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* Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia;
Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia; and
Protein Crystallography Unit, St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR structures.
To better understand the structural correlates of Ag immunodominance
and TCR selection bias, we have solved the crystal structure of the
HLA-B8-FLRGRAYGL peptide complex to a resolution of 1.9 Å. The
structure confirms the importance of P3-Arg, P5-Arg, and P9-Leu as
dominant anchor residues involved in peptide binding to HLA-B8. A
bulged conformation of the bound peptide provides a structural basis
for the critical role of the P7-Tyr residue in T cell recognition. The
peptide also induces backbone and side-chain conformational changes in
HLA-B8 that are transmitted along the peptide-binding groove in a
domino effect. The HLA-B8-FLRGRAYGL complex crystallizes as a dimer in
the asymmetric unit and is oriented such that both peptide ligands are
projected in the same plane suggesting a higher order arrangement of
MHC-peptide complexes that could be involved in formation of the class
I Ag-loading complex or in T cell activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR usage by CTL that
recognize this determinant (11). Indeed, the selection of
the VDJ combination BV6S2/BSD/BJ2S7 (V6.2/DJ2.7)
with the VJ combination AV4S1/AJ14S3 (V4.1/J14.3)
occurs repeatedly in FLR-specific CTL from unrelated individuals with
HLA-B8 (11, 12). Moreover, identical residues are also
encoded by untemplated N region codons present in VDJ and VJ
junctional sequences from these FLR-specific CTL. Accordingly, the
HLA-B8-FLR response may represent the most extreme example yet of TCR
selection bias in an antiviral CTL. This observation has led to the
description of these receptors as "public TCRs" (11).
These public CTL use mainly germline VDJ and VJ sequences, and
their fine specificity is exquisitely sensitive to substitution in the
FLR peptide at P4-Gly, P6-Ala, P7-Tyr, and P8-Gly (13). In
addition, these T cells also react with allogeneic HLA-B*4402
molecules, presumably complexed to an unknown self peptide
(14). In an effort to better understand the structural
basis of the immunodominance of FLR in HLA-B8+
individuals and its recognition by a public TCR, we have determined the
atomic structure of the HLA-B8-FLR complex at 1.9 Å resolution. The
structure confirms the important role of P3-Arg, P5-Arg, and P9-Leu in
anchoring the peptide to the HLA-B8 Ag-binding cleft. In addition,
P6-Ala, P7-Tyr, and P8-Gly form part of the bulged section of the
peptide that results in complete exposure of P7-Tyr, which is
accentuated by the small side chains of P6-Ala and P8-Gly.
Significantly, in comparison to the HLA-B8-GGKKKYKL (HLA-B8-GGK)
series of structures (15), ligation of the FLR determinant
produces backbone and side-chain conformational changes transmitted
along the length of the HLA-B8 peptide-binding cleft. Finally, the
HLA-B8-FLR complex crystallized as a head-to-tail dimer that is
compatible with specific TCR engagement of both HLA-B8-FLR monomers
contained in single plane, suggesting one method of higher order
arrangement of MHC-peptide complexes (MHCp) that might be involved in
formation of the Ag-loading complex or in activation of specific T
cells. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Soluble class I heterodimers containing the FLR peptide were
prepared as described previously (15). Briefly, the
truncated forms (amino acid residues 1–276) of the HLA-B8 H
chain and full-length 2-microglobulin
(2m) were expressed in Escherichia
coli and each protein purified from inclusion bodies. The complex
of H chain/2m/peptide was refolded by diluting
the H chain and 2m inclusion body preparations
into refolding buffer containing a molar excess of peptide ligand. The
refolded complexes were concentrated and purified by anion exchange
chromatography. The complexes were further purified by gel filtration
and Mono-Q anion exchange chromatography (Pharmacia Biotech,
Uppsala, Sweden) to a high level of purity.
Crystallization
Previously, HLA-B8 has been crystallized in complex with a
peptide from HIV (HIV-1 Gag protein p17 residues 24–31 GGKKKYKL
(GGK)), as well as a series of closely related derivatives (K7R, K7Q,
K5R, and K3R), using 30% polyethylene glycol (PEG) 4000, 0.1 M sodium
citrate (pH 6.5), and 0.2 M ammonium acetate (15); the
HLA-B8-GGK peptide crystals belong to space group
P212121
with unit cell dimensions a 
52, b
81, c 112 Å. However, for the HLA-B8-FLR
complex, fine screening around these initial conditions yielded either
phase separation or heavy precipitation, and sparse-matrix screening
did not yield crystals either. Using the most promising conditions,
additive screens one, two, and three (Hampton Research, Riverside,
CA) were used at room temperature. Condition two of additive
screen one (cadmium chloride) yielded a shower of needles, which after
optimization yielded large crystals suitable for crystallographic
studies. Crystals (0.3 x 0.2 x 0.2 mm) were obtained using
the hanging drop vapor diffusion technique at 4°C. The crystals were
grown within 3–5 days by mixing equal volumes of 10 mg/ml HLA-B8-FLR
with the reservoir buffer (10–15% PEG 4000, 0.1 M sodium citrate (pH
5.6), 0.2 M ammonium acetate, and 10 mM cadmium chloride). The crystals
belong to space group
P212121
with unit cell dimensions a = 85.41, b
= 90.10, c = 125.28 Å. A native Patterson map revealed
that the two molecules in the asymmetric unit were related by a
translational component with fractional coordinates 0.09, 0.50, 0.00.
The crystals were flash frozen before data collection using crystals
that had been soaked overnight in the cryoprotectant 20% PEG 400. A
1.9 Å data set were collected using inverse phi geometry, processed,
and scaled using the D*TREK program (16). For a
summary of statistics, see Table I
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The structure was solved by the molecular replacement method, using the program AmoRe within the collaborative computer project no. 4 Suite (17). The previously solved monomeric HLA-B8 structure (Protein Data Bank code: 1AGE) (15), minus the peptide, was used as the search probe. A clear peak in the rotation function yielded two solutions in the translation function that packed well within the unit cell and were related by the translational component identified in the native Patterson map. The two molecules pack as a dimer in the asymmetric unit. Following rigid body fitting in AmoRe, the molecular replacement solution had an Rfactor and correlation coefficient of 54.1 and 45.3, respectively. Unbiased features in the initial electron density map, including that of the FLR peptide confirmed the correctness of the molecular replacement solution.
The progress of refinement was monitored by the
Rfree value (4% of the data) with
neither a sigma nor a low resolution cutoff being applied to the data.
The structure was refined using rigid-body fitting of the individual
domains followed by the simulated-annealing protocol implemented in
Crystallography and Nuclear Magnetic Resonance Systems (version
1.0) (18), interspersed with rounds of model building
using the program "O" (19). Tightly restrained
individual B factor refinement was used, and bulk solvent corrections
were applied to the data set. Throughout refinement,
noncrystallographic symmetry restraints were imposed, apart from
the residues involved in crystal contacts, on the two molecules. Water
molecules were included in the model if they were within
hydrogen-bonding distance to chemically reasonable groups, if they
appeared in Fo-Fc maps contoured at 3.5
, and if they had
a B factor <60 Å2. The loop comprising residues
41–48 in the H chain was disordered and not modeled in the structure.
Very strong peaks in the Fo-Fc difference Fourier indicated
the location of the cadmium sites. The electron density for the bound
peptide was very clear in both monomers. The final model comprises
residues 1–40 and 49–276 of the H chain, residues 1–99 of the
2m, 794 water molecules, and 2 cadmium ions.
The model has an Rfactor of 24.2% and
an Rfree of 28.2% for all reflections
between 20 and 1.9 Å. See Table I
for summary of refinement statistics
and model quality. The coordinates have been deposited in the Protein
Data Bank and will be released upon publication (code: 1 M05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The structure of HLA-B8 (B8*01) complexed to FLR (HLA-B8-FLR) has
been determined to 1.9 Å and to an
Rfactor and
Rfree of 24.2 and 28.3%,
respectively. The final model comprises two HLA-B8-FLR complexes, which
pack as a dimer, 704 water molecules, and two cadmium ions in the
asymmetric unit. The cadmium ion, an essential additive for the
crystallization, was seen to mediate crystal contacts, forming
interactions between the HLA-B8 H chain Glu19 and
2m Glu36 and
Asp38. These cadmium-mediated contacts do not
relate to the dimer-mediated contacts. The root mean square deviation
(r.m.s.d.) between the monomers in the asymmetric unit is 0.32
Å over all C atoms, and unless explicitly stated, structural
analyses have been confined to one monomer. The HLA-B8-FLR complex
comprises two subunits, a H chain and a light chain
(2m), and is similar to that of previously
determined MHC class I structures. Briefly, the H chain contains the
domains that form the Ag-binding cleft comprising an antiparallel
sheet bounded by two long helices and a membrane proximal 3
domain that sits beneath the Ag-binding cleft and makes extensive
contacts with 2m.
Conformation of the bound peptide
The electron density of the bound FLR (Fig. 1A) and the interacting
residues was unambiguous. The peptide is bound in an extended
conformation (Fig. 2), containing a
centrally located, surface-exposed bulge at residues
Ala6 and Tyr7 (Fig. 1B). The average temperature factor for the bound peptide is
26 Å2, whereas the increased mobility of the
P7-Tyr residue (B-factor 46 Å2) is consistent
with the limited number of contacts this residue makes with the HLA-B8
H chain (Table II). The anchor residues
at positions P3, P5, and P9, as well as residues at P2 and P8, are
buried, and thus are unlikely to interact with the TCR, assuming no
dramatic conformational changes occur within the HLA-B8 complex upon
TCR binding. Conversely, the remaining residues of the FLR peptide
display a varying degree of solvent exposure (exposure values: P1, 29
Å2; P4, 51 Å2; P6, 73
Å2; and P7, 65 Å2) and
are thus potentially able to interact with a TCR. This finding is
consistent with previous experiments in which each residue of the FLR
nonamer was substituted with each of the remaining natural amino acids
and then tested for recognition by B8-FLR specific T cell clones
(13). The amino acid substitutions at the nonanchor
residues P1, P2, P4, P6, P7, and P8 that retained recognition by the
V6.2/DJ2.7-V4.1/J14.3 public TCR combination are summarized in
Fig. 1C (13).
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A), making considerable polar and nonpolar
contacts with the HLA-B8 molecule along the length of the peptide
(Table II, Fig. 2) with three salt bridges, 16 hydrogen bonds, and 7
water-mediated hydrogen bonds in total. A notable difference between
the HLA-B8-GGK complexes (15) and the HLA-B8-FLR complex
is the length of the peptide bound within the peptide-binding grooves.
Even though FLR is a nonamer and GKK is an octamer, the start and end
locations of the bound peptide within the groove are identical.
Consequently, the FLR peptide flexes in the middle, where it makes
relatively few contacts with the HLA-B8 molecule. In comparison to the
HLA-B8-GGK complexes, the HLA-B8-FLR structure can be thought of as
containing a residue insertion at position P6, the site at which the
prominent bulge is located (Fig. 3).
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sheet floor as well as flexing within
the helical walls of the peptide-binding groove are clearly evident
between the two complexes (Fig. 4). The structures of the
2m domains in the HLA-B8-FLR and the
HLA-B8-GGK complexes are very similar (r.m.s.d. of 0.40 Å) and are not
discussed further. The overall r.m.s.d. between the H chain HLA-B8-FLR
and the five HLA-B8-GGK complexes ranges between 0.92 Å and 1.00 Å,
with major structural differences (>1 Å) occurring in the following
regions: 13–21, 38–49, 75–76, 86–91, 105–106, 184–200, 206–207,
218–227, 235–240, 248–249, and 250–277. Some of these differences
result from differing crystal packing, dimerization contacts, and
regions of local disorder between the structures. However, some
differences, notably residues 38–49 and 75–76, are a direct result of
peptide-induced conformational changes. Detailed below are the
similarities and differences between the modes of binding by the FLR
and GGK determinants to the common HLA-B8 allele
product.
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In HLA-B8-FLR, the P1 and P2 positions are occupied by a bulky
phenylalanine and a leucine residue, respectively, whereas in the
series of HLA-B8-GGK complexes, these positions are occupied by glycine
residues. Consequently, structural rearrangements are observed within
the corresponding pockets to accommodate the larger P1 and P2 residues
of the FLR peptide, and furthermore some of these structural
rearrangements appear to have a domino effect of conformational change
that propagates along the peptide-binding groove (Fig. 4).
The P1 FLR peptide backbone is tethered by the hydroxyl group of three
tyrosines, Tyr7, Tyr159,
and Tyr171 (Fig. 4A). The partly
exposed side chain of Phe1 points upward, forming
an aromatic cluster with Trp167,
Tyr171, Tyr59, and the
aliphatic moiety of Arg62. In comparison to the
HLA-B8-GGK complex, the Phe1 pushes the charged
side chain Arg62 out of the pocket so that the
guanadinium group now curls away to interact with the
Gln65 (Fig. 4A). The orientation of
Trp167 side chain is also different between the
structures, in that the Trp is pushed upwards 1.9 Å away from the
pocket in the HLA-B8-FLR structure (Fig. 4A). The side chain
of Thr163 also protrudes more into the pocket,
making Van der Waals’ interactions with the Phe side chain.
These changes are likely to be of relevance to TCR recognition. For
instance, HLA-B8+B44+
heterozygous individuals avoid self-reactivity with HLA-B*4402 by
switching TCR usage from the immunodominant V6.2/DJ2.7-V4.1/J14.3
combination (which is alloreactive with B*4402) to other TCR
combinations, some of which are highly sensitive to substitution of
P1-Phe in the FLR sequence (13, 20). This sensitivity at
P1-Phe could result from direct involvement of this residue in TCR
recognition or, more likely, may result from indirect effects resulting
from the reorientation of HLA-B8 1 helix residues
Arg62 and 2 helix residue
Trp167 (Fig. 4A). These
peptide-induced conformational changes imply that a diversity of
"altered self" forms may arise from the complexity of self and
foreign peptides bound to a given HLA isotype.
The side chain of P2 leucine in the FLR peptide forms Van der
Waals’ contacts with Tyr7,
Phe36, Asn63, and
Ile66, and, when compared with the HLA-B8-GGK
structures, further highlights the plasticity of the HLA-B8 binding
pockets (Fig. 4B). The P2 Leu causes
Phe67 to flip away from the pocket into a
position that was previously occupied by Phe36 in
the HLA-B8-GGK series of structures (Fig. 4B). To avoid
steric clashes, this triggers Phe36 and the
Glu45 (a B pocket residue on the outer strand
of the sheet) to reorientate. However, the density for the outer
strand has become disordered in the HLA-B8-FLR structure (residues
41–48; Fig. 4B), suggesting that a compensatory mechanism
may exist to permit the observed structural plasticity within the
peptide-binding groove of HLA-B8.
The change in conformation of the Phe67 side
chain also reorientates the Phe22 side chain, a
residue that is at the base of the P5 pocket (Fig. 4B). The
Phe22 then pushes the position of the
Arg75 side chain away, so it now forms a salt
bridge with Glu19, a residue that represents part
of the cadmium binding site. These conformational changes do not
disrupt the conserved hydrogen bonding between the HLA-B8 H chain and
residues P1/P2 of the FLR peptide.
The anchor residues P3, P5, and P9
The remarkable complementarity at the interface of the floor and
helices of the class I binding cleft with amino acid side chains of the
peptide ligand (21) have defined amino acid residues
responsible for "anchoring" the peptide ligand into the Ag-binding
cleft. Biochemical analysis of peptides purified from mature class I
molecules have helped identify ligand-binding motifs, defining the
so-called "anchor residues" responsible for making conserved and
energetically important contacts with residues lining discrete pockets
in the class I binding cleft (22). The HLA-B8 peptide
anchor residues at positions P3 and P5 are basic amino acids, while the
C-terminal anchor residue is small and hydrophobic (Fig. 1A). In the HLA-B8-FLR structures, both the P3 and P5
positions are occupied by arginine, whereas in the HLA-B8-GGK
structures, these positions are occupied by either a lysine or an
arginine residue (15) (Fig. 3). Both MHCp possess a
leucine at the C terminus that makes very similar contacts with the
HLA-B8 molecule: the leucine side chain sits in a buried hydrophobic
pocket (F pocket), making contacts with Tyr116,
Tyr123, Trp147,
Ser77, Leu95, and
Thr143, while the main chain is tethered by six
hydrogen bonds (Fig. 2).
The guanadinium group of the P3-Arg from the FLR peptide makes a salt
bridge with Asp156 as well as two hydrogen bonds
with Asn114 and Tyr116,
while its aliphatic moiety packs against the side chains of
Tyr99 and Tyr159. The P3
side chains adopt similar conformations between the HLA-B8-FLR and
HLA-B8-GGK structures; however, the orientation of the guanadinium head
group is different, such that one hydrogen bond is lost (to
Tyr116) in the HLA-B8-GGK structure; in addition,
because of its bulkier guanadinium headgroup, the C atom of P3-Arg
from FLR is displaced by 1.1 Å compared with the corresponding Arg in
the HLA-B8-GGK structure (Fig. 3). The P5-Arg side chain, which makes
two salt bridges with Asp9 and
Asp74 and a water-mediated hydrogen bond with
Thr73, represents the seed point whereby the main
chain conformation takes an abrupt detour between the
structures of the two HLA-B8-peptide complexes (Fig. 3). In
comparison to the HLA-B8/HIV structure, which has an Arg at this
position (code AGF), there is 1.0 Å difference in the C position as
well as different main chain conformation (15). The main
chain carbonyl of P5 in the FLR peptide points downwards, making a
hydrogen bond with Thr73, whereas the carbonyl
group of the HIV peptide makes no direct hydrogen bonds to the
HLA-B8.
The positioning of the P5 aliphatic moieties are different between the structures, and the guanadinium groups are in opposite orientations, with small changes within the P5 pocket to accommodate the differences in the orientation of the side chain. These differences can be attributed to the differing position adopted by the P3 residue between the structures.
Potential TCR contacting residues P4, P6, P7, and P8
The P4, P6, and P7 positions display a high degree of solvent
exposure (Fig. 1A and B) representing potential
contact sites with the TCR that are also predicted from functional
studies of T cell recognition (13) (Fig. 1C).
Although the P8 position of the HLA-B8-FLR structure is not solvent
accessible, the long P7 side chains (lysine, glutamine, or an arginine)
in the HLA-B8-GGK structures are solvent accessible (15),
highlighting a different role for P8-Gly in the HLA-B8-FLR structure.
First, the small P8-Gly creates a cavity that allows the B8 H chain
residue Glu76 to protrude into the pocket,
although it does not form any contacts with the glycine. Second,
despite the inaccessibility of P8-Gly, nearly all amino acid
substitutions at this position disrupt CTL recognition by different
clones, including those with the immunodominant
V6.2/DJ2.7-V4.1/J14.3 TCRs, which only tolerate Ala and Ser in
place of Gly at this position (13). It is likely that
large side chains at P8 would interfere with specific TCR contacts made
with the highly exposed neighboring P7-Tyr, and this is supported by
the crystal structure of a V6.2/DJ2.7-V4.1/J14.3 TCR that reveals
a deep cavity in the receptor combining site that is likely to envelope
the P7 tyrosine aromatic side chain (23). Thus, we
speculate that the P8-Gly critically controls productive T cell
recognition by the permissive nature of its buried side chain, which is
conducive to TCR docking over the neighboring P7-Tyr (Fig. 1).
The P4 position is highly solvent exposed in both the HLA-B8-FLR (Gly,
51 Å2) and the HLA-B8-GGK series of structures,
so substitution of this amino acid does not cause a change in
conformation of the HLA-B8 H chain. The P4-Gly of HLA-B8-FLR is also
likely to influence recognition by the V6.2/DJ2.7-V4.1/J14.3 TCR,
because only substitution with P4-Ala or P4-Pro permits recognition by
the immunodominant TCR (13). This may either reflect
direct interaction of this residue with the CDR loops of the TCR,
indirect steric requirements for the interaction of CDR loops with the
MHCp, or steric constraints on the amino acids flanking the bulge
formed between FLR P6-P8 that forms a TCR recognition hotspot. The
P6-Ala residue, which does not have a structural equivalent in the
HLA-B8-GGK structure, is highly solvent exposed and makes minimal
contacts with the HLA-B8 molecule (Fig. 1A). Therefore, this
residue may form an additional component of the TCR footprint
consistent with TCR recognition of FLR substitution analogs that is
restricted to P6-Pro, P6-Ser, and P6-Val.
The P7-Tyr of the FLR peptide is equivalent to the P6-Tyr of HLA-B8-GGK
structures, but the corresponding orientation and location of the
aromatic side chain is dramatically different (Fig. 3). The P7-Tyr
residue was shown to be critical for recognition by virtually all
HLA-B8-restricted, FLR-specific CTL. Substitution of this residue with
any other residue apart from P7-Phe resulted in a dramatic reduction in
responses of CTL bearing V6.2/DJ2.7-V4.1/J14.3 (13).
In the HLA-B8-GGK series of structures, the equivalent P6 tyrosine
folds back onto the peptide backbone and packs against
Val152 of the HLA-B8 H chain (15),
whereas in the bound FLR peptide, it is oriented away from the peptide
backbone, packing against HLA-B8 H chain residues
Ala150 and Val152 and
forming a water-mediated hydrogen bond with
Gln155. Interestingly, in a fully refined 2.3-Å
resolution model (data not shown), the P7-Tyr residue of HLA-B8-FLR was
clearly seen to hydrogen bond directly to the
Gln155 residue implying intrinsic flexibility at
this site, potentially relevant to TCR interaction. In contrast, in the
HLA-B8-GGK structure, Gln155 is oriented away
from the peptide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. Not surprisingly, substitutions in
the anchor residues P3-Arg and P5-Arg, except for those of other basic
amino acids, are poorly tolerated by CTL, suggesting that peptides
altered at these sites were not able to bind to the HLA-B8 molecule
(13). However, changes at the P9 anchor residue are
better tolerated in that small aliphatic side chains, as well as the
larger Phe and His side chains, were still recognized by FLR-specific
CTL (13). The larger residues may be accommodated by local
structural rearrangements within the F pocket, in a manner analogous to
the adjustments at the B pocket (Fig. 4B). Different
FLR-specific CTL, including those expressing the public
V6.2/DJ2.7-V4.1/J14.3 TCR, displayed a significant amount of
tolerance in the type of amino acid present in the P2 position of FLR
(13). This finding is consistent with the hidden nature of
the P2 leucine in the HLA-B8-FLR structure.
The systematic substitution of individual residues in the FLR peptide
indicated that P7-Tyr was the most critical residue for responsiveness
of HLA-B8-restricted, FLR-specific CTL (13). In the
HLA-B8-FLR structure, P7-Tyr is highly solvent exposed and primed to
interact with the TCR. Bulged conformations of peptides bound to other
MHC class I molecules have previously been reported
(24, 25, 26) and occur in longer peptides as a natural outcome
of retaining the conserved hydrogen bonding networks between the
class I H chain and the peptide termini (27).
Substitutions of either the P6-Ala or the P8-Gly flanking the P7-Tyr
adversely affected responses by V6.2/DJ2.7-V4.1/J14.3 and other
FLR-specific CTL (13). The intolerance of many
substitutions at the P6 and P8 positions of FLR suggest that the small
flanking residues on either side of the tyrosine are required to enable
optimal TCR/MHC engagement of the critical P7-Tyr (13).
Interestingly, some HLA-B8-FLR-specific CTL clones that did not express
public TCRs showed more tolerance to substitution at the P8-Gly
position and a greater sensitivity to changes at the P1 position
(13), even though the P1-Phe is only partly exposed in the
unliganded crystal structure of HLA-B8-FLR. Presumably,
Arg62, which interacts with the P1-Phe, moves out
of the way to maximize contacts with the incoming TCR. In comparing the
structures of HLA-B8-FLR and HLA-B8-GGK, Arg62
has already been shown to be mobile and capable of such a
conformational switch. Taken together, the crystal structure confirms
the complementarity of binding between HLA-B8 and FLR and fits the fine
specificity of recognition of this complex by different CTL.
The structure of HLA-B8-FLR also allows us to address how the HLA-B8
molecule is able to bind two different peptides selected from unrelated
pathogens, such as EBV and HIV. The r.m.s.d. for the two complexes is
relatively large, despite the identical primary structure of the HLA-B8
molecules. Superposition of the sheet from the 1 and 2
domains of the H chain from the HLA-B8-FLR and B8-GKK structures
suggests that there is a shift in the juxtapositioning of the helical
jaws as a consequence of the differing bound peptides. These slight
structural changes are likely to be functionally important based on the
effect of naturally occurring mutations at single sites in the GKK
peptide (15). Specifically, T cell clones that were very
sensitive to conservative changes at the solvent-exposed P7 position of
GKK were also sensitive to mutations at the buried positions, P3 and P5
(15). These mutations at the P3 and P5 positions caused a
significant main chain shift in regions of the HLA-B8-GKK 1 helix
(residues 61–66) and the 2 helix (residues 154–163) creating a
form of "altered self." Analogous disruption of T cell recognition
has been observed following substitution of anchor residues in other
class I-restricted T cell responses, implying that peptide-induced
altered self can significantly modulate T cell recognition
(28).
Most HLA-B8-restricted, FLR-specific CTL were very sensitive to
substitutions in residues P6, P7, and P8 of the FLR peptide located at
the bulged region of the peptide that makes minimal contacts with the
HLA-B8 H chain (13). This region of the peptide almost
certainly plays a direct role in T cell recognition, and given the
relative tolerance to buried substitutions in FLR, this may imply a
high level of TCR complementarity with this region of the peptide
ligand. Preliminary data from a structural analysis of HLA-B8-FLR
cocomplexed with the immunodominant V6.2/DJ2.7-V4.1/J14.3 TCR
suggests that a deep cavity in the TCR combining region envelops the
P7-Tyr residue of the FLR peptide (data not shown and Ref.
29). If confirmed, this finding is completely consistent
with the structural predictions made from the HLA-B8-FLR structure and
previous functional studies (13).
It is notable that HLA-B8-FLR complexes form a dimer, mediated via two
hydrogen bonds: Gln54 (H chain) to
Gln2 (2m) and
Ser88 (H chain) to Glu58 (H
chain) (Fig. 5). Importantly, the dimer
contacts are not directly related to the role cadmium plays in
mediating crystal packing. Moreover Gln54 is
conserved in all HLA-B and most HLA-A and
HLA-C alleles; Ser88 is conserved in
most HLA-B and all HLA-A and HLA-C
alleles; and Glu58 is conserved in most
HLA-B and all HLA-A and HLA-C alleles.
However, none of the numerous structures of MHC class I peptide
complexes reveal dimerization of these MHCp structures with the
Ag-binding sites in a planar orientation (30, 31), despite
an apparent intrinsic ability of class I molecules to form homotypic
associations (32). In contrast with MHC class I
structures, superdimers of MHC class II peptide complexes have been
observed in some crystal structures (reviewed in Refs. 30
and 33), and there is evidence to support a functional
role for these dimeric structures in T cell triggering
(34). Thus, while the data is insufficient to suggest a
generalized higher order arrangement of MHC class I peptide complexes,
it is intriguing that the HLA-B8-FLR complexes reported here form
dimers that, when modeled, could accommodate TCR interactions with both
HLA peptide complexes (not shown). The limited contact area between the
dimer subunits suggests a transient interaction that may be stabilized
by coreceptors at the immunological synapse. Alternatively, the
capacity of MHC class I/2m/peptide complexes
to form dimers may be important during the Ag loading of class I
molecules in the endoplasmic reticulum. The loading of MHC class I
molecules with peptides is a carefully managed biochemical process that
is enhanced by the formation of a multimolecular complex thought to
comprise four MHC class I/2m complexes and
four tapasin glycoproteins assembled with each TAP1/TAP2 heterodimer
(35). The thiol oxidoreductase ERp57 and calreticulin are
also incorporated in the complex and appear to influence the efficiency
of peptide loading (35). Assembly of this complex is
associated with formation of a disulfide bond between tapasin and ERp57
(36), interaction of immature N-glycans on MHC class I
molecules with the lectin calreticulin (37), and undefined
protein-protein interactions between different components in the
complex (35). Thus, the dimeric arrangement of the
HLA-B8-FLR complexes described here illustrate a higher order
configuration of class I peptide complexes that might be important in
formation of the Ag-loading complex or in TCR triggering during priming
or effector phases of immune recognition.
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Acknowledgments |
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Footnotes |
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2 L.K.-N. and C.S.C. contributed equally to this work.
3 Current address: Departamento de Fisica e Biofisica, Instituto de Biociencias, UNESP, Distrito de Rubiao Junior, Botucatu, Sao Paolo, Brazil.
4 Address correspondence and reprint requests to Dr. James McCluskey, University of Melbourne, Department of Microbiology and Immunology, Parkville, Victoria 3010, Australia. E-mail address: jamesm1{at}unimelb.edu.au; or Dr. Jamie Rossjohn, Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3168, Australia. E-mail address: Jamie.rossjohn{at}med.monash.edu.au
5 Abbreviations used in this paper: FLR, Epstein Barr nuclear Ag 3 peptide FLRGRAYGL; GGK, HIV-1 Gag protein p17 residues 24–31 GGKKKYKL; HLA-B8-GGK, HLA-B8-GGK complex; HLA-B8-FLR, HLA-B8-FLR complex; PEG, polyethylene glycol; MHCp, MHC-peptide complex; V6.2/DJ2.7, TCR VDJ combination BV6S2/BSD/BJ2S7; V4.1/J14.3, TCR VJ combination AV4S1/AJ14S3; r.m.s.d., root mean square deviation; 2m, 2-microglobulin.
Received for publication June 19, 2002. Accepted for publication August 23, 2002.
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|
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|
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|
|
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|
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|
|
|
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|
|
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|
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