![Molecular surfaces and contacts of pVIPR in the p4 and p6 conformations. (A and B) Molecular surfaces show the central part of the B*2705 peptide binding groove in gray and the pVIPR peptide in the p4 and p6 conformations (color coded as in Fig. 1 [A and B]). The binding groove has been rendered semi-transparent, allowing also the inspection of buried side chains exhibiting conformational differences. In A, the view is TCR-like, straight onto the peptide, whereas in B (rotated by 90 about a horizontal axis), the view is through the 2 helix. The center section of the peptide shows clear shape differences between the p4 and p6 conformations. (C) pVIPR hydrogen bonding in p4 and p6, color coded as in Fig 1 (A and B). Only side chains with different binding modes (residues p3-p7) are shown. The binding groove's secondary structure is represented as gray spirals ( helices) and arrows ( strands) together with selected interacting residues (carbon atoms, gray; oxygens, red; and nitrogens, blue). Hydrogen bonds are depicted as black broken lines, the pArg5Asp116 bidentate salt bridge is depicted as green dotted lines, and water molecules are depicted as dark blue spheres. (D) Electrostatic surfaces of both pVIPR conformations. Red indicates negative, blue indicates positive surface charge, and gray areas are uncharged. The view is looking straight onto the binding groove as in A. The border of the peptides is highlighted in white.](https://www.researchgate.net/profile/Rosa-Sorrentino-2/publication/8909686/figure/fig4/AS:667705287929857@1536204754139/Molecular-surfaces-and-contacts-of-pVIPR-in-the-p4-and-p6-conformations-A-and-B_Q320.jpg)
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The Journal of Experimental Medicine
J. Exp. Med.
The Rockefeller University Press • 0022-1007/2004/01/271/11 $8.00
Volume 199, Number 2, January 19, 2004 271–281
http://www.jem.org/cgi/doi/10.1084/jem.20031690
271
Dual, HLA-B27 Subtype-dependent Conformation
of a Self-peptide
Martin Hülsmeyer,
1
Maria Teresa Fiorillo,
2
Francesca Bettosini,
2
Rosa Sorrentino,
2
Wolfram Saenger,
1
Andreas Ziegler,
3
and Barbara Uchanska-Ziegler
3
1
Institut für Kristallographie, Freie Universität Berlin,14195 Berlin, Germany
2
Dipartimento di Biologia Cellulare e dello Sviluppo, Università ‘La Sapienza,’ 00185 Roma, Italy
3
Institut für Immungenetik, Charité-Universitätsmedizin Berlin, Humboldt-Universität zu Berlin,
14050 Berlin, Germany
Abstract
The products of the human leukocyte antigen subtypes
HLA-B
*
2705
and
HLA-B
*
2709
differ
only in residue 116 (Asp vs. His) within the peptide binding groove but are differentially associated
with the autoimmune disease ankylosing spondylitis (AS);
HLA-B
*
2705
occurs in AS-patients,
whereas
HLA-B
*
2709
does not. The subtypes also generate differential T cell repertoires as
exemplified by distinct T cell responses against the self-peptide pVIPR (RRKWRRWHL).
The crystal structures described here show that pVIPR binds in an unprecedented dual confor-
mation only to HLA-B
*
2705 molecules. In one binding mode, peptide pArg5 forms a salt
bridge to Asp116, connected with drastically different interactions between peptide and heavy
chain, contrasting with the second, conventional conformation, which is exclusively found in
the case of B
*
2709. These subtype-dependent differences in pVIPR binding link the emer-
gence of dissimilar T cell repertoires in individuals with
HLA-B
*
2705
or
HLA-B
*
2709
to the
buried Asp116/His116 polymorphism and provide novel insights into peptide presentation by
major histocompatibility antigens.
Key words: X-ray structure • major histocompatibility antigen • peptide binding modes •
ankylosing spondylitis • residue 116
Introduction
MHC class I antigens are cell surface glycoproteins consisting
of a polymorphic transmembrane heavy chain (HC) nonco-
valently bound to
2
-microglobulin (
2
m; reference 1). The
HC forms a groove with six pockets (A–F) for binding of
proteolytic fragments derived from self- and nonself-proteins
(2, 3). These peptides are presented to TCRs located on
CTLs and initiate a cellular immune response leading to the
destruction of the antigen-presenting cell if the peptide is
derived from a nonself-protein. Several MHC class I alleles
are associated with autoimmune diseases, and allele-specific
binding of foreign or self-peptides by MHC class I molecules
may be crucial to disease pathogenesis (4–8).
The association of the HLA class I allele
HLA-B27
with
ankylosing spondylitis (AS) and related spondyloarthropathies
is among the strongest observed for any
HLA
gene (9, 10).
Population analyses have shown that most of the common
HLA-B27
subtypes, including the frequent
B
*
2705
allele,
are disease associated, whereas two subtypes,
B
*
2706
and
B
*
2709
, are not (11). The B
*
2705 protein differs in only a
single HC amino acid (Asp116) from B
*
2709 (His116)
(12). This residue is located at the floor of the F pocket,
which accommodates the COOH-terminal side chain of
the bound peptide, and both subtypes can be distinguished
by the peptides presented in vivo (13, 14). Despite some
differences, in particular with regard to the COOH-terminal
peptide residue, the majority of the peptides constituting
the B
*
2705 repertoire were found to overlap with those
from B
*
2709 and vice versa (14).
A self-peptide (pVIPR, previously termed VIP1R
400–408
,
RRKWRRWHL) with potential arthritogenic properties
(15) is derived from vasoactive intestinal peptide type 1
receptor and shows high sequence homology to a peptide
Address correspondence to Wolfram Saenger, Institut für Kristallographie,
Freie Universität Berlin, 14195 Berlin, Germany. Phone: 49-30-8385-
3412; Fax: 49-30-8385-6702; email: saenger@chemie.fu-berlin.de; or to
Andreas Ziegler, Institut für Immungenetik, Charité, Universitätsmedizin
Berlin, Humboldt-Universität zu Berlin, 14050 Berlin, Germany. Phone: 49-
30-4505-53501; Fax: 49-30-4505-53953; email: andreas.ziegler@charite.de
Abbreviations used in this paper:
AS, ankylosing spondylitis;
2
m,
2
-micro-
globulin; HC, heavy chain; rms, root mean square.
Dual Self-peptide Conformation in HLA-B27 Antigens
272
from the latent membrane protein 2 (pLMP2, residues 236–
244, RRRWRRLTV) of EBV (16). pVIPR can be pre-
sented by both subtypes, but the resulting T cell repertoires
are distinct;
B
*
2709
individuals very rarely possess pVIPR-
specific T cells, whereas such T cells are more abundant in
peripheral blood of healthy
B
*
2705
persons and are very
frequently present in patients suffering from AS (15). This
indicates efficient thymic elimination of T cells specific
for HLA-B27:pVIPR complexes in
B
*
2709
- but not in
B
*
2705
-positive individuals. Although these findings do not
prove a causative role of pLMP2 and pVIPR in AS, they
open the possibility to correlate structural properties of these
subtypes with the retention (in
B
*
2705
) or elimination (in
B
*
2709
) of pVIPR-specific T cells.
The two crystal structures described here show that
B
*
2705 accommodates pVIPR in two different conforma-
tions, of which one is identical to that seen when complexed
with B
*
2709, whereas the other diverges substantially.
Combined with functional studies in which pVIPR-specific
CTLs were used, these findings suggest a molecular expla-
nation for inappropriate T cell selection by individuals with
a disease-associated
HLA-B27
subtype.
Materials and Methods
HLA-B27–positive Donors.
Six patients with AS and one
healthy donor were enrolled for this paper.
HLA-B27
typing was
performed using the
HLA-B27
high resolution kit (Dynal). Five
patients and the healthy donor were
HLA-B
*
2705
positive, and
one patient (LV) was
HLA-B
*
2702
positive. Lymphoblastoid cell
lines from these individuals were generated by in vitro transfor-
mation of B cells using the standard type 1 EBV isolate B95.8 (17).
Generation of pVIPR-specific Cytotoxic T Lymphocyte Lines.
PBMCs were isolated on a gradient of lymphoprep and depleted
of the CD4
fraction by Dynabeads M-540 CD4 (Dynal). Cells
were incubated at 2
10
4
cells/well in 96-well flat-bottom mi-
croplates, stimulated at an 0.5:1 stimulator/responder ratio with
autologous EBV-transformed B cells prepulsed overnight with
8.5
M pVIPR, and
irradiated (200 Gy). They were grown in
RPMI 1640 medium containing 10% heat-inactivated pooled
human serum, 2 mM
l
-glutamine, 10 U/ml penicillin, and 100
g/ml of streptomycin and in the presence of 20–100 U/ml of
human rIL-2 (Boehringer). 10 d later, cytotoxic T lymphocyte
lines were restimulated as aforementioned. After 1 wk, they were
tested for pVIPR specificity in a standard
51
Cr-release assay using
as targets T2 cells transfected with either B
*
2705 cDNA (T2-
B
*
2705) or B
*
2709 cDNA (T2-B
*
2709), pulsed with 70
M of
the peptide, or used untreated.
Expression, Purification, and Crystallization of the HLA-B27:
pVIPR Complexes.
HLA-B27:pVIPR protein complexes were
produced as described previously (18, 19). The pVIPR peptide
(RRKWRRWHL) was obtained by standard solid phase synthe-
sis and purified by HPLC (Alta Bioscience). The purified protein
complexes were used for crystallization at concentrations of 20
mg/ml (B
*
2705:pVIPR) and 16 mg/ml (B
*
2709: pVIPR), re-
spectively, in TBS buffer (10 mM Tris HCl, pH 7.5, and 150
mM NaCl).
Using hanging drop vapor diffusion, crystals suitable for X-ray
diffraction experiments were obtained from a PEG 8000 pH
screen. As observed earlier for similar HLA-B27 complexes,
streak seeding was indispensible to produce large single crystals.
For B
*
2705:pVIPR, drops made from 1
l of precipitant solution
(0.1 M Tris HCl, pH 8.0, and 16% PEG 8000) and 1
l of pro-
tein solution produced prismatic crystals with a maximum size of
400
250
250
m. Using glycerol as cryoprotectant, the best
dataset in terms of resolution (1.47 Å) was obtained at the BL2
beamline at BESSY-II, Berlin, Germany. In the case of B
*
2709:
pVIPR, drops made of 1
l of precipitant solution (0.1 M Tris
HCl, pH 8.5, and 19% PEG 8000) and 1
l of protein solution
yielded platelike crystals with a maximum size of 300
200
100
m. Cryo-cooled crystals of this complex (glycerol as cryo-
protectant) allowed collection of a 2.2-Å resolution dataset at the
X13 beamline at Deutsches Elektronen Synchrotron. Both
datasets were collected at 100 K and processed with the HKL
package (see Table I; reference 20).
Structure Determination.
The structure of HLA-B
*
2709:
pVIPR was determined by molecular replacement using peptide-
stripped B
*
2709:m9 (PDB code 1k5n) as a search model and pro-
gram molrep (CCP4; reference 21). After rigid body refinement
using refmac (22), the initial model was improved with ARP/
wARP (23), and water molecules were included. Further im-
provement of the structure was achieved by iterative cycles of
manual rebuilding using O (24) and restrained maximum-likeli-
hood refinement with refmac comprising isotropic B factor ad-
justment. After translation, libration, and screw rotation refine-
ment (25), the R value converged at 0.188 (R
free
, 0.244).
As the two HLA-B27:pVIPR complexes crystallized isomor-
phously, initial phases for B
*
2705:pVIPR were calculated from
peptide-stripped B
*
2709:pVIPR with His116 replaced by ala-
nine. This initial model was subjected to rigid body refinement
followed by simulated annealing and energy minimization using
CNS (26). ARP/wARP was used for improvement of the model
and incorporation of water molecules and pVIPR in canonical
conformation. After manual rebuilding, inclusion of alternate
protein conformations, and atom positional refinement (refmac),
there was still residual electron density in the peptide binding
groove, to which an additional, noncanonical conformation of
pVIPR could be fitted with an estimated occupancy of 0.5 for
both peptide chains. Subsequent optimization of atomic displace-
ment parameters decreased the R factor by 2.5% (R
free
, 2%) com-
pared with isotropic refinement, justifying this procedure. Evalu-
ation of anisotropy by parvati (27) showed the expected statistical
distribution of 0.52
0.14 for all atoms of the structure. The R
value converged at 0.128 (R
free
0.178). The equimolar distribu-
tion of the two pVIPR conformations in the B
*
2705 molecule
was confirmed by an occupancy refinement performed with
CNS. With the p6
conformation (see Results) omitted from the
atomic coordinates, the occupancy of the central part of the pep-
tide refined to occupancies
0.5, whereas that of p1, p2, p8, and
p9 stayed with full occupancy. Similar results were obtained
when only the p6
conformation was refined.
In both structures, radiation-decarboxylated Asp and Glu that
showed negative difference electron density were assigned occu-
pancies
1.0. The final models include all 276 residues of the
HC, 100 residues of
2
m (all 99 residues plus NH
2
-terminal
Met), all nonhydrogen atoms of the pVIPR peptide, water, and
glycerol (from the cryoprotectant). All
/ angles lie in allowed
regions of the Ramachandran plot (procheck; reference 28). Both
structures were validated with whatcheck (29). Statistics are com-
piled in Table I. Superimpositions were performed using pro-
fit (30). Figures were generated using Grasp (31), povray
(www.povray.org), molscript (32), povscript1 (33), and raster3D
(34) together with a graphical interface (moldraw) developed by
Hülsmeyer et al.
273
N. Sträter (Institut für Kristallographie, Freie Universität Berlin,
Berlin, Germany; unpublished data). Molecular surfaces were cal-
culated with MSMS (35).
The atomic coordinates and structure amplitudes have been
deposited in the Protein Data Bank (accession codes 1ogt
[B*2705:pVIPR] and 1of2 [B*2709:pVIPR]).
Results
General Features of the HLA-B27:pVIPR Structures. HLA-
B*2705:pVIPR and B*2709:pVIPR crystallized isomor-
phously (same space group and comparable unit cell
constants), showing the typical MHC class I immunoglob-
ulin-like folds (1) and refined to values of Rcryst 12.8%
and Rfree 17.8% at 1.47 Å and Rcryst 18.8% and Rfree
24.4% at 2.20 Å resolution, respectively (Materials and
Methods and Table I). In both structures, pVIPR is bound
in the common canonical conformation (Fig. 1 A) found in
other HLA-B27 molecules, exploiting all six pockets of the
peptide binding groove for interaction with the HC (19,
36, 37). However, B*2705:pVIPR features an additional,
grossly different noncanonical peptide conformation (Fig. 1
B). Both conformations found in B*2705 are present in a
1:1 ratio as shown by occupancy refinement. Despite this
peculiarity in B*2705, the structures of HC and 2m of the
two subtypes are practically indistinguishable (C root
mean square [rms] deviation 0.2 Å), including almost all
side chain atoms. The atoms contributing to the binding
groove occupy the same positions in B*2705 as in B*2709,
and extreme atomic displacement factors (thermal aniso-
tropy), which could mask multiple conformations were also
not detected for B*2705. It has to be emphasized that, due
to the isomorphous crystal structures, the intermolecular
crystal contacts in both B*2705:pVIPR conformations are
comparable, demonstrating that the two different binding
Table I. Data Collection and Refinement Statistics
HLA-B*2705:pVIPR HLA-B*2709:pVIPR
Data Collection
Space group P21P21
Unit cell (Å) a 51.3, b 81.8, c 65.6,
107.6
a 51.2, b 81.6, c 65.3,
107.5
Resolution (Å)a50.0–1.47 (1.53–1.47) 29.1–2.20 (2.28–2.20)
Unique reflectionsa84,476 (8,185) 25,005 (2,495)
Redundancya4.2 (3.3) 3.1 (3.0)
Completeness (%)a97.6 (95.2) 95.8 (96.8)
I/Ia13.3 (2.2) 9.7 (4.1)
Rsyma,b 0.078 (0.314) 0.123 (0.346)
Refinement
Rcrysta,c 0.128 (0.184) 0.188 (0.21)
Rfreea,d 0.178 (0.286) 0.244 (0.27)
Heavy chain No. atoms/average B factor [Å2] 2,355/18.7 2,275/36.9
2m No. atoms/average B factor [Å2] 864/22.8 850/40.8
Peptide No. atoms/average B factor [Å2] 159/16.5 100/35.2
Water No. molecules/average B factor [Å2] 721/35.9 342/50.2
Estimated overall coordinate
error (Å)e0.06 0.22
Rmsdf from ideal geometry
Bond length (Å) 0.01 0.01
Bond angles () 1.4 1.3
PDB entry code 1ogt 1of2
aValues in parentheses refer to the highest resolution shell.
bRsym = h i Ih,i Ih /hiIh,i.
cRcryst = h Fo Fc/ Fo Working set; no cut-off applied.
dRfree is the same as Rcryst, but calculated on 5% of the data excluded from refinement.
eEstimated overall coordinate error based on Rfree as calculated by REFMAC 5.0.
fRoot mean squared deviation from target geometries.
Dual Self-peptide Conformation in HLA-B27 Antigens
274
modes of pVIPR found only in B*2705 are an intrinsic fea-
ture due to the presence of Asp116 and not a crystallo-
graphic artifact.
In addition, there is a metal binding site with an octahe-
dral coordination sphere formed by pHis8-N, His197-N
(of a symmetry-related HC), and four water molecules.
The cation is probably Mn2 as inferred from coordination
distances (38). The electron density and B factors of Mn2
(19.7 Å2 in B*2705 and 42.3 Å2 in B*2709; the B values are
different due to the different resolutions) (Fig. 2, electron
densities for Mn2) suggest that the site is fully occupied.
The presence of Mn2 is a purification artifact that origi-
nates from a lysis buffer.
HLA-B27 Subtype-dependent Peptide Conformations. The
different conformations of pVIPR in the binding groove of
B*2705 are reflected in the backbone torsion angles of the
peptide. In both structures, pVIPR is bound in the canoni-
cal conformation found regularly in crystal structures of
MHC class I–nonapeptide complexes with main chain tor-
sion angles (, ) of the extended -strand type and only
residue p4 (pTrp4) in right-handed -helical conformation
(Fig. 1 A, 85.2 and 32.6). The -helical turn at
position p4 results in a prominent kink directing the main
chain “away” from the floor of the binding groove toward
the solvent. Residues of pVIPR in this conformation
(pVIPR-p4) interact with all six pockets (Fig. 1 E, pock-
ets A–F) provided by the binding groove, as found com-
monly for nonapeptides (1).
The additional peptide conformation found in B*2705 ex-
hibits a pArg6 in -form (Fig. 1 B, 106.4 and
Figure 2. Final electron density of pVIPR conformations
in B*2705 and B*2709. Stereo images of the final 2Fo-Fc
electron density contoured at 1 level, displayed in light
green. The two B*2705:pVIPR conformations are shown
in A, the respective B*2709 complex is shown in B. The
peptides are color coded as in Fig. 1 (A and B): water mol-
ecules as red and Mn2 as green spheres.
Figure 1. pVIPR conformations, atomic
displacement ellipsoids, and B factors. (A)
Superimposition of the canonical pVIPR
conformations (p4) found in B*2705
(blue) and B*2709 (gold). (B) The nonca-
nonical pVIPR conformation (p6, pink)
observed only in B*2705. The peptides are
viewed from the side of the 2 helix together
with a molecular surface covering the floor
and back of the binding groove. The subtype-
specific residue 116 is indicated also
(Asp116, yellow; His116, turquoise); the
bidentate salt bridge to Asp116 is drawn
with green dotted lines in B. The binding
pockets A–F are shown in bold letters.
Atomic displacement ellipsoids for pVIPR-
p4 and -p6 in C and D are colored accord-
ing to the equivalent isotropic temperature
factors B (Å2) (see color bar). (E, left) Sche-
matic description of side chain orientation
when looking from the NH2 to the COOH
terminus of pVIPR. Bottom of peptide
binding groove indicated by “-sheet”
and side for T cell recognition by “TCR”.
(E, right) The orientation of the peptide
side chains in the p4 and p6 conforma-
tions as in E (left). The respective binding
pockets (A–F) are indicated as well. It is
clear from this representation and Fig. 1 (A
and B) that the two pVIPR conformations
show major differences only from pLys3 to
pTrp7.
Hülsmeyer et al.
275
34.7 designated pVIPR-p6) and both pVIPR-p4 and
pVIPR-p6 are half occupied (Materials and Methods and
Table I). pVIPR-p4 is virtually identical in both subtypes
(Fig. 1 A, rms deviation 0.1 Å for nonhydrogen atoms). Be-
tween the p4 peptides in B*2705 and B*2709, there is only
one minor difference in the orientation of the pArg6 guani-
dinium group as it is engaged in different hydrogen bonds.
Conformation-dependent HLA-B27:pVIPR Interactions.
At the peptide NH2- and COOH-termini, pArg1, pArg2,
pHis8, and pLeu9 occupy identical positions in pVIPR-p4
and -p6 (Fig. 1, A, B, and E and Fig. 2 A). The interac-
tions of pArg1 and pArg2 with A and B pocket residues cor-
respond to those observed for the B*2709:s10R complex
(PDB code 1JGD; reference 37); the side chain of pArg1 is
sandwiched between the side chains of HC Arg62 (1-helix)
and Trp167 (2-helix). In addition, the side chains of Arg62
and Glu163 (2-helix) are linked by a water-mediated salt
bridge (Fig. 3, A and B, bridge) that covers the deeply em-
bedded pArg2. At the COOH terminus, pHis8 is solvent
exposed as well (Figs. 2 and 3), and pLeu9 is accommodated
in the F pocket. The carboxy group of pLeu9 forms the
common polar interactions with Tyr84-O, Thr143-O,
and Lys146-N (1), and the aliphatic pLeu9 side chain forms
hydrophobic interactions with the side chains of Leu81,
Leu95, Tyr123, and Trp147 (19). Because the side chain of
pLeu9 is too short to engage in any direct contacts with
Asp116 (B*2705) or His116 (B*2709), there are no signifi-
cant differences in the F pockets between both subtypes.
Figure 3. Molecular surfaces and contacts of pVIPR in
the p4 and p6 conformations. (A and B) Molecular surfaces
show the central part of the B*2705 peptide binding
groove in gray and the pVIPR peptide in the p4 and p6
conformations (color coded as in Fig. 1 [A and B]). The
binding groove has been rendered semi-transparent, allowing
also the inspection of buried side chains exhibiting confor-
mational differences. In A, the view is TCR-like, straight
onto the peptide, whereas in B (rotated by 90 about a
horizontal axis), the view is through the 2 helix. The center
section of the peptide shows clear shape differences between
the p4 and p6 conformations. (C) pVIPR hydrogen
bonding in p4 and p6, color coded as in Fig 1 (A and
B). Only side chains with different binding modes (residues
p3–p7) are shown. The binding groove’s secondary structure
is represented as gray spirals ( helices) and arrows (
strands) together with selected interacting residues (carbon
atoms, gray; oxygens, red; and nitrogens, blue). Hydrogen
bonds are depicted as black broken lines, the pArg5–
Asp116 bidentate salt bridge is depicted as green dotted
lines, and water molecules are depicted as dark blue
spheres. (D) Electrostatic surfaces of both pVIPR confor-
mations. Red indicates negative, blue indicates positive
surface charge, and gray areas are uncharged. The view is
looking straight onto the binding groove as in A. The border
of the peptides is highlighted in white.
Dual Self-peptide Conformation in HLA-B27 Antigens
276
In contrast to the NH2- and COOH-terminal residues,
the central sections of pVIPR-p4 and -p6, pLys3 to
pTrp7, which are principally accessible for recognition by a
TCR, differ drastically with maximal disparity at pArg5,
3.7 Å for C, and 16.5 Å for N2 (Table II and Figs. 1, A,
B, and E and Fig. 2 A and Figs. 3 and 4). In pVIPR-p4,
side chains of pTrp4 and pArg5 are exposed to solvent, but
in pVIPR-p6, pArg5 is locked within the binding
groove, its guanidinium group forming a bidentate salt
bridge with the B*2705 subtype-specific Asp116 at the in-
terface of the C, E, and F pockets (Figs. 1 B and 2 C and
Figs. 3 and 4). In pVIPR-p6, residues pTrp4 and pArg6
flanking pArg5 are maximally exposed to solvent and
stacked (Fig. 1 B and Fig. 2). As a consequence of the
structural differences around pArg5, the solvent-accessible
surface areas of pVIPR-p4 and pVIPR-p6 have different
size (160 Å2 for pVIPR-p4 and 124 Å2 for pVIPR-p6),
shape (Fig. 3, A and B), and charge distribution (Fig. 3 D)
so that several prerequisites for differential recognition by a
TCR are provided.
As both p4 and p6 are roughly half occupied in the
B*2705 structure (Materials and Methods), approximate
energetic equivalence of the two conformations seems
likely. This is suggested by the temperature factors for the
peptide in the two conformations (Fig. 1, C and D) that are
very similar: in B*2705, the average (isotropic) B factor for
all peptide atoms is 14.8 Å2 (range 10.0–25.9 Å2) for p4
and 17.4 Å2 (range 10.8–31.9 Å2) for p6. The highest
flexibility found for pTrp4-p6 (30 Å2) is still relatively
low on an absolute scale, and marked atomic displacement
as indicated by large B factors is only found in the side
chains of pTrp4, pArg6, and pTrp7 for both p4 and p6
(Fig. 1, C and D).
Peptide Conformation-dependent Recognition of HLA-B27
Subtypes by T Cells. Previous analyses of autoreactive
CTL lines from individuals typing as B*2705 or B*2709
have indicated that pVIPR-specific CTLs are frequently
observed in patients with AS, although they occur also in
healthy B*2705 individuals. In contrast, such CTLs are
only rarely found in B*2709 individuals (15). We extended
these studies, in particular in an attempt to identify CTLs
exclusively reactive against B*2705 that might indicate im-
munogenicity also of B*2705-p6. The results shown in
Table III are based on 39 CTL lines, 17 of which are al-
ready published (15) and 22 were newly obtained from six
AS patients (five B*2705 and one B*2702) and one B*2705
healthy control. The CTLs are grouped as preferentially
recognizing peptide-transporter–deficient T2-B*2705 or
T2-B*2709 cells, both subtypes with approximately equal
efficiency, or exclusively T2-B*2705. No CTL recognizing
only T2-B*2709 was observed, although pVIPR was used
at a high concentration (70 M). This experimental setup
was chosen to maximize the chances to detect reactivity
genuinely specific for B*2705:pVIPR complexes. Dose–
response curves for the reactivity of pVIPR-specific CTLs
have already been determined (15); at lower peptide con-
centrations, T2-B*2709 target cells tended to be lysed at
higher efficiency than T2-B*2705 cells.
The majority of the CTLs lysed both T2-B*2705 and
T2-B*2709 equally well (Table III). However, there were
exceptions showing a clear preference for T2-B*2709 cells.
This was not unexpected because it has previously been
found that B*2709 antigens present pVIPR more efficiently
than B*2705 (15). Only one CTL line was found (EP76)
that selectively reacted with pVIPR presented by T2-
B*2705 cells. The complete lack of reactivity against the
B*2709:pVIPR complex, even at the high peptide concen-
tration used, indicates that EP76 is directed against a struc-
ture different from B*2705:pVIPR-p4 because of the ex-
treme structural similarity of B*2709:pVIPR and the p4
binding mode in B*2705 (Fig. 1 A). In contrast, five further
CTLs from the AS-patient EP reacted with both T2-
B*2705 and T2-B*2709 cells.
However, it must be pointed out that oligoclonal CTL
lines were used in these studies. Therefore, it appears possi-
ble that noncross-reactive CTL clones may be concealed in
the population of cells exhibiting cross-reactivity, resulting
in a lower degree of lysis of B*2709 target cells, and leading
to an underestimation of the immunogenicity of the p6
conformation in B*2705. 15.4% of the CTL lines lysed
B*2705:pVIPR targets more efficiently than T2-B*2709:
pVIPR cells (Table III).
Discussion
A fundamental deviation from the single binding mode
found in all previously determined MHC class I:peptide
complexes is the dual conformation of pVIPR in B*2705.
This feature is of considerable interest both from structural
and immunological points of view. Only one exception to
the single binding mode of peptides has been described so
far in the crystal structure of the rat RT1-Aa antigen in
complex with the exceptionally long 13-mer peptide
Figure 4. Conformation-dependent peptide contacts with F-pocket
residues. The F-pocket architecture and intermolecular interactions in
B*2705:pVIPR-p4 (left) and -p6 (right), with relevant part of the peptide
shown (same color code as in Fig. 1 [A and B]). Fully occupied water
molecules are shown in dark blue and partially occupied ones (related to a
specific peptide conformation) are in turquoise. The space occupied by
pArg5-p6 is filled by water molecules in the p4 binding mode. The
view is looking along the binding groove with the peptide COOH terminus
in front.
Hülsmeyer et al.
277
MTF-E. Two entirely different MTF-E conformations
were found that are possibly associated with crystal packing
effects (39). Because they could consequently represent
nonphysiological artifacts, the two conformations and the
reasons why MTF-E does not adopt a single binding mode
are different from those reported here.
The simultaneous occurrence of pVIPR-p4 and
pVIPR-p6 in B*2705 may be static or dynamic. If static,
the analyzed crystals would be composed of approximately
equal amounts of two cocrystallizing asymmetric units: one
containing pVIPR-p4 and the other pVIPR-p6. If dy-
namic, the peptide would change continually from one
conformation to the other within the binding groove, us-
ing fixed NH2- and COOH-terminal amino acids with
pivot points located at or close to the main chain connec-
tions of p2-p3 and p7-p8, respectively (Figs. 2 A and 3 C).
Currently, we cannot distinguish between static and dy-
namic modes of peptide binding, but spectroscopic meth-
ods (40) or X-ray analyses of crystals produced at different
temperatures may eventually provide an answer.
Thus far, the presentation of a peptide by an MHC mole-
cule in a single, defined conformation has been considered a
prerequisite for positive and negative selection of T cells
within the thymus (41). The two drastically different binding
Table II. Comparison of Peptide Coordination in the p4
and p6
Binding Modes of B*2705:pVIPR
p4 conformation p6 conformation
Peptide
residue Atom
Contact
residue Distance Interaction Atom
Contact
residue Distance Interaction
ÅÅ
pArg1 all contacts formed by pArg1-p4 are identical to those observed in -p6 conformation
pArg2 all contacts formed by pArg2-p4 are identical to those observed in -p6 conformation
pLys3
pLys3N* Tyr99OH* 3.02 H bond pLys3N* Tyr99OH* 2.96 H bond
pLys3O‡ pArg6NH1‡ 3.00 H bond pLys3O* Tyr99OH* 3.43 H bond
pLys3NZ ‡ pArg5O‡ 2.78 H bond pLys3NZ‡ pTrp4O‡ 2.82 H bond
pTrp4 solvent exposed solvent exposed
pTrp4O‡ pArg6NH2 ‡ 3.00 H bond pTrp4O‡ pLys3NZ ‡ 2.82 H bond
pTrp4 ‡pArg6 ‡3.3 v.d. Waals
pArg5 solvent exposed
pArg5NH1§ Gln155OE1§ 3.20 H bond pArg5NH1* Asp116OD1 * 3.04 salt bridge
pArg5O‡ pLys3NZ ‡ 2.78 H bond pArg5NH2* Asp116OD2 * 3.10 salt bridge
pArg6 solvent exposed
pArg6NH1¶ Ile66O¶ 2.74 H bond pArg6‡pTrp4‡3.3 v.d. Waals
pArg6NH1‡ pLys3O‡ 3.00 H bond
pArg6NH2 ‡ pTrp4O‡ 3.00 H bond
pTrp7
pTrp7§Val152§3.5 v.d. Waals pTrp7§Leu156§3.5 v.d. Waals
pHis8 all contacts formed by pHis8-p4 are identical to those observed in -p6 conformation
pLeu9 all contacts formed by pLeu9-p4 are identical to those observed in -p6 conformation
Only direct contacts are included, and water-mediated interactions are omitted. Interacting atoms are specified for polar interactions only because
v.d. Waals contacts are too numerous and less specific.
*-sheet floor.
‡Intrapeptide contacts.
§Helix 2.
¶Helix 1.
Dual Self-peptide Conformation in HLA-B27 Antigens
278
modes of pVIPR to B*2705 show that this assumption might
be an oversimplification. A peptide changing its conforma-
tion dynamically within the groove could possibly preclude
high-affinity interaction with the two CDR3 regions of a
TCR (42, 43), whereas a static conformation might allow a
more efficient recognition. The rare occurrence of pVIPR-
specific CTL in B*2709 individuals suggests that the B*2709:
pVIPR complex can indeed serve in establishing tolerance
(15). Conversely, the number of B*2705:pVIPR-p4 and
-p6 complexes might fall below the epitope density thresh-
old required for negative selection (44, 45), leading to the
frequent presence of autoreactive, pVIPR-specific CTL (15).
The CTL lines analyzed here seem to possess relatively
low affinity (15), questioning the presentation of the
VIPR-derived peptide under physiological conditions, and
suggesting the use of VIPR transfectants in cytotoxicity
studies. However, even when testing the reactivity of
CTLs against EBV-derived peptides, lysis of target cells is
rarely observed (15, 46). At least the viral peptides are
surely processed and presented; comparative studies with
transfectants are usually unsuccessful. In addition, a
COOH-terminal pLeu9 as seen in pVIPR does not present
an obstacle for efficient proteasomal processing (47) and
transport to the endoplasmic reticulum, as shown by pep-
tide elution studies from B*2705 and B*2709 molecules
(14). The high number of pVIPR-specific CTLs in some
B*2705 individuals (15) might be explained by selective in-
crease (clonal expansion) due to cross-reaction. This is not
unlikely because TCR cross-reactivity is mandatory for the
control of infections (48–52). In the case discussed here,
the pVIPR cross-reactive nonself antigen could be the
EBV-derived pLMP2 (15, 16, 53). Convincing examples of
molecular mimicry between viral and self-proteins have al-
ready been identified (51, 52).
Unconventional peptide binding modes distantly resem-
bling that observed for the p6 conformation in B*2705:
pVIPR have occasionally been found in other MHC:pep-
tide complexes as well (18, 54, 55), but the involvement of a
strategic subtype-specific residue at the floor of the binding
groove is unprecedented. The detection of p6 in B*2705:
pVIPR but not in the closely related B*2709:pVIPR com-
plex is obviously a consequence of the combined presence
of the allele-specific residue Asp116 (B*2705) and a specific
peptide sequence with Arg at position 5. The larger size and
neutral or positive charge of His116 compared with Asp116
would interfere sterically and electrostatically if the pArg5
side chain were bound to the F pocket (Fig. 1 A). The bind-
ing of the peptide COOH-terminal pLeu9 to the F pocket
brings a further constraint into play: to avoid clashes, it is
likely that the side chain of this residue has to be small and
hydrophobic, but not positively charged to prevent compe-
tition with another basic amino acid (in this instance, pArg5)
that contacts Asp116.
The paucity of peptides with pArg5, such as pLMP2,
which have been found to be presented by HLA-B27
molecules under natural conditions (14, 16), makes it diffi-
cult to provide an estimate of the generality of the p6
conformation, let alone a dual peptide binding mode, in
HLA-B27 molecules. We have found a few further pep-
tides derived from self- as well as microbial proteins that
have in common not only an Arg residue but even the
WRR motif (unpublished data). As it seems conceivable
that some of these peptides share the unorthodox struc-
tural features of pVIPR when complexed with the B*2705
subtype, analyses with these novel peptides are underway
to examine the possibility that the WRR motif represents
a sequence with direct involvement in AS pathogenesis.
With only very few structures of HLA-B27:peptide com-
Table III. Reactivity of CTLs from HLA-B27–positive Subjects towards the pVIPR Peptide in the Context of Either B*2705 or
B*2709 Molecules
Donora
Number
of CTLs
Specific lysis of
T2-B*2709 05b
Specific lysis of
T2-B*2709 05c
Specific lysis of
T2-B*2709 05b
Specific lysis
of T2-B*2705
only
MP 19 9 7 3 0
EP 6 0 4 1 1 (EP76)
AS 4 1 3 0 0
AB 2 0 2 0 0
MD 4 1 2 1 0
LV 2 1 0 1 0
CV 2 0 2 0 0
Total 39 12 (30.7%) 20 (51.3%) 6 (15.4%) 1 (2.6%)
aFive donors (MP, EP, AS, AB, and MD) are HLA-B*2705–positive patients with AS. LV is an HLA-B*2702–positive patient with AS, and CV is a
healthy HLA-B*2705–positive individual. The results summarize the data obtained in two cytotoxicity experiments. Background (specific lysis of
unpulsed T2-B*2705 and T2-B*2709) was 12%.
bThe difference in the percentage of specific lysis between T2-B*2705 and T2-B*2709 cells after background subtraction was 20%.
cThe difference in the percentage of specific lysis between T2-B*2705 and T2-B*2709 cells after background subtraction was 20%.
Hülsmeyer et al.
279
plexes solved to date (Results; references 19, 36, 37), it
seems premature to engage in a speculation on the prereq-
uisites for a dual peptide conformation.
However, the pVIPR-p6 peptide binding mode and its
subtype dependency is not a unique, serendipitous finding.
We have observed recently that the sequence-related
pLMP2 peptide (16) binds in p4-like form to B*2709 but
in p6-like conformation to B*2705, with pArg5 again
forming a bidentate salt bridge with Asp116 (unpublished
data). Therefore, the noncanonical binding mode involving
residue 116 as observed for one of the conformations in
B*2705:pVIPR and even its subtype dependency can be
found in other HLA-B27:peptide complexes as well. Most
AS-associated HLA-B27 alleles encode Asp116, whereas
this residue is Tyr (B*2706) or His (B*2709) in the two
subtypes not associated with AS (7).
A constellation similar to that of the B*2705/B*2709 pair
is found in A*68012 (Asp116) and A*6807 (His116; refer-
ence 56), or in B*3501 (Ser116) and B*3503 (Phe116; ref-
erence 57), but comparative structural studies have not been
performed for these proteins. Among further HLA class I
genes, approximately half of the HLA-A alleles encode
Asp116, and nearly all others encode Tyr116 that occurs
also in a smaller number of HLA-B and HLA-C antigens, in
which Asp116 is rarely represented (58). However, the
mere availability of Asp116 is not sufficient for a basic resi-
due in the middle of a peptide to engage in a p6-like bind-
ing mode. This is exemplified by the HLA-A*0201:MAGE-
A4 complex (59) or HLA-B*4402 and B*4403 complexed
with an HLA-DP–derived peptide (60): these peptides ex-
hibit Arg either at position 5 or 6 that are solvent exposed
and not buried. Other residues on the floor of the binding
groove could principally also be involved in noncanonical
anchoring modes such as the HC residues 97, 99, and 114,
which are, like residue 116, highly polymorphic in HLA
class I molecules (61). For example, HLA-B*3501 presents
an EBV-derived nonapeptide with pAsp5 contacting Arg97
and Tyr99 (18). Clearly, a nonconventional peptide anchor-
ing could be more frequent than currently envisaged.
Polymorphisms within the MHC class I peptide binding
groove as shown here may influence peptide presentation
and T cell repertoire selection (1, 4, 7, 15, 62–64), but a
dual peptide presentation mode as found in B*2705 that
depends on the HLA-B27 subtype-specific amino acid ex-
change is novel. Distinct pVIPR presentation modes and
differential AS-association of B*2705 and B*2709 may be
linked in the course of the development of AS (15), al-
though other factors influencing AS initiation and progres-
sion have to be taken into consideration, among them pos-
sible polymorphisms of HLA-B–linked genes (65–67). Our
results support certain models of AS–pathogenesis that can
incorporate differential peptide display by HLA-B27 sub-
types, such as the arthritogenic peptide theory (4, 7), in
particular in the modified form proposed by us (15), or the
2m-deposition hypothesis (68).
HLA subtype-dependent differential presentation of
peptides in noncanonical conformations due to HLA class I
HC polymorphisms at residue 116 might also be connected
to the pathogenesis of other disease states, such as the rate
of the progression to AIDS in HIV-positive patients (57) or
the outcome of bone marrow transplants between unre-
lated individuals (69). In conclusion, our results provide an
unexpected twist regarding the mode of peptide display as a
single, structurally unaltered MHC molecule can present a
given peptide in two entirely different binding modes, de-
pending only on a buried HC amino acid polymorphism.
The authors thank all patients and the healthy proband for their par-
ticipation in this paper. We are grateful to P.G. Coulie, J.A. López
de Castro, and D.J. Schendel for discussions and comments on the
manuscript; to M. Rühl for excellent technical assistance; and to U.
Müller, S. Popov, and R. Hillig for help at the synchrotron facilities.
This work was financially supported by the Deutsche Forschungs-
gemeinschaft (SFB 449), Berliner Krebsgesellschaft, Monika Kutz-
ner-Stiftung, Berlin, Sonnenfeld-Stiftung, Berlin, Fonds der Chem-
ischen Industrie, COFIN 2001, and Istituto Pasteur Fondazione
Cenci-Bolognetti. Data collection at Deutsches Elektronen Synchro-
tron was supported by a European Community grant to the Euro-
pean Molecular Biology Laboratory Outstation in Hamburg.
Submitted: 1 October 2003
Accepted: 26 November 2003
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