Crystal structures of HLA-A*0201 complexed with melan-A/MART-1 26(27L)-35 peptidomimetics reveal conformational heterogeneity and highlight degeneracy of T cell recognition

Article abstract of DOI:10.1021/jm100683p

There is growing interest in using tumor associated antigens presented by class I major histocompatibility complex (MHC-I) proteins as cancer vaccines. As native peptides are poorly stable in biological fluids, researchers have sought to engineer synthetic peptidomimetics with greater biostability. Here, we demonstrate that antigenic peptidomimetics of the Melan-A/MART-1 _26(27L)-35 melanoma antigen adopt strikingly different conformations when bound to MHC-I, highlighting the degeneracy of T cell recognition and revealing the challenges associated with mimicking native peptide conformation.

Full text of DOI:10.1021/jm100683p

J. Med. Chem. 2010, 53, 7061–7066 7061
DOI: 10.1021/jm100683p
Crystal Structures of HLA-A*0201 Complexed with Melan-A/MART-126(27L)-35 Peptidomimetics
Reveal Conformational Heterogeneity and Highlight Degeneracy of T Cell Recognition†
Celine Douat-Casassus,‡,^ Oleg Borbulevych, ,^ Marion Tarbe,‡ Nadine Gervois,§ Francine Jotereau,§
ꢀ
Brian M. Baker,* and Stephane Quideau*
,
,‡
ꢀ
‡Institut des Sciences Moleꢀculaires (UMR-CNRS 5255) and Institut Europeꢀen de Chimie et Biologie (IECB),
Universiteꢀ de Bordeaux, 2 Rue Robert Escarpit, 33607 Pessac, France, §INSERM, U601, Universiteꢀ de Nantes, 44322 Nantes, France, and
Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame Indiana 46556.
^ These authors have contributed equally to the work.
Received June 7, 2010
There is growing interest in using tumor associated antigens presented by class I major histocompat-
ibility complex (MHC-I) proteins as cancer vaccines. As native peptides are poorly stable in biological
fluids, researchers have sought to engineer synthetic peptidomimetics with greater biostability. Here, we
demonstrate that antigenic peptidomimetics of the Melan-A/MART-126(27L)-35 melanoma antigen
adopt strikingly different conformations when bound to MHC-I, highlighting the degeneracy of T cell
recognition and revealing the challenges associated with mimicking native peptide conformation.
Introduction
Since their first identification in the early 1990s, tumor-
associated antigenic (TAA) peptides have been considered as
promising candidates for the development of cancer vaccines.
However, their weak immunogenicity and their high sensitiv-
ity to proteases have limited the success of synthetic TAA
peptides as immunotherapeutic agents. Effort has therefore
been devoted to modifying naturally occurring TAA peptides
to design more biologically stable analogues for immunother-
apeutic applications.
The Melan-A/MART-1 protein antigen (hereafter referred
to as Melan-A) is up-regulated in about 89% melanoma cell
lines4 and elicits natural CD8þ T cell responses.5 Screening of
peptides derived from Melan-A led to the identification of the
immunodominant peptide epitope comprising residues 26-35
(EAAGIGILTV), for which T cell recognition is restricted by
the human leukocyte antigen (HLA)-A*0201 (HLA-A2). A
drawback of this epitope is its moderate HLA-A2 binding
affinity due to the absence of an optimal residue at the second
anchor residue (P2). Replacement of the P2 alanine by leucine
to yield the altered Melan-A26(27L)-35 peptide (ELAGIGILTV,
referred to as ELA-0)6 resulted in enhanced HLA-A2-binding
affinity and improved immunogenicity with T cells specific for
the native EAA decamer. Consequently, the ELA-0 peptide
has been and continues to be used in numerous clinical trials
for the immunological treatment of melanoma.7 Unfortu-
nately, as with most peptides used in immunological treat-
ments, the ELA-0 peptide exhibits poor stability in biological
fluids.8 In this context, the design of new synthetic, bioresistant
ELA-0 analogues presents an opportunity for the development
of more effective immunologically based therapies targeting
melanoma.
Class I major histocompatibility complex (MHC-I) pro-
teins play a central role in immune surveillance by selectively
binding antigenic peptides derived from intracellular ex-
pressed proteins.1 After transport of the peptide-MHC-I
complex (peptide-MHC-I) to the cell membrane, the cell
surface is surveyed by circulating CD8þ cytotoxic T lympho-
cytes (CTLsa) through their T cell receptors (TCRs). When a
T cell specifically recognizes peptide-MHC-I together with
costimulatory signals, a T-cell-mediated immune response is
initiated, resulting in lysis of the antigen-presenting cell and
initiation and propagation of an immune response. MHC-I
molecules are heterodimers and comprise a membrane-linked
heavy chain, a soluble light chain ( β2-microglobulin), and a
short peptide, typically 8-10 amino acids in length. The
peptide binding site is constructed as a groove formed by
the R1 and R2 helices supported by a β sheet of the heavy
chain. Peptide specificity for MHC-I is conferred by two
dominant anchor residues (P2 and PΩ for the C-terminal
position),2 while TCR antigen specificity is largely determined
by the solvent-exposed side chains of the peptide central core.3
Generally, antigenic peptides bind to MHC-I proteins in an
extended conformation with the peptide central core bulging
out or even zigzagging within the binding groove.
†The crystal structures of HLA-A*0201 complexed with Melan-A/
MART-126(27L)-35 peptidomimetics have been deposited (PDB codes
3O3A, 3O3B, 3O3D, and 3O3E, respectively).
*To whom correspondence should be addressed. For B.M.B.: phone,
(574) 631 9810; fax, (574) 631 6652; e-mail, brian-baker@nd.edu. For
S.Q.: phone, þ33 (0) 540 00 30 10; fax, þ33 (0) 540 00 22 15; e-mail,
s.quideau@iecb.u-bordeaux.fr.
a Abbreviations: AMBA, 3-aminomethylbenzoic acid; CTL, cyto-
toxic T lymphocyte; DMSO, dimethyl sulfoxide; HLA, human leuko-
cyte antigen; MES, 2-(N-morpholino)ethanesulfonic acid; MHC, major
histocompatibility complex; PEG, polyethylene glycol; rmsd, root-
mean-square deviation; RP-HPLC, reverse phase high performance
liquid chromatography; TAA, tumor associated antigen; TLS, transla-
tion, libration, and screw; TCR, T cell receptor.
Over the past few years, attempts have been made to replace
protease-susceptible peptide bonds in the ELA-0 peptide with
their corresponding isosteres or non-natural amino acids.8,9
We recently investigated a strategy involving substitution
of the TCR-contacting central amino acids with protease
r
2010 American Chemical Society
Published on Web 08/31/2010
pubs.acs.org/jmc

7062 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Douat-Casassus et al.
Figure 1. Chemical structures of the four ELA analogues showing the chemical differences in the central TCR-contacting and
N-terminal parts.
resistant, nonpeptidic motifs potentially capable of making
improved interactions with the TCR.10 Through an in silico
design approach, the central GIGI tetrapeptide of ELA-0 was
replaced with pseudopeptidic units equipped with different
organic haptens. To further enhance biostability, some of our
analogues also had a β-alanine at P1 in place of the native
glutamate (Figure 1).8
Notably, despite the drastic modifications made to the
ELA-0 central core, the majority of our synthetic analogues
had similar or even improved binding affinity with the HLA-
A2 molecule. Moreover, some of our peptidomimetics re-
tained reactivity with Melan-A-specific T cell clones.10
Here, we sought to identify how chemical modifications of
ELA-0 alter its presentation by the HLA-A2 molecule with
the aim of gaining further insight into the potential use of
peptidomimetics as immunotherapeutic agents. Toward this
end, we determined the crystallographic structures of our two
most successful ELA-0 based peptidomimetics bound to
HLA-A2 (ELA-1 and ELA-2 in Figure 1). We also explored
the structural consequences of the modifications made
to the N-terminal part as a means to enhance exoprotease
resistance.8
The structures revealed (1) variations in the presentation of
the peptidomimetics, both with respect to each other and with
respect to the native ELA-0 peptide, highlighting the chal-
lenges associated with mimicking the conformations of native
peptides in MHC-I binding grooves, (2) the capacity for
antigen-specific T cell receptors to cross-react with structu-
rally diverse ligands. The structures also revealed that the
modification of peptide N-termini made to enhance protease
resistance can have unintended structural consequences. Be-
yond unveiling the complexity of these structural modula-
tions, our results provide a concrete starting point for
structure-based enhancement of synthetic mimics of natural
antigenic peptides.
Results
The four ELA-0-based peptidomimetics examined are
shown in Figure 1 and termed ELA-1, ELA-1.1, ELA-2,
and ELA-2.1. ELA-1 was referred to as compound 21 in
our previous work, whereas ELA-2 was referred to as com-
pound 6.10 Both ELA-1 and ELA-2 strongly activated two
ELA-0 specific T cell clones. ELA-1.1 and ELA-2.1 are
derivatives of ELA-1 and ELA-2 that either retain the native
glutamate at the N-terminus (ELA-1.1) or include a β-alanine
(ELA-2.1) at this position.
All four peptidomimetic/HLA-A2 complexes crystallized
in the same P21 space group with two molecules per asym-
metric unit, as seen with other HLA-A2 complexes, including
the complex with the native ELA-0 peptide.11,12 The four new
complexes maintained the typical peptide/HLA-A2 architec-
ture, with no perturbation to the secondary structure that
forms the HLA-A2 peptide binding groove. However, im-
portant structural differences were seen in the various struc-
tures as described below. Diffraction data and refinement
statistics for all four structures are shown in Table 1.
Structures of ELA-1 and ELA-1.1 Bound to HLA-A2. In
ELA-1, the central GIGI tetrapeptide is replaced with a
3-aminomethylbenzoic acid (AMBA) spacer combined with
N-(2-aminoethyl)glycine unit bearing a tryptophan-like side
chain that extends away from the backbone by an additional
carbonyl unit. The N-terminal glutamate is also replaced
with a protease-resistant β-alanine. The structure of ELA-1
˚
bound to HLA-A2 was solved to 1.8 A (Table 1). The two
molecules in the unit cell were essentially identical, with all
atoms of the two copies of the constructs superimposing with
˚
an rmsd of 0.3 A.
In the initial in silico design of ELA-1, the AMBA spacer
was predicted to be buried in the base of the peptide binding
groove and the indole acetyl side chain was predicted to point
away from the groove, facilitating interactions with incoming

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7063
Table 1. X-ray Data and Refinement Statistics
protein
ELA-1/HLA-A2
ELA-1.1/HLA-A2
ELA-2/HLA-A2
ELA-2.1/HLA-A2
PDB entry
radiation source (beamline)
space group
3O3A
3O3B
3O3D
3O3E
APS (19BM)
P21
58.2
APS (14BM)
P21
58.1
APS (19BM)
P21
58.2
APS (14BM)
P21
58.2
˚
a (A)
˚
b (A)
˚
c (A)
84.2
84.0
84.3
84.0
84.2
84.1
84.4
84.0
β (deg)
molecules/asymmetric unit
90.0
2
90.0
2
90.1
2
90.1
2
˚
resolution (A)
20-1.8
74605
0.28
20-1.9
61720
0.38
20-1.7
88669
0.30
20-1.9
66944
0.23
total no. of reflections
mosaicity (deg)
completenessa (%)
I/σ
Rmerge (%)
average redundancy
99.5 (96.2)
13.4 (1.7)
9.2 (51.1)
3.6 (3.3)
17.1 (71352)
21.2 (3786)
21.1
96.9 (81.6)
10.9 (2.1)
14.1 (49.7)
3.6 (3.1)
18.6 (58568)
23.8 (3116)
11.6
99.4 (98.3)
17.7 (2.0)
9.1 (49.6)
3.6 (3.2)
18.0 (84177)
21.8 (4435)
16.9
96.5 (72.9)
14.5 (1.7)
10.8 (63.4)
3.6 (2.7)
18.2 (63539)
22.9 (3377)
17.4
Rwork (%) (no. reflections)
Rfree (%) (no. reflections)
2
˚
ÆBæ (all atoms) (A )
Ramachandran plot
most favored (%)
allowed (%)
92.2
7.5
91.6
8.1
93.1
6.6
91.8
7.9
generously allowed (%)
RMS deviations from ideality
0.3
0.3
0.3
0.3
˚
bond (A)
0.016
1.762
0.09
0.018
1.843
0.12
0.016
1.704
0.08
0.017
1.719
0.11
angle (deg)
coordinate error (A)
b
˚
a Numbers in parentheses refer to the highest resolution shell. b Mean estimate based on maximum likelihood methods.
T cell receptors.10 However, the ELA-1/HLA-A2 structure
revealed almost the opposite: the AMBA spacer instead
bulges up and out of the groove, and the indole unit, rather
than pointing away from the peptide binding groove, points
toward the HLA-A2 R1 helix and lies partially underneath
the AMBA spacer (Figure 2a).
conserved hydrogen bonds and salt bridges around the
peptidomimetic N-terminus could significantly impact pep-
tide and TCR binding for other systems.13,15 We thus asked
whether the alterations could be reversed by reverting to a
native glutamate at the first position. The resulting ELA-1.1
peptide is identical to ELA-1 except that the β-alanine at P1
is replaced with glutamate (Figure 1).
As a decameric peptide, the center of the ELA-0 peptide
zigzags throughout the HLA-A2 peptide binding groove. The
center of ELA-1 also zigzags but does so in the opposite
direction, bending toward and above the R1 helix at the AMBA
spacer (Figure 2a). Overall, the path adopted by ELA-1
through the HLA-A2 peptide binding groove diverges sub-
stantially from that of ELA-0, especially in the region replaced
by the synthetic spacer. For example, the distance between the
nitrogen of the AMBA spacer of ELA-1 and that of Gly6 of
˚
The structure of ELA-1.1, determined at 1.9 A (Table 1),
was identical to that of ELA-1 except for the region around
the N-terminus, which indeed showed a return to a “normal”
N-terminal environment, with the hydrogen bond between
the N-terminus and Tyr171, the hydrogen bond between the
Leu2 oxygen and Lys66, and the salt bridge between Lys66
and Glu63 all formed (Figure 2c). Thus, the N-terminal
structural alterations in the ELA-1 structure are all attribu-
table to the presence of the β-alanine.
˚
ELA-0, intended to occupy similar positions, is 6.7 A.
The replacement of the native glutamate with a β-alanine
at the N-terminus had significant structural consequences.
Compared to a standard amino acid, β-alanine introduces an
additional carbon-carbon bond between the first carbonyl
oxygen and the peptidomimetic N-terminus (Figure 1). As
shown in Figure 2b, in the ELA-1/HLA-A2 structure, this
additional bond forces the N-terminus out of the HLA-A2
P1 pocket, resulting in the loss of a hydrogen bond to Tyr171
of the HLA-A2 heavy chain that is conserved in all peptide/
HLA-A2 structures in which the P1 pocket is occupied. The
N-terminus instead points up out of the pocket, displacing
Structures of ELA-2 and ELA-2.2 Bound to HLA-A2. The
ELA-2 peptidomimetic also included the AMBA spacer but
combined it with a reduced peptide bond introduced between
the glycine at P4 and a standard tryptophan at P5 (Figure 1).
Unlike ELA-1, ELA-2 retained the native glutamate at the
N-terminus as it was designed earlier.10 As with ELA-1,
ELA-2 adopted a different path through the HLA-A2 pep-
tide binding groove compared to the ELA-0 peptide. Rather
than zigzagging, the center of ELA-2 simply bulged out such
that the conformation of the ELA-2 central part was some-
what between that of ELA-0 and ELA-1 (Figure 3a). The two
molecules in the ELA-2 asymmetric unit positioned the
AMBA spacer and the preceding peptide bond unit in
different conformations, and in one of the molecules in the
asymmetric unit, the side chains and backbones of Leu8 and
Thr9 could be refined in two conformations (Figure 3b).
Together, this represents a degree of conformational hetero-
geneity present with ELA-2 but not with ELA-1. One of the
˚
the side chain of Lys66 by 3.2 A because of charge and steric
repulsions. This remarkable movement of Lys66 results in
the loss of another conserved peptide-HLA-A2 hydrogen
bond, from Lys66 to the carbonyl oxygen of the leucine at
peptide position 2. The shift in Lys66 also removes a salt
bridge made with Glu63 that is conserved in the majority of
peptide/HLA-A2 complexes.13,14
˚
Although ELA-1 bound well to HLA-A2 and was recog-
nized by Melan-A specific T cell clones, the alterations in
AMBA positions in ELA-2 closely (within 2 A) mimicked the
position of the AMBA spacer in ELA-1 (shown in Figure 3a).

7064 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Douat-Casassus et al.
Figure 2. Structural comparison of the ELA-1 and ELA-1.1 mimetics with the native ELA-0 peptide. (a) Cross-eyed stereo comparison of
ELA-1 with ELA-0 in the HLA-A2 peptide binding groove. ELA-1 diverges substantially from ELA-0, particularly in the central portion of the
molecule. ELA-0 is purple and ELA-1 is green. (b) The nitrogen of the β-alanine in ELA-1 points out of the P1 pocket, forcing a repositioning of
Lys66 and disrupting the traditional N-terminal hydrogen bonding arrangement. Hydrogen bonds are purple, and the clash between β-alanine
and Lys66 is illustrated in red. A hydrogen bond between Lys66 and Glu1 in the ELA-1.1 structure is not shown for clarity. (c) The ELA-1.1
compound, which replaces the β-alanine of ELA-1 with glutamate, restores the normal position of Lys66 and the traditional hydrogen bonding
arrangement. Superimpositions for all panels are through the backbones of the peptide binding domains.
Interestingly, the side chain of Trp5 angled into the same
position as the indole ring in ELA-1, reflecting a preference
for an aromatic side chain at the position 5 pocket in HLA-
A2.16
Surprisingly, the structures of our two most promising
peptidomimetics (ELA-1 and ELA-2) bound to HLA-A2
determined here revealed crucial differences in the central
AMBA-substituted portion of the peptide when compared to
the structure with the ELA-0 peptide, after which they were
modeled. These differences highlight the capacity of our
AMBA-based mimetics to alter their conformation to cor-
rectly position their primary anchors to achieve tight binding
to HLA-A2.
Unlike ELA-1, ELA-2 retained the native glutamate at the
N-terminus. As anticipated from the results with ELA-1 and
ELA-1.1, the normal position of Lys66 and the hydrogen
bonding/salt-bridge network around the N-terminus were
retained in the ELA-2 structure. To verify conclusively that
the rearrangements seen in ELA-1 were due to the use of
β-alanine and not influenced by other modifications, we
examined a version of ELA-2 that replaced the native
glutamate with a β-alanine (ELA-2.1 in Figure 1). The
structure of ELA-2.1 bound to HLA-A2 revealed structural
consequences identical to those seen with ELA-1: loss of the
N-terminal hydrogen bond to Tyr171 and a rearrangement
of Lys66, resulting in the loss of the hydrogen bond to
position 2 and the salt bridge with Glu63 (Figure 3c).
But given the different structures for ELA-1 and ELA-2,
how is it that they are able to stimulate ELA-0-specific T cells?
One important clue may lie in the conformational diversity
observed in the ELA-2/HLA-A2 crystal structure. Several
studies have shown that the adaptability of antigenic peptides
can lead to the formation of stable TCR-pMHC interfaces,
most recently with ligands with little structural homology in
the TCR-free state.18 Alternatively, the TCRs responsible for
cross-recognition between ELA-0 and ELA-1/ELA-2 may
possess flexible CDR loops that permit recognition of dis-
parate structures.19 Indeed, cross-reactivity with structurally
disparate ligands is a hallmark of Melan-A26-35-specific
TCRs, as they readily cross-react with the 27-35 epitope,
which as a nonamer adopts a different path through the HLA-
A2 peptide binding groove as does the 26-35 decamer.20 It is
possible that cross-reactivity between ELA-0 and ELA-1/
ELA-2 proceeds via a combination of peptide and TCR
conformational adaptability, as recently demonstrated with
Discussion
With the growing use of tumor associated antigens as the
basis for the development of vaccines for cancer andinfectious
diseases,17 there has been a concomitant interest in the deve-
lopment of synthetic analogues or peptidomimetics with
greater biological stability or immunological potency. Our
mimetics of the Melan-A26(27L)-35 tumor antigen (ELA-0)
bound well to HLA-A2 and activated Melan-A-specific T
cells,10 demonstrating an important proof of principle and
providing a platform on which further development can be
based.
variants and mimics of the HTLV-1 Tax11-19 peptide.21,22
A
more complete understanding of the mechanism by which
ELA-1 and ELA-2 are recognized will require solution

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7065
Figure 3. Structural comparison of the ELA-1 and ELA-2 peptidomimetics. (a) Cross-eyed stereocomparison of ELA-1 and ELA-2 in the
HLA-A2 peptide binding groove. Unlike ELA-1, ELA-2 does not zigzag but instead bulges straight from the center of the groove. ELA-2 is
yellow and ELA-1 is green. (b) ELA-2 shows conformational heterogeneity in the HLA-A2 binding groove. The AMBA spacer occupies two
different conformations in the two molecules in the asymmetric unit (yellow and cyan). The first molecule in the asymmetric unit (cyan)
displayed alternative conformations for the backbones and side chains of Leu8 and Thr9. (c) The structure with the ELA-2.1 mimetic, which
replaced the N-terminal glutamate of ELA-2 with β-alanine, revealed an altered position for Lys66 and disrupted N-terminal hydrogen
bonding arrangement, similar to that seen in the structure with ELA-1. ELA-2 is yellow, and ELA-2.1 is pink. Hydrogen bonds are purple, and
the clash between β-alanine and Lys66 is illustrated in red. A hydrogen bond between Lys66 and Glu1 in the ELA-2 structure is not shown for
clarity. Superimpositions for all panels are through the backbones of the peptide binding domains.
of crystal structures of the TCR-peptide-MHC ternary
complexes.
structural mimics of antigenic peptides. Nonetheless, our
results provide a concrete starting point for the structure-
based enhancement of synthetic mimics of Melan-A/MART-1
epitopes capable of being recognized by human TCRs in a
HLA-A2-restricted context.23
A second key result revealed by the crystallographic struc-
tures is the structural consequences of using a β-alanine at the
N-terminus. β-Alanine was used to enhance resistance to
exoproteases. However, the structures showed unambigu-
ously that β-alanine disrupted the conformation at the
N-terminus, resulting in the loss of hydrogen bonds and
altering the position of Lys66. Although this did not appear
to negatively affect the binding of our compounds to HLA-A2,
nor did it disrupt recognition by two different T cell clones, this
should not be expected in all cases. Lys66 is a key amino acid in
several interfaces that TCRs form with HLA-A2, and altera-
tions to Lys66 have been linked to a loss in recognition in other
cases.14 Thus, caution should be used when considering
β-alanine or similar non-native amino acids at the N-terminus
of peptidomimetics.
Experimental Methods
Synthesis. We have reported the synthesis of ELA-1 and
ELA-2 in a previous publication.10 The compounds were de-
termined to be >95% pure by RP-HPLC. Detailed procedure
for the solid phase synthesis of their derivatives ELA-1.1 and
ELA-2.2 is described in the Supporting Information. Both
peptidomimetics were obtained with purities of >95%.
Protein Expression, Purification, and Crystallization. The
extracellular domains of HLA-A2 and β2-microglobulin were
expressed in Escherichia coli as inclusion bodies, refolded with
excess peptidomimetic dissolved in DMSO, and chromatogra-
phically purified as described previously.24 The HLA-A2 com-
plexes were crystallized at 4 ꢀC from a precipitant solution of
24% PEG3350, 0.1 M KCl or NaF, buffered with 0.025 M MES
at pH 6.5 using sitting drop/vapor diffusion. Seeding was used to
obtain higher quality single crystals.
Data Collection, Structure Solution, and Refinement. For data
collection, crystals were transferred to mother liquor supple-
mented with 20% of glycerol and flash-frozen in liquid nitrogen.
Diffraction data were collected at the indicated beamlines
(Table 1). Data collection statistics are given in Table 1. Oscilla-
tion frames were integrated and scaled with HKL2000.25 Struc-
tures were solved by molecular replacement with MOLREP26
using PDB code 2GT919 as a search model with the coordinates
for the peptide excluded. Rigid body refinement, TLS refinement,
Conclusions
In summary, we have determined the crystallographic
structures of two promising peptidomimetics of the Melan-
A26-35 tumor antigen to HLA-A2. Although the conforma-
tions in the peptide binding groove diverged from that of the
native peptide, HLA-A2 binding and, for at least two T cell
clones, TCR recognition were maintained.10 This structural
analysis underscores the degeneracy present in TCR recogni-
tion of ligand. Furthermore, such degeneracy beingdifficultto
predict, this work highlights the difficulties in designing close

7066 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Douat-Casassus et al.
and restrained refinement were performed with Refmac527 as
incorporated in CCP4-6.0.2. The TLS groups for refinement were
chosen as previously described.21 Once refined, TLS parameters
were included in all subsequent refinement steps. Anisotropic and
bulk solvent corrections were performed throughout refinement.
Water was added using ARP/wARP.28 Graphical evaluation of
the model and fitting to maps were performed using Coot29 and
XtalView.30 The quality of the structures during refinement was
monitored with the Coot validation engine and Procheck.31
Structure factors and coordinates have been submitted to the
Protein Data Bank; PDB codes are listed in Table 1.
rational design of antigenic peptide mimetics that activate tumor-
specific T-cells. J. Med. Chem. 2007, 50, 1598–1609.
(11) Sliz, P.; Michielin, O.; Cerottini, J. C.; Luescher, I.; Romero, P.;
Karplus, M.; Wiley, D. C. Structures of two closely related but
antigenically distinct HLA-A2/melanocyte-melanoma tumor-
antigen peptide complexes. J. Immunol. 2001, 167, 3276–3284.
(12) Borbulevych, O. Y.; Insaidoo, F. K.; Baxter, T. K.; Powell, D. J.,
Jr.; Johnson, L. A.; Restifo, N. P.; Baker, B. M. Structures of
MART-126/27-35 peptide/HLA-A2 complexes reveal a remarkable
disconnect between antigen structural homology and T cell recog-
nition. J. Mol. Biol. 2007, 372, 1123–1136.
(13) Baxter, T. K.; Gagnon, S. J.; Davis-Harrison, R. L.; Beck, J. C.;
Binz, A. K.; Turner, R. V.; Biddison, W. E.; Baker, B. M. Strategic
mutations in the class I major histocompatibility complex HLA-A2
independently affect both peptide binding and T cell receptor
recognition. J. Biol. Chem. 2004, 279, 29175–29184 and references
cited therein.
(14) Gagnon, S. J.; Borbulevych, O. Y.; Davis-Harrison, R. L.; Baxter,
T. K.; Clemens, J. R.; Armstrong, K. M.; Turner, R. V.; Damirjian,
M.; Biddison, W. E.; Baker, B. M. Unrevealing a hotspot for TCR
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Acknowledgment. The authors thank the Servier Labora-
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tories and the Societe Franc-aise de Chimie Therapeutique for
financial support and M.T.’s research assistantship. O.Y.B.
was supported by the Walther Cancer Foundation. This work
was supported by Grant GM067079 from NIGMS, NIH (U.S.)
and Grant RSG-05-202-01-GMC from the American Cancer
Society. Results shown in this report are derived from work
performed at Argonne National Laboratory, Structural Biology
Center at the Advanced Photon Source. Argonne is operated by
UChicago Argonne, LLC, for the U.S. Department of Energy,
Office of Biological and Environmental Research under Contract
DE-AC02-06CH11357. Use of the BioCARS Sector 14 was
supported by the NCRR, NIH, U.S., under Grant RR007707.
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to rethink the use of peptides in vaccine design. Nat. Drug Dis-
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Supporting Information Available: Detailed procedure of the
solid phase synthesis of the four peptidomimetics and X-ray
electron density images. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Jr.; Johnson, L. A.; Restifo, N. P.; Baker, B. M. Structures of
MART-1(26/27-35) peptide/HLA-A2 complexes reveal a remark-
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