Antagonist HIV-1 Gag Peptides Induce Structural Changes in HLA B8

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 ​(Table1,1, 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. ​Fig.11 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 ​Table1.1. 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. ​Fig.2).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

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 Δ B (angles)		4.0		4.6		4.7		4.9		5.0
RMS Δ 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 = Σ ∣ I − < I >∣ / Σ < I > × 100, bR-factor = Σ ∣ Fobs − Fcalc ∣ / Σ Fobs × 100 for each data set.

Open in a separate windowOpen in a separate windowOpen in a separate windowOpen in a separate windowOpen in a separate windowFigure 1CTL 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.Open in a separate windowFigure 2Crystal 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 α2 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 (13–18, 24–33). 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. ​(Fig.3).3). The yellow arrow indicates the lateral shift in the position of the peptide backbone and consequent repositioning of a portion of the α1 helix spanning residues 61–66 (for this view, the shift is primarily into the plane of the paper). This region of the α1 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 α2 helix may be limited by the disulphide bond between residues 164 on the α2 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).