL showed
% 35% activity at a concentration of 200 l
M
,anincreasein
inhibitory activity compared to linear and c yclic 12-mer ER
peptides. However, the VY cyclic hexapeptide (cYT) lost
activity upon truncation. Similar trends were observed in
the heterotypic adhesion assay of cyclic hexapeptides
(Fig. 5). The cEL peptide showed increased activity (50%
at 90 l
M
), whereas cYT showed drastically diminished
inhibitory activity compared to 12-mer VY peptides.
NMR structure determination
The three-dimensional structures of the cyclic peptide were
determined based on the NMR data of the cyclic peptides.
The one dimensional
1
H NMR spectrum of the peptides
cVY, cER, cEL and cYT showed good dispersion of the
chemical shifts and the coupling patterns, indicative of a
stable major conformer at the experimental temperature.
The structure of peptide cER. NMR data of cER
indicated the possibility of the b-turn structure in peptide
cER. The d
NN
(i, i +1) cross peaks between Gly4-Ser5 and
the stronger d
aN
(i, i +1) cross peaks between Arg3-Gly4
suggesting a possible b-turn at Glu2-Arg3-Gly4-Ser5
(Fig. 6A). The two consecutive d
NN
(i, i +1) cross peaks
between Leu7-Val8 and Val8-Ala9 suggest a tight b-turn
at Leu7-Val8-Ala9-Glu10. The temperature-dependent
coefficient of the chemical shift data indicated that the
NH of Glu10 (Dd/DT ¼ )3.0 p.p.b.ÆK
)1
) is intramolecular
hydrogen bonded, suggesting a stable b-turn of Leu7-
Val8-Ala9-Glu10 [23]. The temperature coefficient of
chemical shift of Ser5 amide resonance showed a value
>)3.0 p.p.b.ÆK
)1
suggesting an open b-turn conformation
at Glu2-Arg3-Gly4-Ser5. From ROE-restrained MD
simulations and energy m inimization, four families o f
conformers that satisfied the NMR data were obtained.
An average structure was taken from e ach f amily t o
represent the family. Based on ROE violation > 0.2 A
˚
and
allowed values of /, w in the Ramachandran map, only one
family of structure th at was consistent with NMR data was
chosen to represent the conformation of peptide cER. A
family of low energy structures that were consistent with
NMR data representing the conformation of cER is shown
in Fig. 7A. The structure exhibits a well-defined b-turn
spanning residues Glu2 to Ser5. The rmsd of the backbone
atoms o f t he 12 structures in the chosen family was
compared with the average structure in the same family. It
was found that the rmsd of all the backbone atoms in the
peptide was 1.02 A
˚
, while that of residues at turn region
Glu2-Arg3-Gly4-Ser5 was 0.32 A
˚
, indicating the stable
nature of the b-turn conformation. The /, w angles around
Arg3-Gly4 and Val8-Ala9 of the structures showed the
possibility of a type II b-turn at Glu2-Arg3-Gly4-Ser5
residues and a type III b-turn at Leu7-Val8-Ala9-Glu10,
respectively [31]. Therefore, the structure of peptide cER
consists of two b-turns, located at the N- and C-termini. A
comparison of the b-turn structure of cER with the similar
region in the crystal structure of rat and human CD2 was
carried out. In the rat CD2 crystal structure, the b-turn
structure was exhibited by residues Arg37-Gly38-Ser39-
Thr40. The peptide cER displayed a b-turn structure with
shift in one re sidue compared to the protein from which it is
derived. In ratCD2, the type of b-turn observed at Arg37-
Gly38-Ser39-Thr40 i s a type II¢ b-turn whereas in cER
peptide t he b-turn is type II [31]. This is due to the position
of Gly amino acid in the b-turn which is flexible. In human
CD2, similar region (Fig. 1) has a b-turn is around Thr38-
Ser39-Asp40-Lys41 a nd the turn o bserved was type I
b-turn. An additional b-turn was observed in the cER
peptide structure at the Leu7-Val8-Ala9-Glu10 sequence
compared with the corresponding part in rat CD2
(Fig. 7 A).
The structure of peptide cVY. Several lines of NMR
evidence we re consistent with the existence of a b-turn in
the cVY peptide at Ser4-Thr5-A sn6-Gly7: (a) the Gly7
enantiotopic protons showed Dd-values > 0.4 p.p.m.
indicating the rigidity around this residue; (b) the d
NN
(i,
i + 1) cross peaks and medium range distance d
aN
(i,i+1)
cross peaks between Thr5-Asn6 and Gly7-Thr8 (Fig. 6B);
(c) the
3
J
HNa
of Thr5 and Asn6 were close to those expected
for a type I b-turn (i.e.
3
J
HNa
values of 4 Hz and 9 Hz are
expected for the i +1 and i +2 turn r esidues, respectively);
(d) the temperature dependence of the chemical shift data
indicates that the NH of Gly7 (Dd/DT ¼ )2.9 p.p.b.ÆK
)1
)
was solvent shielded or intramolecular hydrogen-bonded.
Molecular modeling studies resulted in seven families of
peptide cVY structures that best fi t the ROE and dihedral
angle data. The family/cluster of structures that had ROE
violation of £ 0.2 A
˚
was used to represent the final structure.
To check the convergence, the structures in each family were
superimposed on the average structure i n each family. All
structures presented a well-defined b-turn spanning residues
Ser4-Gly7 [31]. Lack of convergence was observed in the
first residue and the last three residues in the peptide
sequence. The average rmsd of the b ackbone atoms of
12 structures compared to the average structure was
0.98 ± 0.35 A
˚
, while the average rmsd at the residue
Ser4-Thr5-Asn6-Gly7 was 0.34 ± 0.06 A
˚
, indicating the
Fig. 6. Summar y of ROEs for pe ptides cER (A) and cVY (B). The
thickness of bars indicate the intensity o f ROE cross-peaks, and were
assigned as strong, medium a nd weak.
Ó FEBS 2004 Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2879
stable nature of the b-turn conformation. A representative
structure o f p eptide cVY families are shown in the Fig. 7B.
The /, w angles around Thr5-Asn6 showed that the
structure of the peptide deviated slightly from the ideal
type I b-turn [31]. A comparison of the b-turn structure of
cVY with the similar region in the crystal structure of rat
and human CD2 was carried out. T he type-I b-turn
observed in cVY around S er4-Thr5-Asn6-Gly7 was similar
to that in rat CD2 crystal structure (Ser87-Thr88-Asn89-
Gly90). In human CD2, the similar region Asp87-Thr88-
Lys89-Gly90 exhibits a type I b-turn. Superimposition of b-
turn regions from rat CD2 and human CD2 crystal
structure with the b-turn region of cVY peptide indicated
that the rmsd o f the backbone ato ms for four residues was
less than 1 A
˚
. Thus, the overall backbone and side chain
topologies of the turn region mimic those of rat CD2.
Structure of cyclic hexapeptides – cEL peptide. The
chemical shifts of amide resonances of peptide cEL were
well dispersed over a region of 1.2 p.p.m. indicating the
stable conformation of the peptide. The Gly3 enantiotopic
H
a
protons were well separated in chemical shift, indicative
of stable conformation around Gly3. The NH-NH region of
the NOESY data showed connectivity between the amides
of Glu1-Leu6, Glu1-Arg2, Gly3-Ser4, S er4-Thr5 and Arg2-
Lue6 which is suggestive of a b-turn in the peptide and
the proximity of amide protons due to compact nature of
the structure. However, t he coupling constant of most of the
amide protons was in the range of 6–8 Hz, suggestive of
rapidly interconverting conformers that coexist in solution.
The temperature coefficients of chemical shift of amides
Glu1 and Thr5 were n ear 3.8–4.0 p.p.b.ÆK
)1
which may be
due to intramolecular hydrogen bonding or solvent-shielded
amide protons of Glu1 and Thr5. Molecular modeling
studies indicated that the peptide exhibits two b-turns, i.e.
one at Arg2-Gly3-Ser4-Thr5 and the other at Thr5-Leu6-
Glu1-Arg2. A representative structure of cEL is shown in
the Fig. 8A. The b-turn at Arg2-Gly3-Ser4-Thr5 was type
II¢ b-turn as observed in the case of rat CD2 crystal
structure. Superimposition of backbone atoms of the
residues in the b-turn region of rat and human CD2
(similar region) with cEL peptide b-turn region (Arg2-Gly3-
Ser4-Thr5) indicated t hat the rmsd of the backbone atoms
was 0 .67 A
˚
with rat CD2 and 1.2 A
˚
with human CD2. Thus,
the peptide mimics the b-turn region of the protein from
which it is derived from. The peptide model also showed
intramolecular hydrogen bonds between NH of Thr5 and
CO of Arg2, as well as NH of Arg2 and CO of Thr5.
Structure of cyclic hexapeptides – cYT peptide. The
NMR data of the cYT peptide wer e indicative of its flexible
nature. The chemical shift dispersion of amides was less
than 1 p.p.m., and the Gly5 H
a
enantiotopic protons had a
degenerate chemical shift usually indicative of flexible
structure. Amide region of t he NOESY data suggested
weak intensity N OE connectivities between Tyr1-Ser2, Ser2-
Thr3, Asn4-Gly5 and Gly5-Thr6. Most of the coupling
constants were in the range of 6–8 Hz. Ser2 NH showed a
temperature coefficient of chemical shift value of
2.2 p.p.b.ÆK
)1
which may be due to the hydrogen-bonded
amide of Ser2. The ROE-based molecular modeling data on
cYTresultedinastructureshowninFig.8B.Thedihedral
angles around Thr6 and Tyr1 exhibited the dihedral a ngles
of a type I b-turn, and Gly5 exhibited c-turn dihedral angles.
The overall structure of the peptide was open/flexible as
indicated i n t he superimposed 12 structures shown in
Fig. 8B.
Docking
Recently, it has been shown that
AUTODOCK
can be used to
dock peptide to proteins without prior knowledge of the
binding site [32]. Peptides derived from CD2 presum-
ably modulate cell-adhesion by binding to CD58, hence
inhibiting CD2–CD58 interaction. Therefore, docking
studies of peptides to CD58 protein were carried out in
order to understand peptide–protein interactions by using
autodock [29]. In the docking of CD2 peptides to CD58
Fig. 7. Superposition o f 12 NMR-MD derived
structures for the cyclic peptides with average
structure for (A) cER and (B) cVY. Only heavy
atoms are shown for clarity. The residues
which are involved in b-turn conformation are
labeled.
2880 L. Jining et al. (Eur. J. Biochem. 271) Ó FEBS 2004
protein, the g rid was centered at three possible binding sites,
with a 110 · 110 · 110 A
˚
cubic area to cover the whole
CD58 protein. Three positions were chosen on the protein
surface of CD58 (Fig. 2A), i.e. (a) position 1, which is a
CC¢ sheet and the interface of CD2–CD58 interaction; (b)
position 2, the top cavity where the turn region from CD2
interacts with C D58; (c) position 3, the bottom c avity where
a turn region of CD2 interacts with C D58. First, a
randomized rigid docking (blind docking) was performed
and the conformers with lowest energy or in significant
clusters were chosen to perform further docking studies with
flexible docking.
Peptide cER–CD58 complex. The automated molecular
docking calculations produced several possible binding sites
and c onformations for the peptide. The c onformation
corresponding to the low energy of docking was chosen as
the possible binding site. The results from the docking
studies of cER peptide-CD58 protein are s hown in Table 2.
Although, differen t starting positions were chosen for the
cER peptide on the CD58 protein surface, the final low
energy docked conformers of the peptide were near the top
cavity region on the protein. Thus, the most probable
binding site of cER peptide on CD58 is possibly near the top
cavity. Table 3 lists the residues involved in intermolecular
hydrogen bonding in the cER peptide and CD58 protein
interface. It is very clear that most of the residues that
exhibit b-turn structure in the peptide (Glu2-Arg3-Gly4-
Ser5) were involved in hydrogen bonding with the protein
receptor (CD58). The Ser5 residue in the turn region of
peptide cER interacts with the key r esidue Asp33 of CD58
that is important in adhesion. Thr6, the flanking residue of
the b-turn region also forms a hydrogen bond with Asp33
which was shown to be important in CD2–CD58
interaction.
Peptide cVY–CD58 complex. Docking studies of the c VY
peptide and CD58 protein revealed that structures w ith low
energy of docking were around the CC¢ sheet of CD58
protein (Table 4, Fig. 2A). The CC¢ sheet is the interface of
CD2–CD58 interaction. Different starting positions yielded
low docked energy conformations in the CC¢ sheet region,
and hence the most possible binding site may be near the
CC¢ sheet. T he amino acid residues that are involved in the
cVY peptide–CD58 protein interaction are shown in
Fig. 8. Super position of 12 NMR-MD derived
structures for the cyclic hexapeptide w ith
average structure for (A) cEL and (B) cYT.
Only heavy atoms are shown for clarity.
Table 2. Peptide cER: CD58 docking results starting from the potential binding sites out of 100 runs. O n ly the c lus ter s with t he lowest e n erg y of
docking are listed.
Starting position
Final, low energy position
of the peptide after docking Cluster Rank
Lowest docked
energy (kcalÆmole
)1
)
Number of
conformations
in the cluster
CC¢ sheet Top cavity 1 )15.7 1
2 )15.5 5
Top Cavity Top cavity 1 )15.2 2
2 )13.2 1
Bottom cavity Top cavity 1 )15.5 1
2 )14.9 2
Table 3. Amino acid residues forming hydrogen bonds in the cER–
–
CD58
interface. The residues in the turn region of peptide cER and in CD58
which are imp ortant for the CD2–CD58 interac tion are shown in bold
italic typeface.
Peptide cER
()15.5 kcalÆmol
)1
) CD58
Residue Atom Residue Atom
Ser5 H
c
Lys30 O
Phe11 O Lys30 H
f
Ser5 HN Gln31 O
d
Arg3 O Gln31 H
e
Ser5 O Asp33 HN
Thr6 O Asp33 H
d
Arg3 NH1 Ser69 O
Phe11 O Ser70 H
c
Arg3 H
e
Glu72 O
e
Ó FEBS 2004 Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2881
the Table 5. It is very clear that the residues in the b-turn
(Ser4-Thr5-Asn6-Gly7) region of the cVY peptide are
involved in hydrogen bonding interaction with key
residues Asp33 and Lys29 of CD58.
Cyclic hexapeptide–CD58 docking
The NMR-derived cyclic hexapeptide struc tures were used
to perform docking studies of peptide–CD58 protein
interaction. Docking studies of cEL starting from different
possible positions on CD58 resulted in low energy structures
that were clustered around the top cavity of CD58 protein.
The lowest energy docked structure indicated that the Arg2
side chain of the peptide formed intermolecular hydrogen
bonding with key residue Lys34 on CD58. Ser4 (backbone
NH) and Thr5 (side chain) in the peptide were also involved
in intermolecular hydrogen bonding with Gln31 and Glu72
of the CD58 protein, respectively. Thus, the involvement
of key residue Lys34 in CD58 protein with hydrogen
bonding to peptide may result in inhibition of CD2–CD58
interaction.
The cYT peptide did not show binding site specificity.
The lowest energy clusters obtained after docking calcu-
lations were near the starting position of the peptide. The
low energy docked structures also indicated that Gly5
carbonyl carbon and Ser2, Asn4 side chains were involved
in intermolecular hydrogen bonding with the protein.
However, none of the hydrogen bonds were with the key
residues that are essential for CD2–CD58 interactions on
the CD58 protein. This supports the low biological
activity of cYT observed in the E-rosetting and hetero-
typic adhesion assays.
Discussion
Inhibition of CD2–CD58 interaction has important impli-
cations in controlling immune responses in autoimmune
diseases. I n this study, we d esigned 12-mer linear and cyclic
peptides (lVY, cVY, lER, and cER) as well as cyclic
hexapeptides (cEL a nd cYT) that were derived from the rat
CD2 sequence. Initially, the design of small molecular
inhibitors was based on the crystal structure of rat CD2 [33–
37]. The CD58 (LFA-3) binding ability o f CD2 is known to
reside in domain-1 of CD2 protein. CD2 peptide mapping
and mutagenesis indicated that the binding surface of CD2
consists of b-sheet formed by strands GFCC¢C¢¢.Thecrystal
structure of CD2 (Fig. 2B) indicated that the rather flat
b-sheet surface does not provide a complementary shape to
bind to CD 58, and h ence does not have well-defined
epitopes t o design small molecular inhibitors. The structure
of CD2 is similar to CD4 and other IgSF molecules. In the
D1 domain of CD4, the b-turn near CC¢ appears to be
important fo r binding to its receptor [38]. b-Turn peptides
based on C D4 have been shown to be effective in inhibiting
CD4 interactions [38]. Analysis of the crystal structure of
CD2 revealed that on either side of t he binding surface of
CD2, there are b-turns whic h stabilize the b-strands. Thus,
we hypothesized that these b-turns may serve as good
surface epitopes for the design of peptides to inhibit CD2–
CD58 interactions. Meanwhile, the crystal structure of
human CD2–CD58 became available [30]. Examination of
the CD2–CD58 crystal structure indicated that the interface
of the CD2–CD58 complex has poor shape complement-
arity in the center region of interaction (Fig. 2A). Most of
the interaction is via salt-bridges with c harge n eutralization
and hydrogen bonds. Furthermore, the b-strand surface of
CD2 that interacts with CD58 is stabilized by b-turns on
either side. These b-turn regions seem to be important in
holding the CD2–CD58 interface intact with b-sheet and
salt-bridges. Rat CD2 and human CD2 share sequence
similarity (Fig. 1). The residues in the b-turn of rat CD2
sequence are Arg37-Gly38-Ser39-Thr40 and Ser87-Thr88-
Asn89-Gly90, while those in human CD2 are at Thr38-
Ser39-Asp40-Lys41 and at Asp87-Thr88-Lys89-Gly90.
Lys41 of the b-turn at Thr38-Ser39-Asp40-Lys41 is involved
in the hydrogen bonding interaction with CD58. Similarly,
in the b-turn at Asp87-Thr88-Lys89-Gly90, the Gly90
backbone carbonyl carbon is involved in hydrogen bonding
interaction with CD58. The flanking residue Tyr86 of the
b-turn at Asp87-Thr88-Lys89-Gly90 has been shown to be
Table 4. Peptide cVY: CD58 docking results starting from the potential binding sites out of 100 runs. Only the clusters with the lowest docked energy
are listed.
Starting position
Final, low energy position
of the peptide after docking Cluster Rank
Lowest docked
energy
(kcalÆmole
)1
)
Number of
conformations
in the cluster
CC¢ sheet CC¢ sheet 1 )10.7 2
2 )10.0 5
Top cavity CC¢ sheet 1 )10.4 1
2 )10.0 1
Bottom Cavity CC¢ sheet 1 )9.7 2
4 )9.4 1
Table 5. Amino acid residues forming hydrogen bonds in the cVY–
CD58 in ter face. The residue s in the turn region of peptide cVY an d in
CD58 which are important for t he CD2–CD58 interactions are shown
in bold italic t ypeface.
Peptide cVY
()10.7 kcal/mol) CD58
Residue Atom Residue Atom
Tyr3 O Lys29 Hz
Thr5 O
c
Lys29 Hz
Asn6 H
d
Asp33 O
d
2882 L. Jining et al. (Eur. J. Biochem. 271) Ó FEBS 2004
important in CD2–CD58 interaction [3]. Therefore, we
hypothesized that the stable b-turn conformation mimick-
ing the native CD2 surface-binding region with CD58 may
be important for inhibitory activity of the peptide. Since
human CD2 shares sequence homology with rat CD2, we
designed the peptid e based on the b-turn region o f rat C D2
sequence. These peptides have been shown to inhibit cell–
cell adhesion. Several lines of evidence suggest that peptides
from rat CD2 may bind to human CD58. Human-CD2 and
rat-CD2 have high 3D structure similarity. Comparison of
3D structu res o f hCD2 from CD2–CD58 complex [30] and
the structure of rat CD2 [36] using D S modeling (Accelrys
Inc., Sandiego, CA, USA) suggested that the two structures
overlap very well. The pairwise r.m.s.d of 95 residues
(covering the interaction region of hCD2–hCD58) of
hCD2 and rat CD2 was 0.9 A
˚
.Theb-turn regions of
hCD2 (Thr38-Ser39-Asp40-Lys41 and Asp87-Thr88-
Lys89-Gly90) o verlap with b-turn regions of rat C D2
(Arg37-Gly38-Ser39-Thr40 and Ser87-Thr88-Asn89-
Gly90). In CD2–CD58 complex, the residues in the b-turn
and flanking residues o f this b-turnhavebeenshowntobe
important in interface contact of hCD2–hCD58. The
peptides designed from rat-CD2 sequence are in the b-turn
region and they overlap very well with b-turn regions of
human CD2. Point mutation data indicated that flanking
residue of th e b-turn region in human CD2 (Tyr86) is
important in binding to CD58. Similarly Gly90 in hCD2
makes a contact with hCD58. Glu36 a flanking residue of
the b-turn (Glu36-Arg37-Gly38-Ser39-Thr40) is also an
important residue in the interface of hCD2–hCD58. Com-
parison of hCD2 and rat CD2 3D structures indicated that
Tyr86 and Glu36 side chains overlap in hCD2 and rat
CD2 a nd oriented in the same direction in both the
structures.
To stabilize the b-turn structure in the designed
peptides, cyclic versions of the peptides were synthesized.
We chose to cyclize the peptides by disulfide bonds with
the introduction of amino acids penicillamine (Pen) and
cysteine at the two ends of the peptide sequence.
Cyclization by disulfide bond is relatively easy and yields
good yield after purification. Penicillamine with two bulky
methyl groups is known to stabilize disulfide bonds [39].
Penisusedintheposition1becauseinpreviousworkwe
have been successful in improving conformational stability
of the cyclic peptides by using Pen at position 1 [24,40].
Initially, 12-mer peptides were designed (Table 1). After
preliminary examination of biological activity of 12-mer
linear and cyclic peptides, the peptides were truncated to
six amino acid residues in order to elucidate the minimum
number of critical amino acids necessary for biological
activity. These hexapeptides were cyclized by amide bonds
to stabilize the structure. In the hexapeptides, cyclization
by disulfide bond w as not d esigned since a ddition of
penicillamine and cysteine to form a disulfide bond will
increase the number of amino acids in the peptide. The
information obtained from truncating the peptides will
also be useful in the design of future generation pharma-
cophores. A control peptide (Table 1) was designed to
compare the importance of primary and secondary
structures in the designed peptides. The control peptide
sequence was chosen from the Ôhot-spotÕ [3] region of the
hCD2–hCD58 interface on t he CD2 p rotein. The
sequence w as then r eversed and the important amino
acidTyr86aswellasTyr81wasreplacedbyAlato
generate the control peptide sequence shown in Table 1.
The abilities of our designed peptides (except peptide
lVY) to inhibit binding of anti-CD58 to CD58 protein
expressed on the surface of MOLT-3 cells suggested that
they may interrupt the interaction between CD2–CD58.
In addition, cyclic peptide cVY exhibited higher activity
than the linear peptide derived from the same region,
supporting the conformational dependence of the peptide
inhibition. Two different methods of inhibition of cell
adhesion were used to evaluate the biological activities of
peptides. Both the assays indicated that peptides designed
from CD2 were potent inhibitors of cell-adhesion. While
the 12-mer cyclic peptide cVY showed % 45–50% inhibi-
tion in the E-rosetting and heterotypic adhesion assays,
linear and cyclic ER peptides showed similar inhibition
activity. In the case of the cVY peptide, cyclization
seemed to improve the activity of the p eptide. T hus,
cyclization s tabilizes the b-turn conformation in the
peptide which may be necessary for biological activity
exhibited by the peptides. Comparing the antibody
binding assay and cell adhesion assay, there is a difference
in the trend of activities of the peptides particularly in the
case of linear peptide lVY. This peptide showed enhance-
ment in the a ntibody b inding assay, whereas i n cell-
adhesion inhibition assay, the peptide inh ibited cell-cell
adhesion. While all the three methods used to evaluate
biological activities of the p eptides in this s tudy were
expected to generate similar results, there were differences
due to the nature o f d ifferent cell types u sed i n t he
experiments. The binding sites on the surface of these cells
may be unique. MOLT-3 and Jurkat cells are derived
from lymphocytes, while sheep blood cells are erythro-
cytes. Furthermore, CD58 may have different epitopes to
bind to antibody in the antibody binding assay. Compar-
ing the concentration of peptides used in inhibition studies
by all the three methods, the antib ody binding inhibition
assay was the least sensitive and required higher concen-
trations of peptides to observe inhibition. Also, the
peptide lVY is a linear and may exhibit random structure.
Overall, the E-rosetting and heterotypic adhesion assays
provide concrete evidence that the designed peptides can
inhibit the mechanism of cell–cell adhesion.
Truncation of the 12-mer peptide cER to the cyclic
hexapeptide cEL resulted in higher biological activity. This
provides the evidence that amino acids in the b-turn region
and stable conformation of the peptide are important for
biological activity. Truncation of the cVY peptide to the
hexapeptide cYT resulted in drastically reduced biological
activity suggesting the lack of stable structure and amino
acids important for biological activity of the peptide .
This suggests that b-turn conformation of the residues in
the b-turn region may be important in the inhibitory
mechanism, in addition to the primary structure of the
peptide.
To confirm our hypothesis that the b-turn structures are
important for the inhibitory activities of the peptides, the
structures of the cyclic peptides were determined by NMR.
The results proved that the stable b-turn conformation
exists in the cyclic peptides. The b-turn regions in these
peptides appeared to be more stable with flexibility at the
Ó FEBS 2004 Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2883
terminal regions, thus reinforcing our hypothesis that the
Ôactive coreÕ is located in this turn region or b-turn exposes
the important residues to the receptor. Moreover, the
modeling of the peptide-protein complex suggested that
the residues in the b-turnregionplayedakeyroleinthe
interaction. Molecular modeling studies predicted that the
b-turn in the cyclic peptides closely mimics the conforma-
tional feature of b-turn in CD2 protein. In addition, the
binding sites of the peptides to CD58 protein predicted by
autodock were near the CD2–CD58 interface, either in the
topcavityofCD58orneartheCC¢ sheet which explained
the inhibitory activity of the cyclic peptides. There was no
direct correlation between low energy docked structure of
the peptide from docking studies and biological activity in
terms of cell-adhesion inhibition by two methods explained.
Docking studies using autodock do not take into account
the presence of CD2 receptor in competing with peptides
from CD2 to bind to CD58. It provides only probable
binding sites with relatively low energy of interaction
between peptide and protein analyzed in the stud y. How-
ever, docking studies were i n agreement with a ntibody
binding inhibition assay. Comparison of docking energies of
the two cyclic peptides indicated that the cER peptide had
low docked energy compared with the cVY peptide.
Biological activity of the peptides by antibody b inding
assay suggested that cER peptide inhibits antibody binding
better than cVY peptide. However, E-rosetting assay and
heterotypic cell adhesion assay indicated that cVY exhibits
cell-adhesion inhibition better than cER. The cER peptide
may potentially bind to the CD58 protein as indicated by
the docking studies. The lowest energy docked structure was
at the top cavity. On the other hand, cVY peptide has
relatively high energy compared to cER, but the position of
docking is near the CC¢ sheet which involves many salt
bridges and hydrogen bonds in CD2–CD58 interaction.
Thus, in terms of inhibition of cell adhesion, cVY has more
potential since it may directly interrupt the binding mech-
anism of CD2–CD58. Inhibition of cell adhesion involves
interrupting the interaction of key residues at t he protein–
protein interface. If we consider hydrogen bonding interac-
tion between the p eptide and CD58, the cER peptide f orms
hydrogen bonds with Asp33 in the lowest energy docking
position. Asp33 is one of the key residues in CD2–CD58
protein–protein interaction [3]. The peptide cVY is invol-
ved in hydrogen bonding interaction with Asp33 and Lys29
on CD58 in the lowest docked energy structure. Both
Asp33 and Lys29 are very important residues in CD2–
CD58 interaction. Mutational studies have indicated that
removal of these two interactions can result in loss of
CD2–CD58 interaction. This correlates with higher inhib-
itory activity of the cVY peptide compared to the cER
peptide.
In the case of cyclic hexapeptide cEL, the lowest
energy docked structure showed that cEL hydrogen
bonds with Lys34, whereas cYT is not involved in
hydrogen bonding with key residues that are important
for CD2–CD58 interaction. This correlates with the
higher inhibitory activity of the cEL peptide and the very
low inhibitory activity of the cYT peptide compared to
other peptides.
The flanking residue of the b-turn, i.e. Tyr3 of the cVY
peptide appears to be important in exhibiting inhibitory
activity. Mutation of Tyr86 to alanine in the native protein
reduces the affinity of the CD2–CD58 complex by more
than 1000-fold. Thus, Tyr86 on the CD2 surface has been
defined as a Ôhot-spotÕ [3], and may represent a useful target
for the design of small molecules. This may explain the
higher inhibitory activity of the cVY peptide (which
contains Tyr3) compared to the cER peptide in the
E-rosetting and heterotypic adhesion assays. However, in
the case o f cyclic hexapeptide cYT, although it contains an
important residue Tyr1, the peptide exhibits low inhibition
activity in E-rosettin g and heterotypic adhesion assays. The
peptide cYT is expected to mimic the region with Tyr86 in
the protein. NMR and molecular modeling studies sugges-
ted that cYT does not acquire a stable b-turn structure.
Cyclization may change the orientation of Tyr1 in the small
cyclic peptide which may result i n the loss of activity.
Docking studies of cYT peptide resulted in nonspecific
binding to CD58 surface at three positions.
In conclusion, we have designed peptides from the b-turn
region of the CD2 protein that are critical for inhibiting
CD2–CD58 interaction. NMR studies indicated that the
cyclic peptides acquire b-turn structures in solution. Cell
viability assays clearly suggested that the peptides are not
toxic to cells tested in the studies, and represent potential
lead compound s for immunomodulation. Thus, the de-
signed peptides have been shown to mimic the epitopes on
CD2 protein that interact with CD58.
Acknowledgements
We gratefully acknowledge the Super Computer and Visualization
Unit, National University of Singapore for the use of computational
facilities; Prof. Arthur J. Olson, the Scripps Research Institute , USA,
for providing us with the AutoDock software. This research was funded
by an a cademic research grant ( R-148-000-026-1 12) from the National
University of Singapore, S ingapore.
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Supplementary material
The following material is available from http://blackwell
publishing.com/products/journals/suppmat/ejb/ejb4198/
ejb4198.htm
Appendix 1. Chemical shift, coupling constants and tem-
perature dependence of amide proton resonance data at
298K for peptides, cER, cVY, cEL.
Ó FEBS 2004 Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2885