By
From the * Laboratory of Molecular Biophysics, The Rex Richards Building, Oxford OX1 3QU
United Kingdom; Molecular Immunology Group, Nuffield Department of Clinical Medicine,
Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU United Kingdom;
and § Oxford Centre for Molecular Sciences, New Chemistry Building, Oxford OX1 3QT
United Kingdom
In the cellular immune response, recognition by CTL-TCRs of viral antigens presented as peptides by HLA class I molecules, triggers destruction of the virally infected cell (Townsend, A.R.M., J. Rothbard, F.M. Gotch, G. Bahadur, D. Wraith, and A.J. McMichael. 1986. Cell. 44:959-968). Altered peptide ligands (APLs) which antagonise CTL recognition of infected cells have been reported (Jameson, S.C., F.R. Carbone, and M.J. Bevan. 1993. J. Exp. Med. 177:1541-1550). In one example, lysis of antigen presenting cells by CTLs in response to recognition of an HLA B8-restricted HIV-1 P17 (aa 24-31) epitope can be inhibited by naturally occurring variants of this peptide, which act as TCR antagonists (Klenerman, P., S. Rowland Jones, S. McAdam, J. Edwards, S. Daenke, D. Lalloo, B. Koppe, W. Rosenberg, D. Boyd, A. Edwards, P. Giangrande, R.E. Phillips, and A. McMichael. 1994. Nature (Lond.). 369:403- 407). We have characterised two CTL clones and a CTL line whose interactions with these variants of P17 (aa 24-31) exhibit a variety of responses. We have examined the high resolution crystal structures of four of these APLs in complex with HLA B8 to determine alterations in the shape, chemistry, and local flexibility of the TCR binding surface. The variant peptides cause changes in the recognition surface by three mechanisms: changes contributed directly by the peptide, effects transmitted to the exposed peptide surface, and induced effects on the exposed framework of the peptide binding groove. While the first two mechanisms frequently lead to antagonism, the third has more profound effects on TCR recognition.
Residues 24-31 (GGKKKYKL) of the HIV-1 Gag
protein p17, a region overlapping the nuclear localization site (1), have been mapped as an HLA B8-restricted
epitope capable of eliciting a CTL response in HIV-1 seropositive individuals (2). Variations in the genetic sequence
encoding these residues have been detected in viruses isolated from patients making a CTL response to this epitope
(2, 3). Our present study focuses on four peptides which are
related to the index peptide (GGKKKYKL) by single residue changes corresponding to naturally occurring variant epitope
sequences, each of which has occured in more than one
HLA B8 positive, HIV infected patient (Table 1, denoted
as 3R, 5R, 7R, and 7Q). The index and variant peptides bind
HLA B8 with similar affinities in vitro (4). A number of CTL
clones and lines specific for this epitope have been generated from two HIV positive donors. Fig. 1 shows data from
two clones and a line demonstrating the effects that these
substitutions can have in terms of recognition and antagonism. The differences between the index and the four variant
HLA B8-peptide complexes have been analysed in a series of x-ray crystallographic structure determinations at 2.3 Å resolution or better. Crystallographic statistics for each of
the complexes are detailed in Table 1. In line with the
binding motif deduced from several epitopes and pooled
peptide sequences (5), the index peptide (residues P1-P8) is
anchored in the HLA B8-binding groove by buried lysine
residues at peptide positions P3 and P5 and by the COOHterminal (P8 or PC) leucine residue (see Fig. 2). Conversely, the sidechains of residues P4, P7 and P6, contribute to the surface exposed for TCR recognition. The APLs
thus encompass changes at residues directly exposed to TCR
recognition (P7) and at buried anchor residues (P3 and P5).
Table 1.
Statistics for Crystallographic Structure Determination
Antagonist Assays.
CTL lines and clones were derived and
maintained from donors 008 and 84 by stimulation with the index peptide as previously described (4). Peptides were synthesized
by standard Fmoc chemistry and purity checked by HPLC. Targets were either autologous or HLA matched Epstein-Barr virustransformed B cell lines pulsed with 300 µ Ci51Cr. In antagonist
assays, targets were prepulsed with a suboptimal concentration
(100 nM) of index peptide. Target cells (5 × 103) were then
plated into round-bottomed wells containing experimental peptide or media controls. Effector cells, media, or 5% Triton X-100
were then added to appropriate wells to a total volume of 166 µl.
Supernatant (20 µl aliquots) was then sampled 4 h later to measure
experimental release, spontaneous release, and maximal release.
Specific lysis (SL)1 was then calculated as 100 × (experimental lysis Production and Crystallization.
Production and crystallization
of soluble forms of HLA B*0801-peptide complexes have been
described (6). Briefly, residues 1-276 of the HLA B8 heavy chain
and X-ray Data Collection.
For each complex, a diffraction data set
was collected from a single, cryocooled crystal at the Synchrotron
Radiation Source (SRS), Daresbury using a MAR-research imaging plate system (30 cm diam, Structure Determination and Analysis.
The crystal structure determination, by molecular replacement, of HLA B8 complexed
with the index peptide is detailed elsewhere (Reid, S.W., K.J.
Smith, A. McMichael, J. Bell, D.I. Stuart, and E.Y. Jones, manuscript in preparation). For each variant complex, rigid-body refinement was carried out in the program X-PLOR (10) using the
B8-index complex with peptide coordinates removed as an initial
model. Difference Fourier maps calculated on the basis of these
phases (using programs in the CCP4 suite (11)) showed unambiguous electron density for the entire peptide backbone and all peptide side chains with the exception of the P7 lysines in the 3R
and 5R variants. Electron density maps were displayed and model
coordinates fitted on an Evans and Sutherland (Salt Lake City,
UT) ESV workstation using the interactive computer graphics program FRODO (12). Further rounds of refinement by conjugate gradient minimization, restrained temperature factor, and
simulated annealing using standard X-PLOR protocols (10), interspersed with rebuilding to 2|Fo|-|Fc| All clones tested were highly sensitive to
the arginine substitution at P3 (Fig. 1, a and b), a variant we
have detected in three patients. Only one clone (4) was
able to lyse targets pulsed with high concentrations of peptide. Over 40 other clones and lines from five patients were
unable to recognize the 3R variant (reference 4 and data
not shown). The 3R variant complex is distinguished from
other B8-index and variant peptide complexes by concerted mainchain shifts in the position of portions of the Recognition of the 5R complex varied
considerably between clones. This variant acted as an antagonist towards clone 20 (Fig. 1 c) while clone 18 failed to
distinguish it from the index peptide (Fig. 1 a). The 5R
variant again represents a change of an anchor residue. The
sidechain of the index P5 lysine is deeply buried in the distinctive C pocket of HLA B8 (Fig. 2 and Reid, S.W., K.J.
Smith, A. McMichael, J. Bell, D.I. Stuart, and E.Y. Jones, manuscript in preparation) hydrogen bonding a triad of residues (Asp 9, Asp 74, and Ser 97) at the base of the binding
groove. The arginine residue in the 5R variant complex
also binds within this pocket, maintaining a comparable
hydrogen bond network, but the differences in size and hydrogen bonding geometry of arginine and lysine necessitate
an alteration in the direction of the P5 C The 7R and 7Q variants represent altered
peptide ligands (APLs) with changes at a residue which is
potentially able to interact directly with a TCR. This leads
to abolished functional recognition by clones 18, 20, and
the donor 84 line. The TCRs from line 84 specifically interact with these variants, both of which act as potent antagonists (Fig. 1 d ). In the index complex, the lysine sidechain of the P7 residue is fully exposed to solvent and
relatively mobile, the sidechain amide interacts indirectly
with other protein groups via a water mediated, hydrogen
bond network to the sidechains of HLA residues glutamic
acid 76 and asparagine 80 on the There are a range of conceivable mechanisms by which
minor changes in the sequence of the peptide could affect
TCR recognition, from localized surface perturbations of
amino acid sidechains to concerted mainchain shifts in the
MHC class I-peptide complex. Our analysis of the structural differences between HLA complexes for a series of
APLs demonstrates that subtle local changes are sufficient
to affect TCR recognition. The 7Q and 7R variants, which retain some form of specific recognition for many
CTL clones are, somewhat surprisingly, the only variants
that show chemical differences in the residues directly exposed to the TCR. In this case, however, the surface perturbations are localized and restricted to sidechain atoms
(Figs. 2 and 3). Conversely, amino acid changes at anchor
residues, which result in indirect perturbation of surface characteristics, can have more potent effects. Such effects
clearly do not imply that the residue concerned directly
contacts the TCR, as sometimes assumed. The simple difference in orientation of the P4 residue in the 5R variant
appears sufficient to abolish recognition for one CTL
clone, but does not alter recognition by the second. The
effect of the subtle, but more extensive perturbation in the
3R variant (Fig. 3) appears much more drastic in that recognition is abolished for virtually all CTLs. In contrast to
the other variants, the 3R changes are predominantly to
mainchain atoms. An explanation of these results could be
that sidechains in the contact surface of the 7Q, 7R, and
5R variants may be remolded during the binding of the
TCR. Indeed, altered rigidity of a portion of the surface
could lead to the change in recognition of the 7Q and 7R
variants. The 3R variant produces the most extreme effect on TCR recognition by subtle, but extensive and irreversible (Fig. 3), alterations in the positions of mainchain atoms.
The changes seen are consistent with the hypothesis that
altered peptide ligands produce antagonism by very small
changes, mainly in sidechains which modulate the interaction with the TCR, possibly leading to an increased offrate. Conversely, such subtle changes in the surface of the
MHC class I-peptide complex appear unlikely to change the
geometry of the interaction with the TCR. Thus, these
data favor the altered avidity model for antagonism (for review see reference 19). Indeed, increased TCR off-rates for
antagonist peptide-MHC class II complexes have recently
been reported (20). The more extensive conformational
changes, including MHC mainchain movements seen with
the 3R variant, tend to abolish recognition. This result indicates a direct role for regions of the HLA molecule flanking the peptide in TCR recognition. The combination of
our findings with those of Lyons et al. (20) clearly favors
the models for antagonism whereby the T cell signaling process could amplify affinity differences between agonist
and antagonist MHC-peptide complexes (21, 22)
Data Set
Abbreviated name
B8/GGKKKYKL
Index
B8/GGKKKYRL
7R
B8/GGKKKYQL
7Q
B8/GGKKRYKL
5R
B8/GGRKKYKL
3R
Data collection site
SRS (9.6)
SRS (9.6)
ESRF (BL 4)
ESRF (BL 4)
SRS (9.6)
Total data collected (°)
90.5
120
180
110
90
Unit cell (Å3)
50.6 × 81.4 × 110.7
50.7 × 81.2 × 110.6
50.4 × 80.9 × 109.2
50.6 × 81.3 × 110.1
51.0 × 81.6 × 111.6
Resolution range (Å)
14-2.05
14-2.3
14-2.1
14-2.2
14-2.2
Number of
observations
142,749
119,501
305,517
140,466
88,881
Number of unique
reflections
29,118
25,985
25,086
26,227
27,424
Completeness (%)
98.5
97.6
93.7
96.3
97.7
I/sig(I)
7.3
7.2
5.1
5.7
8.5
Rmerge (%)a
8.3
8.8
8.6
7.6
8.9
R-factor (%)b
18.1
17.1
18.4
18.1
19.5
Number of protein
atoms
3,146
3,148
3,146
3,148
3,140
Number of water
molecules
398
315
364
302
298
Rms bond length
deviation (Å)
0.011
0.011
0.012
0.013
0.012
Rms bond angle
deviation (°)
1.6
1.6
1.7
1.7
1.6
Average B-factor
(mainchain) (Å2)
17.5
16.5
21.1
14.9
21.1
RMS
B (angles)
4.0
4.6
4.7
4.9
5.0
RMS
B (bonds)
2.7
3.4
3.1
3.3
3.4
A diffraction data set was collected for each complex according to the protocol described in Materials and Methods. RMS deviations from ideal values for bond lengths and angles are based on the stereochemical parameters of Engh and Huber (23). For restrained B factor refinement, RMS deviations in B factors are quoted between bond and bond-angle related atoms. aRmerge =
| I
< I >| /
< I > × 100, bR-factor =
| Fobs
Fcalc | /
Fobs × 100 for each data set.
Fig. 1.
CTL recognition and antagonism by naturally occurring p17 variants. Recognition of variant peptides by two donor 008 clones (18 and 20)
(a and b) at an ET of 8:1. (c) Inhibition of killing by clone 20, at an ET of 8:1, by the 3R and 5R variants shown to be encoded for by this provirus (5).
Gag p24 (residues 261-269, GEIYKRWII) was used as a control HLA-B8 restricted peptide. (d ) Inhibition of killing by line 84, at an ET of 4:1, by 7R
and 7Q. Influenza nuclear protein (residues 380-388, ELRSRYWAI) was used as a HLA B8 restricted control peptide.
[View Larger Versions of these Images (14 + 13 + 10 + 12K GIF file)]
Fig. 2.
Crystal structures of the HLA B8-index and variant peptide complexes. The index peptide (P1-P8; GGKKKYKL) in the HLA B8 binding groove (top right) is viewed through the 2 helix with surface delineating the peptide volume in blue and the HLA B8 in green. The basic features of peptide binding are comparable to those observed in other MHC class I-peptide complexes (13, 24). From top left to bottom right, three close up
views depict details of the differences between the index versus 3R, index versus 5R and index versus 7R plus 7Q complexes, respectively. The mainchain of the HLA B8 index complex is shown schematically in green, the peptide and representative HLA B8 sidechains in cyan, and the equivalent residues of the variant complexes are colored red in the 3R and 5R panels, red for 7R, and gold for 7Q in the joint P7 variants panel. Hydrogen bonds are
depicted by appropriately colored dashed lines. In the 3R variant panel, the P5 sidechain is omitted for clarity. The most significant, concerted differences in HLA B8 mainchain positions are observed for the 3R variant (Fig. 3). The yellow arrow indicates the lateral shift in the position of the peptide backbone and consequent repositioning of a portion of the
1 helix spanning residues 61-66 (for this view, the shift is primarily into the plane of the paper).
This region of the
1 helix has previously been observed to flex to accommodate different peptide binding requirements (17, 29). Direct expansion of the
D pocket by movement of the
2 helix may be limited by the disulphide bond between residues 164 on the
2 helix and 101 on the floor of the binding
groove. The figure was produced using programs SYBYL (Tripos Assoc., St. Louis, MO), MOLSCRIPT (34) (with modifications by R. Esnouf), and
RASTER3D (35).
[View Larger Version of this Image (65K GIF file)]
Fig. 3.
Mainchain structural differences between index and variant complexes. Ribbon representations of the index complex are coloured according
to differences in pairwise C superpositions of index and variant complex HLA B8
1 and
2 domains plus peptide (188 structurally equivalent residues). Regions colored green show the least variation in C
position (
0.2 Å) while those in red correspond to changes of
0.5 Å. (a) The 3R variant
complex (overall RMS deviation 0.23 Å). (b) The 5R variant complex (overall RMS deviation 0.26 Å). (c) The 7R variant complex (overall RMS deviation 0.1 Å). (d) The 7Q variant complex (overall RMS deviation 0.1 Å). From these comparisons we estimate that the likely positional errors on main
atom coordinates in all of these structures is less than 0.2 Å. The peptide mainchain position is significantly altered for the 3R variant over residues P1-P4
(0.4-1.1 Å) and residues P3-P5 for the 5R variant (0.4-0.8 Å). For the HLA B8 residues, significant concerted shifts (0.4-0.5 Å) are observed for residues
61-66 of the
1 helix in the 3R variant complex. Changes also occur at residues 159, 162, and 163 of the
2 helix (0.5 Å). For 5R, changes occur at residues 154, 155, and 163 of the
2 helix (0.4-0.5 Å). Figures were produced using programs MOLSCRIPT (34) (with modifications by R. Esnouf ) and
RASTER3D (35). Structural superpositions were made using the program SHP (36).
[View Larger Version of this Image (46K GIF file)]
spontaneous release)/(maximal release
spontaneous release). Inhibition was determined as 100 × (SL without antagonist
SL with antagonist)/(SL without antagonist).
2m were separately expressed in Escherichia coli and refolded
in the presence of the appropriate peptide (Table 1). All the complexes crystallized in closely related unit cells (space group
P212121 with one molecule per asymmetric unit; 6) facilitating a
series of structure determinations. Note that for the index, 7R,
7Q, and 5R complex crystals, the peptide used for refolding was
predominantly a 9 mer (with an additional P9 lysine residue).
Subsequent electron density maps indicate that the crystals contained molecules bearing an 8-mer peptide, and sufficient numbers of 8-mer peptides were found, in analysis of original samples
by mass spectrometry, to be consistent with selective crystallization of 8-mer complexes.
= 0.87 Å), or at the European
Synchrotron Radiation Facility (ESRF) (Grenoble, France) using
a XRII/CCD detector (
= 0.76 Å) (7). ESRF data sets were
corrected for spatial distortion and nonuniformity of response
over the detector system using program FIT2D (8). All data sets
were auto-indexed and integrated with the program DENZO (9).
calc maps, resulted in
the current models for the complexes. A bulk solvent correction,
as implemented in version 3.1 of X-PLOR (10), allowed all measured data to 2.3 Å resolution, or better, to be incorporated into
the refinement and map calculations. In the final stages of refinement, ordered water molecules were added to the complexes.
The final electron density maps are of high quality and omit maps
confirm all the conformational changes discussed in the text including the position of the P7 lysines that were initially unclear.
The mobility of these sidechains is reflected in the refined B-factors.
P3 Variant.
1
and
2 helices which flank the binding groove. In both index and variant complexes, the P3 anchor residue is buried
in the D pocket which is situated at the
2 side of the
binding groove. In HLA B8, as in other HLA class I alleles
(13), one side of the D pocket is formed by residues
Tyr-159, Leu-160, and Tyr-99 while the other side opens
into the central portion of the binding groove (Reid, S.W.,
K.J. Smith, A. McMichael, J. Bell, D.I. Stuart, and E.Y.
Jones, manuscript in preparation). This open pocket allows the P3 lysine residue of the index peptide to hydrogen
bond to residue Asp-156. The shifts in HLA and peptide
mainchain positions between index and variant are triggered by the steric requirements of accommodating the
larger P3 arginine residue in place of the index lysine while
conserving this hydrogen bond (Figs. 2 and 3). The orientations and mobilities (as judged by crystallographic B-factors) of exposed peptide and HLA sidechains are similar in the two complexes. The major differences in the surface
exposed to TCR recognition are therefore the mainchain
shifts in the peptide (0.4, 0.6, 1.1, and 0.8 Å for residues
P1-P4 C
positions) and flanking helices (0.4-0.5 Å for
residues 61-66, 0.5 Å for residues 159, 162, and 163 C
positions). These small changes produce the dramatic alterations in recognition by CTLs.
-C
bond of
some 26° (Fig. 2). This is simply accomplished by a distortion of the P4-P5 peptide mainchain, and the resultant perturbations at the surface of the complex are limited primarily to an altered orientation of the exposed P4 sidechain (Fig. 2).
1 helix (Fig. 2 ). In contrast, for both 7R and 7Q variants, the P7 sidechain is tethered by two direct hydrogen bonds to one or both of these
1 sidechains: the 7R arginine via two of its guanadinium
NH groups to glutamic acid 76, and the 7Q glutamine to
both glutamic acid 76 and asparagine 80. Thus, in addition
to direct changes in charge and shape at the P7 sidechain
position, the mobility of sidechains in this portion of the
surface is altered between index and variant complexes.
Address correspondence to E. Yvonne Jones, Laboratory of Molecular Biophysics, The Rex Richards Building, South Parks Road, Oxford OX1 3QU, United Kingdom.
Received for publication 30 May 1996
This work was supported by the Medical Research Council (MRC) and the Wellcome Trust. The Oxford Centre for Molecular Sciences is supported by the Biotechnology and Biological Sciences Research Council and MRC. E.Y. Jones is supported by the Royal Society, A.J. McMichael and D.I. Stuart by the MRC.We thank M. Pitkeathly and S. Shah for peptide synthesis, T. Willis for protein sequencing and amino acid analysis, C. Robinson for mass spectrometry, E. Garman for help with crystal freezing conditions, the staff at the Synchrotron Radiation Source Daresbury and the European Synchrotron Radiation Facility and European Molecular Biology Laboratory outstation Grenoble for help with x-ray data collection, R. Bryan and R. Esnouf for computing facilities and computer software and S. Lee for help with the preparation of figures. Atomic coordinates for the HLA B8/p17 index and variant peptide complexes have been deposited with the Protein Data Bank, Brookhaven National Laboratory, USA. Prerelease coordinates are available from S.W. Reid or E.Y. Jones; e-mail addresses SCOTT@ BIOP.OX.AC.UK. and YVON@BIOP.OX.AC.UK.
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