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

Eur. J. Biochem. 271, 2873–2886 (2004) Ó FEBS 2004

doi:10.1111/j.1432-1033.2004.04198.x

Design, structure and biological activity of b-turn peptides
of CD2 protein for inhibition of T-cell adhesion
Liu Jining1, Irwan Makagiansar3, Helena Yusuf-Makagiansar3, Vincent T. K. Chow2, Teruna J. Siahaan3
and Seetharama D. S. Jois1
1

Department of Pharmacy and 2Department of Microbiology, National University of Singapore, Singapore; 3Department 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 interactions has been shown to be therapeutically useful. Synthetic 12-mer linear and cyclic peptides,
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

Accessory molecules, CD2–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 be
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, erythrocytes, endothelium,
platelets, and granulocytes. It has been found that this
heterophilic adhesion facilitates initial cell–cell contact
before specific antigen recognition, and also enhances
T-cell receptor (TcR) triggering by fostering interaction
with peptide-class II major histocompatability complex
(pMHC). The affinity of CD2–CD58 interaction is relatively low (Kd % 1 lM), with very rapid koff and kon 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

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:
Abbreviations: AET, 2-aminoethylisothiouronium hydrobromide;
BCECF-AM, bis-carboxyethyl-carboxyfluorescein, acetoxymethyl;
FITC, fluorecein isothiocynate; hCD2, human CD2; hCD58, human
CD58; MEM-a, minimum 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)

cyclic peptides exhibit b-turn structure in solution and closely mimic the b-turn structure 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).

pMHC with a 50- to 100-fold greater efficiency than its
absence [4]. In addition, endothelial cells (EC) in rheumatoid 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 the treatment of autoimmune
diseases.
It has been shown that blockade of the CD2–CD58
interaction [7,8] and/or modulation of the CD2 costimulatory 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 their
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.-hostdisease. 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, interfere
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 protein epitopes to modulate
CD2–CD58 interaction. We designed linear and cyclic peptides (Table 1) from the b-turn regions of rat CD2 protein
(Fig. 1), and evaluated their ability to inhibit cell adhesion
using antibody, E-rosetting and heterotypic-adhesion


Ó FEBS 2004

2874 L. Jining et al. (Eur. J. Biochem. 271)
Table 1. Peptides used in this study that are derived from rat CD2
protein. The sequence number refers to the residues from the second
position in the peptide to eleventh position. Pen1 and Cys12 were
introduced for cyclization purpose.

purchased from Biodesign International (Saco, ME, USA)
and Immunotech, respectively.

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 MOLT-3 cells were maintained in suspension in
RPMI1640 medium supplemented with 10% (w/v) heatinactivated fetal bovine serum and 100 mgỈL)1 of penicillin/
streptomycin. Caco-2 cells were maintained in minimum
essential medium-a containing 10% (w/v) fetal bovine
serum, 1% (v/v) nonessential amino acids, 1 mM

Na-pyruvate, 1% (v/v) L-glutamine and 100 mgỈL)1 of
penicillin/streptomycin. Caco-2 cells were used between
passages 50 and 60. Sheep blood in Alsever’s solution was
purchased from TCS Biosciences Ltd., Singapore.

Code

Name

Sequence number
in the native protein

lER
cER
lVY
cVY
cEL
cYT
Control
peptide

PenERGSTLVAEFC
Cyclo(1,12) PenERGSTLVAEFC
PenVYSTNGTRILC
Cyclo (1,12) PenVYSTNGTRILC
Cyclo (1, 6) ERGSTL
Cyclo (1, 6) YSTNGT
KGKTDAISVKAI

36–45

36–45
85–94
85–94
36–41
85–90
91–80a

a
Sequence from human CD2. The sequence was reversed, Tyr81
and Ty86 were replaced by Ala.

inhibition assays. In order to understand structure–function relationship of peptides, we also carried out detailed
NMR, molecular modeling and docking studies of peptide-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 bombardment mass spectrometry (FABMS). The HPLC chromatogram 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 using automatic solid-phase peptide synthesizer (Pioneer, Perspective
Biosystem, Foster, CA, USA) using Fmoc chemistry with
PAL resin. The 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) with a linear gradient of
solvent A (0.1% (v/v) trifluoroacetic acid/H2O) 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 as in the 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 CD2 (FITC-anti-CD2) were

Cell lines

CD2 detection and flow-cytometry assay
To detect CD2 expression, 106 Jurkat cells were washed
with 0.5% (w/v) BSA/NaCl/Pi, and incubated with FITCCD2 monoclonal antibody (mAb) for 1 h at 37 °C. After
washing three times with 0.5% (w/v) BSA/10 mM Hepes/
NaCl/Pi, the cells were fixed using 1% (v/v) paraformaldehyde/NaCl/Pi and analyzed with a flow cytometer (FACScan apparatus, Becton Dickinson) equipped with the CELL
QUEST software. Ten thousand cells were counted for every
sample during acquisition.
Inhibition of antibody binding
MOLT-3 cells were grown and activated with 0.2 lM of
phorbol 12-myristate-13-acetate (PMA) (Sigma) in 75 cm2
tissue culture flasks at 37 °C in a saturating humidified
atmosphere of 95% air and 5% CO2. Cells were pelleted

at %100 g for 5 min, and re-suspended in serum-free
medium to reach a cell count of 2.5 · 106 per mL.
Peptide stock solution was prepared in phosphate
buffered saline (NaCl/Pi) and 0.25% (v/v) dimethylsulfoxide. Cell suspensions and peptide solutions (80, 200
and 500 lM) were aliquoted into a 48-well cell culture
cluster and incubated at 37 °C for 1 h. At the end of
incubation, unbound peptide was removed by washing
with 10 mM Hepes/NaCl/Pi. FITC-anti-CD58 was added
to the cell pellets, followed by incubation on ice for
45 min. After washing three times with 10 mM Hepes/
NaCl/Pi, the cells were fixed using 2% (v/v) paraformaldehyde/NaCl/Pi and analyzed with a flow cytometer
(FACScan, Becton Dickinson) equipped with CELL QUEST
software. Binding of FITC-anti-CD58, following incubation with Fc 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 every 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 enhancement to the positive control.


Ó FEBS 2004

Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2875

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 structure; *, residues in the interface;
b-turn regions are in bold letters; designed 12
amino acid residue peptide sequences are
underlined.

E-rosetting
Sheep red blood cells (SRBCs) were isolated by centrifuging sheep blood in Alsever’s solution at 200 g for 5 min.
SRBCs were washed three times with NaCl/Pi and
incubated with four volumes of 2-aminoethylisothiouronium hydrobromide (AET) solution (Sigma) at 37 °C for
15 min. The cells were washed three times in NaCl/Pi, 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 dilutions of peptides in NaCl/Pi 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· 106 per mL) was added to the
mixture, and incubated for another 15 min. The cells were
pelleted by centrifugation (200 g, 5 min, 4 °C) and then
incubated at 4 °C for 1 h. The cell pellet was gently
resuspended, and the E-rosettes counted with a haemocytometer [16]. 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. The
inhibitory activity was calculated by the following Eqn (1):

inhibition %ị
ẳ ẵnegative E- rosette %peptide À negative E-rosette %blank Þ=
E-rosette %blank Š 100
1ị

where, negative E-rosette %peptide ẳ (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 were
plated onto 48-well plates at % 2 · 104 cellsỈwell)1. When
the cells reached confluency, the monolayers were washed
once with MEM-a. Jurkat cells were labeled the same day as
the adhesion assay by loading with 2 lM fluorescent dye biscarboxyethyl-carboxyfluorescein (BCECF-AM) at 37 °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 labeled Jurkat cells (1 · 106
cellsỈwell)1) were added onto the monolayers. After incubation at 37 °C for 45 min, nonadherent Jurkat cells
were removed by washing three times with NaCl/Pi, and
the monolayer-associated Jurkat cells were lysed with 2%
(v/v) Triton X-100 in 0.2 M NaOH. Soluble lysates were


Ó FEBS 2004

2876 L. Jining et al. (Eur. J. Biochem. 271)

transferred to 96-well plates for reading with a microplate
fluorescence analyzer.
Cell viability assay
Peptides which exhibited effects on Jurkat/Caco-2 adherence were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay [17] to determine if
their effects were due to frank toxicity. A final concentration
of 180 lM 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 mgỈmL)1 MTT at 37 °C
for 3 h. The MTT-labeled cells were lysed by dimethylsulfoxide 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% H2O/10% D2O. For pH titration experiments, the pH
of the solution was varied by the addition 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 spectrometers 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 Overhauser spectroscopy (ROESY) [20] and NOESY [21] experiments 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. Coupling constants (3JHNa) were measured from the DQF-COSY
spectrum. Intensities were assigned as strong, medium and
weak with upper and lower boundaries of distance for dNa
(i, i), daN (i, i +1) and dNN (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].

performed for 20 ps to explore several possible conformations that the peptide can acquire. The trajectory from high
temperature dynamics was analyzed for similarities between 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 Matrix Analysis of Relaxation for Discerning the Geometry 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 (sc) of 0.25, 0.35, 0.45, 0.55 and 0.65 ns were
performed with coupling constants calculated from starting
model and observed 3JHNa. The correlation time 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 GlyHa
protons in peptide cVY and PheHb protons in peptide
cER, respectively. For cyclic hexapeptides NOESY crosspeaks were observed at 200 ms mixing time and GlyHa
protons were used for calibration. After high temperature
dynamics with NOE constraints the folded peptide was
cyclized by peptide bond to arrive at the starting structures
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 with solvent molecules using the steepest
descents and conjugate gradient methods until the rms
˚
derivative was 0.3 kcalỈmol)1ỈA)1. The resulting structures
were analyzed again by MARDIGRAS, and the final structures chosen when two criteria were fulfilled: (a) the
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
JHNa [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.

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 was cyclized by

forming disulfide bonds. The cyclic structure obtained was
slowly heated to 900 K in steps of 100 K dynamics for 5 ps
duration at each step. At 900 K, MD simulations were

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].


Ó FEBS 2004

Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2877

Fig. 2. Ribbon diagram of crystal structure of 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 studies are shown in the figure. The residues of hCD2 that are in b-turn
region are shown as red sticks. Tyr86 from CD2 is shown in green. Residues from CD58 that are important in the interaction of CD2–CD58 are
shown in the 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 CD2 in docking studies. (B) Crystal structure of ratCD2. Residues in the b-turn region are shown as sticks
and labeled.

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 hindrance between
peptide and protein, and to relax the hydrogen added to the
protein, the peptide and protein were merged and minimized
before docking. Atomic solvation parameters and fragmental volumes were assigned to the protein atoms by the
auxiliary program, ADDSOL. Affinity grid files were generated 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 of 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 the surface of MOLT-3 cells
was evaluated. Figure 3 shows that the peptide lVY
enhanced the binding of FITC-anti-CD58 in a concentration-dependent manner, while the peptides lER, cVY and
cER inhibited antibody binding. Compared with the two
cyclic peptides, linear peptide lER displayed less inhibitory
activity, inhibiting only 6% at 500 lM. The peptide cER
showed better inhibitory activity (18% at 200 lM) compared to peptide cVY (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 on their
surface. The ability of Jurkat cells to express CD2 was


2878 L. Jining et al. (Eur. J. Biochem. 271)

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 lM PMA to

induce CD58 expression. FITC-anti-CD58 was added to the peptidetreated cells, followed by a further incubation. Binding of FITC-antiCD58, following incubation with Fc blocker was used as a positive
control. Median values of fluorescence intensity were taken as the
binding intensities. As many as 104 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 represented as their relative inhibition or enhancement
to the positive control. Each data point represents the mean of triplicate assay at different peptide concentration (lM).

Fig. 4. Inhibition of E-rosette formation by synthetic peptides derived
from CD2 protein. Peptides were added to AET-treated Sheep Red
Blood Cells (expressing CD58 protein) first and then an equal amount
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 counting the E-rosettes. 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.

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 lM. When the concentration of the peptide was
decreased, the inhibitory activity of the peptide was
correspondingly decreased. Even at 50 lM, peptide cVY
displayed nearly 30% activity. Among the four peptides

(12-mers) studied, cVY showed the highest inhibitory

Ó FEBS 2004

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 by washing with
NaCl/Pi and the monolayers associated Jurkat cells were lysed with
Triton X-100 solution. Soluble lysates are transferred to 96-well plates
for reading in a microplate fluorescence analyzer. Values are showed in
the percentage inhibition of peptide-treated cells and expressed as the
mean of three independent experiments.

activity of 40% at 100 lM 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 showed increased activity compared
to its linear counterpart lVY. Correspondingly, a control
peptide showed less than 5% inhibitory activity by the
E-rosetting assay.
As a third method, inhibition of adhesion between
Caco-2 cells and Jurkat cells was used to evaluate the
biological activity of peptides designed. Caco-2 cells express
CD58 while Jurkat cells express CD2 protein. The inhibitory activity observed between Caco-2 cells and Jurkat cells
provides evidence that the peptides designed from CD2 can
inhibit the adhesion between the heterotypic cells. The
inhibitory activities of designed CD2 peptides were measured by using fluorescently labeled Jurkat-cells by fluorescence 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 lM
concentration. The cyclic peptide cVY showed % 50%
inhibition at 90 lM concentration. However, as the peptide
concentration was decreased to 10 lM, 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 involved in the biological activity and to study
the effect of reducing the chain length of peptides on


Ó FEBS 2004

Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2879

biological activity, cyclic hexapeptides were 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 lM, an increase in
inhibitory activity compared to linear and cyclic 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 lM), 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 1H 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 dNN (i, i +1) cross peaks between Gly4-Ser5 and
the stronger daN (i, i +1) cross peaks between Arg3-Gly4
suggesting a possible b-turn at Glu2-Arg3-Gly4-Ser5
(Fig. 6A). The two consecutive dNN (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 ẳ )3.0 p.p.b.ặK)1) is intramolecular
hydrogen bonded, suggesting a stable b-turn of Leu7Val8-Ala9-Glu10 [23]. The temperature coefficient of
chemical shift of Ser5 amide resonance showed a value
>)3.0 p.p.b.ỈK)1 suggesting an open b-turn conformation
at Glu2-Arg3-Gly4-Ser5. From ROE-restrained MD
simulations and energy minimization, four families of

conformers that satisfied the NMR data were obtained.
An average structure was taken from each family to

Fig. 6. Summary of ROEs for peptides cER (A) and cVY (B). The
thickness of bars indicate the intensity of ROE cross-peaks, and were
assigned as strong, medium and weak.

˚
represent the family. Based on ROE violation > 0.2 A and
allowed values of /, w in the Ramachandran map, only one
family of structure that 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 of the 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-Ser39Thr40. The peptide cER displayed a b-turn structure with
shift in one residue compared to the protein from which it is
derived. In ratCD2, the type of b-turn observed at Arg37Gly38-Ser39-Thr40 is a type II¢ b-turn whereas in cER
peptide the 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 Thr38Ser39-Asp40-Lys41 and the turn observed 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. 7A).
The structure of peptide cVY. Several lines of NMR
evidence were consistent with the existence of a b-turn in
the cVY peptide at Ser4-Thr5-Asn6-Gly7: (a) the Gly7
enantiotopic protons showed Dd-values > 0.4 p.p.m.
indicating the rigidity around this residue; (b) the dNN (i,
i +1) cross peaks and medium range distance daN (i, i +1)
cross peaks between Thr5-Asn6 and Gly7-Thr8 (Fig. 6B);
(c) the 3JHNa of Thr5 and Asn6 were close to those expected
for a type I b-turn (i.e. 3JHNa values of 4 Hz and 9 Hz are
expected for the i +1 and i +2 turn residues, respectively);
(d) the temperature dependence of the chemical shift data
indicates that the NH of Gly7 (Dd/DT ẳ )2.9 p.p.b.ặK)1)
was solvent shielded or intramolecular hydrogen-bonded.
Molecular modeling studies resulted in seven families of
peptide cVY structures that best fit 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 in 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 backbone 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


Ó FEBS 2004

2880 L. Jining et al. (Eur. J. Biochem. 271)

Fig. 7. Superposition of 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.

stable nature of the b-turn conformation. A representative
structure of peptide 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. The type-I b-turn
observed in cVY around Ser4-Thr5-Asn6-Gly7 was similar
to that in rat CD2 crystal structure (Ser87-Thr88-Asn89Gly90). In human CD2, the similar region Asp87-Thr88Lys89-Gly90 exhibits a type I b-turn. Superimposition of bturn regions from rat CD2 and human CD2 crystal
structure with the b-turn region of cVY peptide indicated
that the rmsd of the backbone atoms 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
Ha 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, Ser4-Thr5 and Arg2Lue6 which is suggestive of a b-turn in the peptide and
the proximity of amide protons due to compact nature of
the structure. However, the 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 near 3.8–4.0 p.p.b.ỈK)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-Leu6Glu1-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-Gly3Ser4-Thr5) indicated that 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 were indicative of its flexible
nature. The chemical shift dispersion of amides was less
than 1 p.p.m., and the Gly5 Ha enantiotopic protons had a
degenerate chemical shift usually indicative of flexible
structure. Amide region of the NOESY data suggested
weak intensity NOE connectivities between Tyr1-Ser2, Ser2Thr3, 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.ỈK)1 which may be due to the hydrogen-bonded
amide of Ser2. The ROE-based molecular modeling data on
cYT resulted in a structure shown in Fig. 8B. The dihedral
angles around Thr6 and Tyr1 exhibited the dihedral angles
of a type I b-turn, and Gly5 exhibited c-turn dihedral angles.
The overall structure of the peptide was open/flexible as
indicated in the 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 presumably 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


Ó FEBS 2004

Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2881

Fig. 8. Superposition of 12 NMR-MD derived
structures for the cyclic hexapeptide with
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. Only the clusters with the lowest energy of
docking are listed.

Starting position

Final, low energy position
of the peptide after docking

CC¢ sheet

Top cavity

Top Cavity

Top cavity

Bottom cavity

Top cavity

protein, the grid 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 CD58; (c) position 3, the bottom cavity where
a turn region of CD2 interacts with CD58. 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 conformations for the peptide. The conformation
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 shown in Table 2.
Although, different 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-Gly4Ser5) were involved in hydrogen bonding with the protein
receptor (CD58). The Ser5 residue in the turn region of
peptide cER interacts with the key residue Asp33 of CD58
that is important in adhesion. Thr6, the flanking residue of
the b-turn region also forms a hydrogen bond with Asp33

Cluster Rank

Lowest docked
energy (kcalỈmole)1)

Number of
conformations
in the cluster

1
2
1
2
1
2

)15.7
)15.5
)15.2
)13.2
)15.5
)14.9

1
5
2
1
1
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 important for the CD2–CD58 interaction are shown in bold
italic typeface.
Peptide cER
()15.5 kcalỈmol)1)

CD58

Residue

Atom

Residue

Atom

Ser5
Phe11
Ser5
Arg3

Ser5
Thr6
Arg3
Phe11
Arg3

Hc
O
HN
O
O
O
NH1
O
He

Lys30
Lys30
Gln31
Gln31
Asp33
Asp33
Ser69
Ser70
Glu72

O
Hf
Od
He

HN
Hd
O
Hc
Oe

which was shown to be important in CD2–CD58
interaction.
Peptide cVY–CD58 complex. Docking studies of the cVY
peptide and CD58 protein revealed that structures with 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. The amino acid residues that are involved in the
cVY peptide–CD58 protein interaction are shown in


Ó FEBS 2004

2882 L. Jining et al. (Eur. J. Biochem. 271)

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.

CC¢ sheet

CC¢ sheet

Top cavity

CC¢ sheet

Bottom Cavity

Cluster Rank

CC¢ sheet

Table 5. Amino acid residues forming hydrogen bonds in the cVY–
CD58 interface. The residues in the turn region of peptide cVY and in
CD58 which are important for the CD2–CD58 interactions are shown
in bold italic typeface.
Peptide cVY
()10.7 kcal/mol)

CD58

Residue

Atom

Residue

Atom

Tyr3
Thr5
Asn6

O
Oc
Hd

Lys29
Lys29
Asp33

Hz
Hz
Od

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 structures 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 calculations 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 heterotypic adhesion assays.

Lowest docked
energy
(kcalỈmole)1)

Number of
conformations
in the cluster

1
2
1
2
1
4

Starting position

Final, low energy position

of the peptide after docking

)10.7
)10.0
)10.4
)10.0
)9.7
)9.4

2
5
1
1
2
1

Discussion
Inhibition of CD2–CD58 interaction has important implications in controlling immune responses in autoimmune
diseases. In this study, we designed 12-mer linear and cyclic
peptides (lVY, cVY, lER, and cER) as well as cyclic
hexapeptides (cEL and 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 of 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¢¢. The crystal
structure of CD2 (Fig. 2B) indicated that the rather flat
b-sheet surface does not provide a complementary shape to
bind to CD58, and hence does not have well-defined

epitopes to 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 for binding to its receptor [38]. b-Turn peptides
based on CD4 have been shown to be effective in inhibiting
CD4 interactions [38]. Analysis of the crystal structure of
CD2 revealed that on either side of the binding surface of
CD2, there are b-turns which 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 complementarity in the center region of interaction (Fig. 2A). Most of
the interaction is via salt-bridges with charge neutralization
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-Thr88Asn89-Gly90, while those in human CD2 are at Thr38Ser39-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

Ó FEBS 2004

Design of peptides for T-cell adhesion inhibition (Eur. J. Biochem. 271) 2883

important in CD2–CD58 interaction [3]. Therefore, we
hypothesized that the stable b-turn conformation mimicking 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 peptide based on the b-turn region of rat CD2
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 structures of hCD2 from CD2–CD58 complex [30] and
the structure of rat CD2 [36] using DS 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. The b-turn regions of
hCD2 (Thr38-Ser39-Asp40-Lys41 and Asp87-Thr88Lys89-Gly90) overlap with b-turn regions of rat CD2
(Arg37-Gly38-Ser39-Thr40
and
Ser87-Thr88-Asn89Gly90). In CD2–CD58 complex, the residues in the b-turn
and flanking residues of this b-turn have been shown to be
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 the 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. Comparison of hCD2 and rat CD2 3D structures indicated that
Tyr86 and Glu36 side chains overlap in hCD2 and rat
CD2 and 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].
Pen is used in the position 1 because in previous work we
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 was not designed since addition 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 pharmacophores. 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 the CD2 protein. The

sequence was then reversed and the important amino
acid Tyr86 as well as Tyr81 was replaced by Ala to
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% inhibition 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 peptide. Thus,
cyclization stabilizes 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 enhancement in the antibody binding assay, whereas in celladhesion inhibition assay, the peptide inhibited cell-cell
adhesion. While all the three methods used to evaluate

biological activities of the peptides in this study were
expected to generate similar results, there were differences
due to the nature of different cell types used in the
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 erythrocytes. Furthermore, CD58 may have different epitopes to
bind to antibody in the antibody binding assay. Comparing the concentration of peptides used in inhibition studies
by all the three methods, the antibody binding inhibition
assay was the least sensitive and required higher concentrations 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

2884 L. Jining et al. (Eur. J. Biochem. 271)

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-turn region played a key role in the
interaction. Molecular modeling studies predicted that the
b-turn in the cyclic peptides closely mimics the conformational 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
top cavity of CD58 or near the CC¢ 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 study. However, docking studies were in agreement with antibody
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 binding

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 mechanism of CD2–CD58. Inhibition of cell adhesion involves
interrupting the interaction of key residues at the protein–
protein interface. If we consider hydrogen bonding interaction between the peptide and CD58, the cER peptide forms
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 involved 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 inhibitory 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 of cyclic hexapeptide cYT, although it contains an
important residue Tyr1, the peptide exhibits low inhibition
activity in E-rosetting and heterotypic adhesion assays. The
peptide cYT is expected to mimic the region with Tyr86 in
the protein. NMR and molecular modeling studies suggested 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 in 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 compounds for immunomodulation. Thus, the designed 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 academic research grant (R-148-000-026-112) from the National
University of Singapore, Singapore.

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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 temperature dependence of amide proton resonance data at
298K for peptides, cER, cVY, cEL.


2886 L. Jining et al. (Eur. J. Biochem. 271)

Appendix 2. The backbone dihedral angles (in deg.) at
residues Glu2-Arg3-Gly4-Ser5 and at residues Ser4-Thr5Asn6-Gly7 for conformations of peptide cVY.
Appendix 3. Peptides cEL and cYT: CD58 docking results
starting from the potential binding sites out of 100 runs.
Fig. S1. Amide region of 500 MHz ROESY spectra of cER
cyclic peptide in 90% H2O/10% D2O.
Fig. S2. Amide region of 500 MHz ROESY spectrum of
cVY cyclic peptide in 90% H2O/10% D2O.

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Fig. S3. Amide region of 500 MHz ROESY spectrum of
cEL cyclic peptide in 90% H2O/10% D2O.
Fig. S4. Amide region of 500 MHz NOESY spectrum of
cVT cyclic peptide in 90% H2O/10% D2O.
Fig. S5. Measurement of CD2 expression on the surface of
Jurkat cells by flow cytometry.
Fig. S6. Cell viabilities of peptide-treated Caco-2 and Jurkat
cells.