Synthetic 12-mer linear and cyclic peptides, and cyclic hexapeptides based on rat CD2 protein, were designed to modulate CD2–CD58 interaction.. Docking studies of CD2 peptides and CD58 p
Trang 1Design, 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 and2Department 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
thera-peutically 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
cyclic peptides exhibit b-turn structure in solution and clo-sely 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)
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
relat-ively 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
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 the treatment of autoimmune diseases
It has been shown that blockade of the 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 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.-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, 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 pep-tides (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
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, 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)
Trang 2inhibition assays In order to understand
structure–func-tion relastructure–func-tionship 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 using
auto-matic 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 k¼ 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
purchased from Biodesign International (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 MOLT-3 cells were maintained in suspension in RPMI1640 medium supplemented with 10% (w/v) heat-inactivated fetal bovine serum and 100 mgÆL)1of 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 mgÆL)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
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 FITC-CD2 monoclonal antibody (mAb) for 1 h at 37C After washing three times with 0.5% (w/v) BSA/10 mM Hepes/ NaCl/Pi, the cells were fixed using 1% (v/v) paraformalde-hyde/NaCl/Piand analyzed with a flow cytometer (FAC-Scan apparatus, Becton Dickinson) equipped with theCELL QUESTsoftware 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 37C 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) dimethylsulf-oxide Cell suspensions and peptide solutions (80, 200 and 500 lM) were aliquoted into a 48-well cell culture cluster and incubated at 37C 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) paraformal-dehyde/NaCl/Pi and analyzed with a flow cytometer (FACScan, Becton Dickinson) equipped withCELL QUEST
software Binding of FITC-anti-CD58, following incuba-tion 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 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 the second
position in the peptide to eleventh position Pen1 and Cys12 were
introduced for cyclization purpose.
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 91–80a
a Sequence from human CD2 The sequence was reversed, Tyr81
and Ty86 were replaced by Ala.
Trang 3Sheep 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/Pi and
incubated with four volumes of
2-aminoethylisothiouro-nium hydrobromide (AET) solution (Sigma) at 37C 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 37C 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, 4C) and then
incubated at 4C for 1 h The cell pellet was 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 the percentage of E-rosette cells The
inhibitory activity was calculated by the following Eqn (1):
inhibitionð%Þ
¼ ½ðnegative E- rosette %peptide negative E-rosette %blankÞ=
where, negative E-rosette %peptide¼ (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 · 104cellsÆwell)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 lMfluorescent dye bis-carboxyethyl-carboxyfluorescein (BCECF-AM) at 37C for 1 h Peptide dissolved in MEM-a was added at various concentrations to Caco-2 cell monolayers After incubation
at 37C for 30 min, the labeled Jurkat cells (1 · 106 cellsÆwell)1) were added onto the monolayers After incu-bation at 37C 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 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 structure; *, residues in the interface;
b-turn regions are in bold letters; designed 12
amino acid residue peptide sequences are
underlined.
Trang 4transferred 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 due to frank toxicity A final concentration
of 180 lMpeptide 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 37C
for 3 h The MTT-labeled cells were lysed by
dimethylsulf-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% 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
spec-trometers equipped with a 5-mm broad-band inverse probe,
at a proton frequency of 300.3414 MHz and 500.134 MHz,
respectively, usingXWINNMRversion 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 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]
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
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 Geometry of
an Aqueous Structure (MARDIGRAS) [27], which takes into account TOCSY contributions for the calculated intensities
in the ROESY spectrum.MARDIGRASruns 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 cross-peaks 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 kcalÆmol)1ÆA˚)1 The resulting structures were analyzed again byMARDIGRAS, and the final struc-tures 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
3JHNa[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
AUTODOCKprogram [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]
Trang 5Hydrogen atoms were added to the protein usingINSIGHTII
(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
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 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 concentra-tion-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) com-pared 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
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.
Trang 6measured 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
activity of 40% at 100 lMconcentration 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 inhib-itory 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 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 lM
concentration The cyclic peptide cVY showed 50% inhibition at 90 lMconcentration 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
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 was added to the
peptide-treated cells, followed by a further incubation Binding 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 104cells 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
tripli-cate 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.
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/P i 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.
Trang 7biological 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 dimensional1H 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/DT¼)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)1suggesting 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
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-Ser39-Thr40 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 Arg37-Gly38-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 Thr38-Ser39-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) the3JHNaof Thr5 and Asn6 were close to those expected for a type I b-turn (i.e.3JHNavalues 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 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 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
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.
Trang 8stable 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-Asn89-Gly90) In human CD2, the similar region
Asp87-Thr88-Lys89-Gly90 exhibits a type I 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 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
Haprotons 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
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, 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.ÆK)1which 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 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 Haenantiotopic 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, 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)1which 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 thatAUTODOCKcan 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 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.
Trang 9protein, 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-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 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
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
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 Cluster Rank
Lowest docked energy (kcalÆmole)1)
Number of conformations
in the cluster
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 kcalÆmol )1 ) CD58
Trang 10the 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
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 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 complement-arity 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-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
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