The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 Å) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace the terminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elements at that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable, ΔTm = 16°C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infected individuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a “closed conformation” of the peptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does not functionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes.

Short peptides (∼9 residues) from proteins degraded in the cytoplasm of vertebrate cells are bound by class I MHC molecules in the endoplasmic reticulum and transported to the cell surface for recognition by the Ag-specific receptors (TCR) of T cells as part of the immune system’s surveillance for foreign Ags (1, 2). Any one class I molecule, of the many different alleles expressed in the population, is capable of forming very stable complexes, with half-lives of tens of hours, with a large number of different short peptides. These long-lived MHC/peptide complexes, which can mark cells for destruction by CTL, appear to be kinetic traps for peptides. In vitro, the off-rates of peptides from MHC molecules are very slow (3, 4), whereas peptide association rates vary between experimental systems (5, 6, 7, 8, 9). The kinetics of MHC assembly suggests a two step process involving a conformational change of the MHC molecule from a short-lived, receptive, “open” binding state to a long-lived, “closed” conformation (5, 10, 11, 12, 13).

X-ray crystal structures of MHC molecules have revealed the structure of the closed conformation with peptides bound (14, 15, 16). Some side chains of bound peptides (anchor residues) are held in pockets in the peptide-binding groove that are polymorphic in the different MHC allelic products, providing a sequence-dependent element to peptide binding (16, 17, 18). In class I MHC molecules the charged N and C termini and main chain of the bound peptide are held, through a network of hydrogen bonds and salt bridges, to polar residues conserved in all human and murine class I MHC molecules (19, 20, 21, 22, 23, 24). This network of conserved hydrogen bonds at both termini of peptides provides an independent peptide sequence-independent element to peptide binding. Because these peptide N- and C-terminal contacts to class I MHC molecules are conserved for both all bound peptides and all class I alleles, whereas the peptide side chain contacts to the polymorphic pockets of class I molecules vary considerably with different peptides and different MHC alleles, the conserved interactions at the peptide termini have been proposed to have an important role in forming the shared property of long half-life peptide-bound conformations of class I molecules (19).

The stability of class I MHC molecules, which are heterodimers of a polymorphic heavy chain (Hc)3 and β2-microglobulin (β2m), is strongly dependent on the presence of a bound peptide. In vitro, in the absence of bound peptides, β2m dissociates and Hc aggregates (17, 22, 25, 26, 27, 28, 29), whereas in vivo in the endoplasmic reticulum peptide-free class I molecules are stabilized by chaperonins and the peptide transport and loading proteins, TAP and Tapasin (30, 31). Thermal denaturation studies of MHC/peptide complexes in which either the peptide N-terminal amino group or C-terminal carboxylate group was substituted by a methyl group showed a decrease in the Tm of the MHC/peptide of 22°C, indicating a decrease in stability of ∼4.6 kcal/mol, whereas substituting both peptide anchor residues with alanine showed a decrease of only 5.5°C or ∼1.2 kcal/mol (28, 32). These thermodynamic data support the suggestion that the conserved interactions between the N and C termini of bound peptides may dominate in the formation and stabilization of peptide/MHC complexes.

In this paper we have studied the effect of removing the N-terminal amino acid of an antigenic peptide (Tax9) on the stability and structure of its interaction with HLA-A2, and on its recognition by T cells as an HLA-A2/Tax8 complex. Tax9 is the dominant antigenic peptide inducing cytotoxic T cells in human T cell lymphotrophic virus-1 (HTLV-1)-infected individuals with the neural degenerative disorder HAM/TSP (33). The x-ray structure of the HLA-A2 complex with Tax9 (34) was previously refined to 2.5-Å resolution, whereas the complexes of HLA-A2/Tax9 with two human αβTCRs, A6 and B7, were determined at 2.6-Å resolution (35, 36, 37). Although HLA-A2/Tax8 activates cytotoxic T cells in HTLV-1-infected individuals (38), we find that it does not cross-react functionally with HLA-A2/Tax9-specific T cells in cell lysis assays and has a low affinity for a HLA-A2/Tax9-specific TCR. The stability of the HLA-A2/Tax8 complex is markedly reduced, as expected for loss of the conserved interactions at the N-terminal peptide-binding site, but the structure of the complex is remarkably similar with water molecules substituting for some of the peptide interactions in the binding site.

The extracellular region of the HLA-A2 Hc and β2m were expressed separately in Escherichia coli as inclusion bodies (39). The inclusion bodies were refolded together in the presence of excess Tax8 (LFGYPVYV) or Tax9 (LLFGYPVYV) peptide. Briefly, milligram amounts of β2m, Hc, and peptide were injected into 500 ml of a refolding buffer (100 mM Tris, 400 mM arginine-HCl, 2 mM NaEDTA, 0.5 mM oxidized glutathione, and 5 mM reduced glutathione, pH 8.3). The final concentrations of the constituents of the complexes were 2 μM β2m, 1 μM Hc, and 50 μM peptide (Tax9). A significantly larger excess of Tax8 peptide (100–150 μM) was required to produce that complex in sufficient quantity for biochemical studies, and the resulting yield was very low (∼5%) compared with typical yields with Tax9 (20–25%). The ternary complexes were purified by ion-exchange and gel filtration chromatography as described previously (34).

Crystals of Tax9-A2 were obtained by vapor diffusion from hanging drops containing equal volumes of protein (5 mg/ml; 25 mM MES, pH 6.5) and 13–20% polyethylene glycol (PEG) 6000 (25 mM MES, pH 6.5). Tax8-A2 crystals were obtained by seeding with Tax9-A2 crystals. A 3-μl solution of seed crystals was incubated with a 3-μl solution containing 3 mg/ml Tax8-A2 and 400 μM Tax8 peptide in 25 mM MES (pH 6.4) and 0.1% NaN3. The drop was equilibrated against 13% PEG 6000, 25 mM MES, and 0.1% NaN3 at 18°C.

Crystals of Tax8-A2 were transferred to a 20% glycerol solution in steps of 4–10% and flash-cooled in a stream of cryo-cooled nitrogen gas. Data were collected on a Mar345 detector (Mar Research, Hamburg, Germany) mounted on an Elliot GX-13 x-ray generator (GEC Avionics, London, U.K.). The structure of Tax8-A2 was refined by using the previously determined structure of Tax9-A2 as the starting model (Protein Data Bank code 1hhk; Ref. 34). A subset (10%) of the reflection data were flagged (R-free) and excluded from refinement protocols. As the crystals of Tax8-A2 and Tax9-A2 were isomorphous, the same set of flagged reflections previously used for monitoring Tax9-A2 refinement was used in the R-free data set for Tax8-A2. Because the structure of Tax9-A2 was previously refined to only 2.5-Å resolution (without waters), the data were extended to 1.8 Å and the model refined to that resolution with the inclusion of water molecules.

The starting model of Tax8-A2, stripped of water molecules and the peptide was initially subjected to rigid-body fitting using the program CNS (40). Electron density maps clearly indicated the position of Tax8 within the peptide-binding groove of HLA-A2. The structure was further refined by multiple cycles of energy minimization and model building using the programs CNS and O (40, 41). All reflections between 50 Å and the resolution limits of the data sets (2.14 Å for Tax8-A2, 1.8 Å for Tax9-A2) were used during refinement and electron density map calculations (Table I). Water molecules were gradually introduced during the course of model building and were selected from >3ς peaks in difference (FoFc) electron density maps within 2.5–3.6 Å of hydrogen bond donors or acceptors.

Table I.

Crystallographic dataa

Tax8-A2Tax9-A2
Space group P1 P1 
Molecules in AU 
Cell parameter a = 50.34 Å, b = 62.86 Å, c = 74.84 Å a = 50.56 Å, b = 63.79 Å, c = 75.08 Å 
 α = 82.01°, β = 76.1°, γ = 77.96° α = 81.58°, β = 75.66°, γ = 77.38° 
Total reflections 60,478 137,168 
Rmerge (%) 4.7 4.2 
Resolution (Å) 50-2.15 50-1.80 
Unique reflections 38,048 72,184 
Completeness (%) 78.0 85.3 
FF > 3.0 (2.26-2.15 Å) 38.3% (1.94-1.80 Å) 59.9% 
 (2.26-2.15 Å) 33.2% (1.94-1.80 Å) 45.4% 
Tax8-A2Tax9-A2
Space group P1 P1 
Molecules in AU 
Cell parameter a = 50.34 Å, b = 62.86 Å, c = 74.84 Å a = 50.56 Å, b = 63.79 Å, c = 75.08 Å 
 α = 82.01°, β = 76.1°, γ = 77.96° α = 81.58°, β = 75.66°, γ = 77.38° 
Total reflections 60,478 137,168 
Rmerge (%) 4.7 4.2 
Resolution (Å) 50-2.15 50-1.80 
Unique reflections 38,048 72,184 
Completeness (%) 78.0 85.3 
FF > 3.0 (2.26-2.15 Å) 38.3% (1.94-1.80 Å) 59.9% 
 (2.26-2.15 Å) 33.2% (1.94-1.80 Å) 45.4% 
a

Rmerge = ΣhkljIhklj − <Ihklj>‖Σhklj<Ihkl> × 100%.

During the course of refinement, it was observed that the N-terminal leucine residue (Leu2) of Tax8 has a backbone conformation that is distinct from that residue in Tax9, resulting in a net shift of 1.0 Å in the position of the α-amino group of Tax8. Simulated annealing omit maps, in which the peptide and surrounding water molecules were removed, confirmed that the N terminus was both well positioned and ordered in both molecules of the asymmetric unit. The conformation of Leu2 precludes formation of a salt bridge with Glu63 of the B pocket; in the Tax9 structure, the identical amide nitrogen hydrogen bonds to Glu63. To further test the validity of the Tax8 model, one further cycle of positional refinement was performed in which the α-amino group was manually re-fitted by adjustment of torsion angles to within 3.2 Å of the side-chain of Glu63 (Oε1). Following crystallographically restrained energy minimization in the CNS program, it was again observed that the N terminus shifted away, to a position 3.6 Å from Glu63 (Oε1), but to within 3.29 Å of the hydroxyl group of Tyr159. The statistics from the final cycle of refinement are shown in Table II. Coordinates and structure factors have been submitted to the Protein Data Bank (A2-Tax8 and A2-Tax9 PDB codes 1DUY and 1DUZ, respectively).

Table II.

Refinement statistics

Tax8Tax9
Resolution (Å) 50-2.15 50-1.8 
Rfree (%)a 25.41 (2.26-2.2 Å) 28.7% 25.02 (1.86-1.8 Å) 30.7% 
Rcryst (%)a 19.18 (2.26-2.2 Å) 21.6% 19.77 (1.86-1.8 Å) 24.9% 
rms deviations from ideality   
Bonds (Å) 0.01 0.03 
Angles (°) 1.6 2.4 
Dihedrals (°) 25.3 26.2 
Improper (°) 0.9 1.6 
Luzzati e.s.d. (Å)b 0.23 0.21 
Protein atoms 6306 6322 
Water molecules 496 607 
Ramachandran mapc   
Most favored (%) 89.3 93.2 
Disallowed (%) 
Tax8Tax9
Resolution (Å) 50-2.15 50-1.8 
Rfree (%)a 25.41 (2.26-2.2 Å) 28.7% 25.02 (1.86-1.8 Å) 30.7% 
Rcryst (%)a 19.18 (2.26-2.2 Å) 21.6% 19.77 (1.86-1.8 Å) 24.9% 
rms deviations from ideality   
Bonds (Å) 0.01 0.03 
Angles (°) 1.6 2.4 
Dihedrals (°) 25.3 26.2 
Improper (°) 0.9 1.6 
Luzzati e.s.d. (Å)b 0.23 0.21 
Protein atoms 6306 6322 
Water molecules 496 607 
Ramachandran mapc   
Most favored (%) 89.3 93.2 
Disallowed (%) 
a

Rcryst (Rfree) = (ΣhFoFc‖/(ΣFo). Rfree calculated using 10% of the reflections, randomly generated and excluded from refinement protocols.

b

Estimated coordinate accuracy of the structures from the method of Luzzati (51).

c

Analyzed using the program PROCHECK (52).

The thermal stability of peptide-MHC complexes was monitored by CD spectroscopy using a Jasco J-710 instrument (Jasco International, Tokyo, Japan) equipped with a Peltier temperature regulator. Solution conditions were 20 mM phosphate and 75 mM NaCl (pH 7.4). Protein concentrations were ∼0.15 mg/ml. The spectrum between 250 and 190 nm indicated a predominantly β-sheet conformation. Temperature denaturation was monitored at the minimum of 218 nm between 10 and 90°C using a gradient of 1°C/min. Scans were repeated twice with fresh protein and the data points averaged. Rather than constraining the data to a two-state unfolding model, the data in the transition region were simply fit to a nine-order polynomial equation, and the apparent Tm was determined from the maximum of the first derivative of the fitted curve.

The 2G4 T cell clone has a TCR (A6-TCR) specific for Tax9 complexed with HLA-A2, and the interaction of this receptor with a number of altered peptides complexed with HLA-A2 has been previously investigated (37, 42). The binding of Tax8-A2 to the A6-TCR was investigated here using an equilibrium BIAcore assay as described previously (37). Briefly, recombinant A6-TCR with a single free thiol at the C terminus of the β-chain was coupled to a CM5 sensor chip using standard thiol coupling. Multiple concentrations of Tax8-A2 were injected at a flow rate of 10 μl/min, and the responses at equilibrium (∼400 s after injection) were determined. The temperature of the sample was maintained at 4°C, and Tax8-A2 dilutions were made from a highly concentrated stock immediately before injection to minimize dissociation of the Tax8 peptide from HLA-A2. The responses from injections over a mock surface were subtracted from the data, and all injections were repeated twice. Equilibrium responses were fit against the concentration of injected Tax8-A2, assuming a single-site binding model. Solution conditions were 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% polysorbate-20 (pH 7.4) at 25°C.

T cell-mediated cytotoxicity was quantitated by a time-resolved fluorometric assay using HLA-A2-transfected cells as targets as previously described (36). Effector cells were the A6-TCR-expressing CD8+ T cell clone 2G4 that recognizes the Tax11–19 peptide presented by HLA-A2 and was isolated from a patient with HAM/TSP (43).

For the antagonism assay, HLA-A2-transfected Hmy2.C1R cells were treated with 100 μg/ml mitomycin C (Sigma, St. Louis, MO) for 2 h at 37°C, washed three times with PBS, and then pulsed with 1000 nM of candidate antagonist peptides for 2 h at 37°C. The cells were washed, and 1 × 105 cells were incubated with 1 × 105 2G4 T cells for 1 h at 37°C. Tax peptide was added at concentrations of 0.1–10 nM, and the cells were incubated for 48 h. Supernatants were collected and assayed for IFN-γ content as previously described (36).

The structures of Tax8-A2 and Tax9-A2 were refined to reasonable agreement between observed and calculated structure factors, as well as good stereochemistry and geometry (Table II). Although data extending to 2.15 Å were used in the refinement of Tax8-A2, the data were incomplete in the highest resolution shell (Table I). The number of observed reflections corresponds to a 100% complete data set to 2.30-Å resolution. The overall structure of the Tax8-A2 complex is identical to Tax9-A2 and is shown in Fig. 1. The quality of the structures permits a detailed discussion of the peptide-binding groove and its bound solvent.

FIGURE 1.

Molecular structure of Tax8-A2. The overall architecture is identical with the Tax9-A2 structure. Upper, The various domains are differentiated by color. The position of the peptide (red) and three water molecules that occupy the A site of the groove are shown. The transmembrane anchor would extend down from the green (α3) domain of Hc into the lipid bilayer. Lower, View of Tax8 within the MHC groove. The view is looking down from the “top” of the upper panel. The images were created with MOLSCRIPT (53 ).

FIGURE 1.

Molecular structure of Tax8-A2. The overall architecture is identical with the Tax9-A2 structure. Upper, The various domains are differentiated by color. The position of the peptide (red) and three water molecules that occupy the A site of the groove are shown. The transmembrane anchor would extend down from the green (α3) domain of Hc into the lipid bilayer. Lower, View of Tax8 within the MHC groove. The view is looking down from the “top” of the upper panel. The images were created with MOLSCRIPT (53 ).

Close modal

The conformation and position of Tax8 within the peptide-binding groove of HLA-A2 is identical to the structure observed in Tax9-A2 (Fig. 2). Hydrogen bonds and hydrophobic contacts are preserved from Phe3 to Val9 of Tax8 and the corresponding MHC binding pocket, indicating that loss of the position 1 Leu (P1) does not induce global shifts or conformational changes in the peptide or MHC. Following alignment of the HLA Hc, the root-mean-square difference in the coordinates of the common atoms of Tax8 and Tax9 was 0.36 Å. However, the α-amino group of Leu2 (Tax8) is positioned about 1 Å from the equivalent P2 amide nitrogen of Tax9 (see Materials and Methods), making a hydrogen bond with the hydroxyl of Tyr159 rather than Glu63 as in Tax9 (compare Fig. 3, a and b). The distance between the side chain of Glu63 and the P2 amino group of Tax8 is 3.9 Å, which may still permit a favorable electrostatic interaction.

FIGURE 2.

Least-squares superimposition of Tax8-A2 and Tax9-A2. Only the peptides and conserved water molecules are shown. Tax9 is colored green to distinguish the molecule from Tax8. Superimposition was performed using only the backbone atoms of the Hc (1100 atoms), giving a root-mean-square deviation of 0.33 Å. The image was created with MOLSCRIPT (53 ).

FIGURE 2.

Least-squares superimposition of Tax8-A2 and Tax9-A2. Only the peptides and conserved water molecules are shown. Tax9 is colored green to distinguish the molecule from Tax8. Superimposition was performed using only the backbone atoms of the Hc (1100 atoms), giving a root-mean-square deviation of 0.33 Å. The image was created with MOLSCRIPT (53 ).

Close modal
FIGURE 3.

Hydrogen bonding interactions at the N-terminal anchor region. a, Tax8 is represented as a ball-and-stick model. The peptide bond vectors are green, and atom color corresponds to atom type. The A2 side chains and secondary structure elements are light blue; water molecules are magenta. The peptide is truncated at Phe3. b, Hydrogen bonding interactions at the N-terminal region of Tax9. The color scheme is the same as in a and a similar orientation is shown. The images were created with MOLSCRIPT (53 ).

FIGURE 3.

Hydrogen bonding interactions at the N-terminal anchor region. a, Tax8 is represented as a ball-and-stick model. The peptide bond vectors are green, and atom color corresponds to atom type. The A2 side chains and secondary structure elements are light blue; water molecules are magenta. The peptide is truncated at Phe3. b, Hydrogen bonding interactions at the N-terminal region of Tax9. The color scheme is the same as in a and a similar orientation is shown. The images were created with MOLSCRIPT (53 ).

Close modal

In the Tax8 complex two water molecules, Wat-1 and Wat-2, partially fill the space occupied by the P1 peptide residue, Leu1, in the Tax9 complex (Fig. 3). Wat-1 provides a bridge from the N terminus of Tax8, via hydrogen bonds, to the carboxylate group of Glu63 (Fig. 3). Wat-2 is in the position that is occupied by the α-amino group of Leu1 in Tax9 (Fig. 3, a and b). This water provides a bridge from the hydroxyl group of Tyr7, in the β-sheet forming the floor of the peptide-binding groove, via hydrogen bonds to Tyr171 in α-helix forming one side of the binding site (Fig. 3). In the Tax9 complex, the same hydrogen bonded bridge between elements of secondary structure is made through the amino group at Leu1 of the bound peptide (Fig. 3,b). Wat-2 is also within hydrogen bonding distance of Wat-1 (distance of 2.82 Å; Fig. 3,a) and a third water (Wat-3) found in both the Tax8 and Tax9 complexes (Fig. 3).

The hydrogen bonds in the peptide N-terminal region of the binding groove for the Tax8-A2 and Tax9-A2 structures are summarized in Table III. Although in the Tax8 complex there is a loss of three hydrogen bonds that went directly from the bound peptide to the MHC molecule relative to Tax9 complex, the shift in the nitrogen position of Tax8 and the addition of two water molecules creates a network of hydrogen bonds that both satisfies all of the hydrogen bonding acceptors and donors of the conserved MHC residues and cross-links together the same secondary structure elements as in the Tax9 complex.

Table III.

Hydrogen bonds in the peptide N-terminal region of the binding groovea

PeptideWaterHeavy Chain
Tax9-A2 structure    
Leu1 (N)  Tyr7 (OH) (2.82 Å) 
Leu1 (N)  Tyr171 (OH) (2.84 Å) 
Leu1 (O)  Tyr159 (OH) (2.71 Å) 
Leu2 (N)  Glu63 (OE1) (2.91 Å) 
Leu2 (O)  Lys66 (NZ) (3.02 Å) 
Tax8-A2 structure    
Leu2 (N) Wat-1 (3.04 Å)   
 Wat-1 Glue63 (3.02 Å) 
Leu2 (N)  Tyr159 (OH) (3.29 Å) 
Leu2 (O)  Lys66 (NZ) (3.32 Å) 
 Wat-2 Tyr7 (OH) (2.53 Å) 
 Wat-2 Tyr171 (OH) (2.72 Å) 
PeptideWaterHeavy Chain
Tax9-A2 structure    
Leu1 (N)  Tyr7 (OH) (2.82 Å) 
Leu1 (N)  Tyr171 (OH) (2.84 Å) 
Leu1 (O)  Tyr159 (OH) (2.71 Å) 
Leu2 (N)  Glu63 (OE1) (2.91 Å) 
Leu2 (O)  Lys66 (NZ) (3.02 Å) 
Tax8-A2 structure    
Leu2 (N) Wat-1 (3.04 Å)   
 Wat-1 Glue63 (3.02 Å) 
Leu2 (N)  Tyr159 (OH) (3.29 Å) 
Leu2 (O)  Lys66 (NZ) (3.32 Å) 
 Wat-2 Tyr7 (OH) (2.53 Å) 
 Wat-2 Tyr171 (OH) (2.72 Å) 
a

Only hydrogen bonds differing between the Tax8-A2 and Tax9-A2 structures are listed.

The absence of the P1 peptide residue apparently causes some small conformational changes in HLA-A2. The indole ring of Trp167 and the side chain of Glu163 are about 1 Å closer to the space that would be occupied by Leu1 of Tax9 (data not shown). Despite these observed structural differences at the binding site for the peptide N terminus, the side chain of the P2 peptide residue, Leu2, occupies the identical hydrophobic pocket in both Tax8 and Tax9 complexes, forming contacts with Met45 and Phe9 in the B pocket (16).

In the middle portion of the peptide, three buried water molecules form part of the interface between Tax8 and Hc (Wat-4, Wat-5, Wat-6; Fig. 4). These waters are also present in the structure of Tax9-A2 refined here, but were not visible in the previously published, lower resolution (2.5 Å) structure (34). The carbonyl oxygen of Tyr5 is linked via Wat-4 to the ε-amino group of Arg97. The carbonyl oxygen of Val7 is linked via Wat-6 to the carboxylate oxygen of Asp77. These water-mediated, hydrogen bond bridges are formed with the part of the peptide that is central to recognition by TCRs (35, 37). Upon binding of both the A6- and B7-TCRs, a conformational change takes place in the Tax9 peptide such that the side chain of Pro6 becomes buried in the pocket occupied by two of these water molecules (35), and the Val7 side chain projects outward toward the TCR. Consequently, two water molecules, Wat-4 and Wat-5, must be displaced upon TCR binding. Another water molecule, Wat-7, bridges the C-terminal carboxylate of the peptide via hydrogen bonds to the side chain of Thr80 (Oγ1) in both the Tax8-A2 and Tax9-A2 structures.

FIGURE 4.

Water-mediated hydrogen bonds between Tax8 and the MHC groove. Water molecules (magenta) are represented as spheres, and the side chains of A2 are green. The image was created with MOLSCRIPT (53 ).

FIGURE 4.

Water-mediated hydrogen bonds between Tax8 and the MHC groove. Water molecules (magenta) are represented as spheres, and the side chains of A2 are green. The image was created with MOLSCRIPT (53 ).

Close modal

The thermal denaturation curves of Tax8-A2 and Tax9-A2 were determined by monitoring the loss of secondary structure using CD. The mid-point of the transitions were 49°C for Tax8-A2 and 65°C for Tax9-A2 (Fig. 5), indicating a significant destabilization of the peptide-A2 complex following loss of the N-terminal residue of the peptide. A second transition observed in the denaturation curves beginning near 80°C corresponds to the unfolding of β2m (27, 32). Consequently, the early loss of secondary structure observed for Tax8-A2 is attributed to loss of the peptide and unfolding of the Hc, as previously shown (27, 32). Due to linkage between peptide binding and stable folding of the Hc/β2m heterodimer, it follows that Tax8 binds the A2 molecule with a much lower affinity than Tax9 (28, 44). These results are consistent with peptide-binding studies of the murine class I MHC H2-Kd, in which deletion of the N-terminal residue of a nonomer peptide significantly reduced the stability of the MHC/peptide complex (45).

FIGURE 5.

CD thermal denaturation profiles of Tax8-A2 and Tax9-A2. The apparent Tm values given by the inflection points are indicated by solid diamonds. The y-axis for both data sets was normalized for illustrative purposes and does not indicate percent completion of the unfolding transition (see Materials and Methods).

FIGURE 5.

CD thermal denaturation profiles of Tax8-A2 and Tax9-A2. The apparent Tm values given by the inflection points are indicated by solid diamonds. The y-axis for both data sets was normalized for illustrative purposes and does not indicate percent completion of the unfolding transition (see Materials and Methods).

Close modal

Binding affinity of Tax8-A2 to the A6-TCR, specific for HLA-A2/Tax9, was measured using an equilibrium plasmon resonance experiment. The A6-TCR clone, isolated from a patient with the HTLV-1-associated autoimmune disorder HAM/TSP, is a class I-restricted TCR specific for Tax9 bound to HLA-A2 (43). Equilibrium binding experiments (Fig. 6 A) revealed that the association of the A6-TCR with Tax8-A2 is about 16-fold weaker (KD = 15 ± 2 μM) relative to the full-length Tax9 peptide-MHC complex (KD = 0.9 ± 0.1 μM; Ref. 37). Although we attempted to minimize such contributions during the experiment, it is possible that this result is influenced slightly by dissociation of Tax8 from the MHC (see Materials and Methods).

FIGURE 6.

T-cell receptor binding and T cell activation. A, Equilibrium binding of Tax8-A2 to the A6-TCR performed using a BIAcore assay as described. B, Sensitization of HLA-A2 target cells by Tax9 (▴) and Tax8 (□) to lysis by the A6-TCR bearing CTL clone 2G4 (E:T = 2.5:1). A control peptide (influenza M1 57–68) that lacks biological activity with the A6-TCR is shown as a reference (▪). C, Tax8 does not antagonize A6-bearing T-cells for IFN-γ production by Tax9. Tax-P6A, a known antagonist, is a positive control (37 ), and M1 58–66 is a negative control.

FIGURE 6.

T-cell receptor binding and T cell activation. A, Equilibrium binding of Tax8-A2 to the A6-TCR performed using a BIAcore assay as described. B, Sensitization of HLA-A2 target cells by Tax9 (▴) and Tax8 (□) to lysis by the A6-TCR bearing CTL clone 2G4 (E:T = 2.5:1). A control peptide (influenza M1 57–68) that lacks biological activity with the A6-TCR is shown as a reference (▪). C, Tax8 does not antagonize A6-bearing T-cells for IFN-γ production by Tax9. Tax-P6A, a known antagonist, is a positive control (37 ), and M1 58–66 is a negative control.

Close modal

The biological activity of the Tax8 peptide was measured in a specific lysis (cytotoxicity) assay (Fig. 6,B). The data show that, compared with the wild-type Tax9 peptide, Tax8 has a very limited ability to activate A6-TCR-bearing T cells (barely detectable lysis at a million-fold increase in peptide concentration). In addition, no TCR antagonism (vs Tax9) was detected with Tax8 peptide (Fig. 6 C). However, Tax8 could sensitize targets cells for lysis by some Tax-specific CTL (38).

Until now, all the structures of class I MHC molecules with bound peptides determined by x-ray crystallography, whether the peptides were 8-mer, 9-mer, or 10-mer, have had a peptide residue in the P1 binding site making the conserved array of hydrogen bonds to nonpolymorphic MHC residues (Fig. 3 b). In the human class I molecule HLA-B35, a short peptide was accommodated by stretching out a kink usually found in longer peptides (46), and in murine class I molecules the shape of the bottom of the binding site is conducive to the binding of shorter peptides (21, 47). The Tax8/Tax9 pair of peptides were studied here, because it seemed likely that the 8-mer would bind just like the 9-mer, except with the P1 site empty. This offered the opportunity to see whether the loss of the conserved hydrogen bonds, which both held bound peptides in the site and tethered the secondary structures of the binding site together, would affect the conformation of the binding site. In particular, we hoped to see whether the binding site might adopt a partially “open state” as proposed to exist from the slow binding and dissociation kinetics of peptides.

In the Tax8 complex, as a result of no peptide residue occupying the P1 binding site, we observed a loss of three hydrogen bonds directly from the bound peptide to the MHC molecule, relative to the Tax9 complex. However, the shift in the primary amine position of Tax8, and the addition of two water molecules, created a new network of hydrogen bonds that satisfies all of the hydrogen bonding acceptors and donors of the conserved MHC residues (Fig. 3). Furthermore, the new network of hydrogen bonds in the Tax8 complex cross-links together the same secondary structure elements as in the Tax9 complex. Thus, although the structure indicates that the Tax8 peptide might be expected to bind more weakly than Tax9, due to the loss of the three conserved hydrogen bonds from peptide to MHC, the restoration of a hydrogen bonding network using water molecules apparently serves to stabilize the “closed conformation” of the MHC binding site.

In previous studies with a different peptide, the influenza virus matrix peptide (GILGFVFTL), the N-terminal amino group of the peptide was replaced, synthetically, by a methyl group (32). The modified N terminus prevented the formation of two of the hydrogen bonds that group normally makes with conserved residues of the MHC molecule (28). This loss of interactions resulted in a decrease of the thermal denaturation temperature of the peptide/HLA-A2 complex of 21°C, corresponding to a ΔΔG° value of about 4.6 kcal/mol (28, 32). A structure of the complex revealed that the substituted methyl group rotated away from the hydrogen bonding groups of the MHC molecule and was replaced by a water molecule occupying the position vacated by the peptide N terminus (28) and located identically to water Wat-2 observed in the Tax8 complex (Fig. 3 a). The bound water molecule formed a similar set of hydrogen bonds to the conserved MHC residues as the full-length 9-residue matrix and Tax peptides, but did not provide any bridging hydrogen bonds to the modified peptide. Apparently the loss of these two hydrogen bonds to the N-terminally modified peptide resulted in the large (∼4.6 kcal/mol) decrease in stability of the peptide/MHC complex.

The thermal denaturation temperature of the Tax8 complex was observed in this paper to be 16°C lower than that of the Tax9 complex. This difference also presumably results partly from the loss of direct hydrogen bonds between the peptide and MHC molecule. Although the bridging water molecules Wat-1 and Wat-2 form a network of hydrogen bonds, the new hydrogen bonds do not directly link the peptide to the MHC molecule (Fig. 3 a). Thus, as in the earlier study of the modified matrix peptide/MHC complexes, eliminating the conserved hydrogen bonds from the peptide N terminus to the MHC molecule in the Tax8 complex also very substantially decreased the stability of the MHC molecule. In both cases, despite drastic changes in the stability of the MHC molecules, when hydrogen bonding interactions with the peptide were removed, the conformation of the peptide-binding site of the destabilized molecules was not changed. Instead, in both cases, water molecules were observed to bind and created networks of hydrogen bonds that apparently maintained the binding site structure. This suggests the possibility that even an empty binding site, devoid of peptide, which is quite unstable relative to peptide/MHC complexes (27), might maintain this closed conformation as the result of binding water molecules at its ends. “Closed-empty” and “closed-full” binding sites may look similar and dominate at equilibrium, with “open-empty” and “open-full” states being more transient states separated from the closed states by the high free energy barriers responsible for the slow association and dissociation kinetics. It is these transient states that enzyme-like molecules must stabilize in vivo to accelerate peptide loading.

HTLV-1 Tax8 peptide-specific HLA-A2-restricted CTL cell lines have been generated from PBL of patients with HTLV-1-associated neurological disease (38). The x-ray structure determined here of the Tax8/HLA-A2 complex establishes the P1 peptide pocket is empty, so that the TCR of Tax8-specific CTL must bind to a peptide/MHC complex with no P1 peptide side chain.

The x-ray structures of two TCR specific for Tax9/HLA-A2 have been determined complexed with Tax9/HLA-A2 (35, 36, 37). In both cases the major contacts between TCR and peptide are with peptide residues P4 to P8, but the TCR does cover and make one atomic contact to the P1 sidechain (Leu1). If we model a TCR interaction with Tax8/HLA-A2 by superimposing the MHC/peptide coordinates from the Tax8/MHC structure with the Tax9/MHC/TCR structure, then the solvent accessible surface area of the MHC/peptide buried by the TCR would be decreased by 194 Å2 (20%) as the result of deleting P1. However, a Tax8-specific TCR might fill in this cavity with either a different CDR1α sequence or conformation.

In published studies, it is the kinetic off-rate of the TCR from the MHC/ligand complex that most often correlates with the nature of the T cell response (48). The kinetic off-rate of A6-TCR with Tax9/HLA-A2 is 0.093 s1 (37, 42), in the range of a typical agonist. The kinetic off-rate of the A6-TCR with Tax8/HLA-A2 was too fast to measure by surface plasmon resonance, consistent with the extremely low T cell response observed (Fig. 6, B and C).

The affinity for a Tax9 specific αβTCR ectodomain for the Tax8/HLA-A2 complex was measured as 16-fold lower than its affinity for Tax9/HLA-A2 (Fig. 6,A). This affinity difference is at least qualitatively consistent with the modeling that indicates a potential cavity in the interface. A 16-fold magnitude of affinity difference in other TCR/peptide/MHC complexes has been observed to be sufficient to alter T cell responses dramatically (37, 42, 49, 50). Qualitatively consistent with this expectation, we observed markedly reduced (>106 reduction) activity in Tax8-induced cell lysis of a Tax9-specific T cell line (Fig. 6,B) and no antagonism of Tax9 activity at 1 μM Tax8 peptide concentration (Fig. 6 C). Quantitatively, however, the absolute affinity, KD = 15 ± 2 μM, of A6-TCR for the Tax8/HLA-A2 ligand, is in the range that often either signals as a partial agonist or an antagonist (37, 42), rather than as a null as observed here. This comparison suggests that the Tax8/HLA-A2 complex may have unusual properties as a cell surface ligand for a T cell. Apparently the decreased stability of the Tax8/MHC complex (Tm = 49°C), coupled with the 16-fold decrease in TCR affinity, combine somehow to decrease the effectiveness of the Tax8/MHC complex as a ligand in cellular assays (since neither alone prevents either partial agonist or antagonist responses).

We thank R. Crouse, A. Haykov, and C. Garnett for technical support.

1

A.R.K. is supported by a Human Frontiers Long Term Fellowship. B.M.B. is supported by a fellowship from the Cancer Research Institute. D.C.W. is an investigator of the Howard Hughes Medical Institute.

3

Abbreviations used in this paper: Hc, heavy chain; β2m, β2-microglobulin; Tm, melting temperature; HTLV-1, human T cell lymphotrophic virus-1; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; CD, circular dichroism.

1
Falk, K., O. Rötzschke, S. Stevanovic, G. Jung, H.-G. Rammensee.
1991
. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules.
Nature
351
:
290
2
Townsend, A., H. Bodmer.
1989
. Antigen recognition by class I-restricted T lymphocytes.
Annu. Rev. Immunol.
7
:
601
3
Buus, e. a..
1986
. Isolation and characterization of antigen-Ia complexes involved in cell recognition.
Cell
47
:
1071
4
Stern, L., D. C. Wiley.
1992
. The human class II MHC protein HLA-DR1 assembles as empty heterodimers in the absence of antigenic peptide.
Cell
68
:
465
5
Springer, S., K. Doring, J. C. Skipper, A. R. Townsend, V. Cerundolo.
1998
. Fast association rates suggest a conformational change in the MHC class I molecule H-2Db upon peptide binding.
Biochemistry
37
:
3001
6
Matsumura, M., Y. Saito, M. R. Jackson, E. S. Song, P. A. Peterson.
1992
. In vitro peptide binding to soluble empty class I major histocompatibility complex molecules isolated from transfected Drosophila melanogaster cells.
J. Biol. Chem.
267
:
23589
7
Burshtyn, D. N., D. H. Barber.
1993
. Dynamics of peptide binding to purified antibody-bound H-2Db B2 M complexes.
J. Immunol.
151
:
3082
8
Boyd, L. F., S. Kozlowski, D. H. Margulies.
1992
. Solution binding of an antigenic peptide to major histocompatibility complex class I molecule and the role of β2-microglobulin.
Proc. Natl. Acad. Sci. USA
89
:
2242
9
Olsen, A. C., L. O. Pederson, A. S. Hansen, M. H. Nissen, M. Olsen, P. R. Hansen, A. Holm, S. Buus.
1994
. A quantitative assay to measure the interaction between immunogenic peptides and purified major histocompatibility complex molecules.
Eur. J. Immunol.
24
:
385
10
Sadegh-Nasseri, S., S. McConnell.
1989
. A kinetic intermediate in the reaction of an antigenic peptide and I-Ek.
Nature
337
:
274
11
Sadegh-Nasseri, S., L. J. Stern, D. C. Wiley, R. N. Germain.
1994
. MHC class II function preserved by low-Affinity peptide interactions preceding stable binding.
Nature
370
:
647
12
Rabinowitz, J., et al
1998
. Formation of a highly peptide-receptive state of class II MHC.
Immunity
9
:
699
13
Joshi, R. V., J. A. Zarutskie, L. J. Stern.
2000
. A three-step kinetic mechanism for peptide binding to class II proteins.
Biochemistry
39
:
3751
14
Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, D. C. Wiley.
1987
. Structure of the human class I histocompatibility antigen, HLA-A2.
Nature
329
:
506
15
Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, D. C. Wiley.
1987
. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens.
Nature
329
:
512
16
Saper, M. A., P. J. Bjorkman, D. C. Wiley.
1991
. Refined structure of the human histocompatibility antigen HLA-A2 at 2.6 Å resolution.
J. Mol. Biol.
219
:
277
17
Garrett, T. P. J., M. A. Saper, P. J. Bjorkman, J. L. Strominger, D. C. Wiley.
1989
. Specificity pockets for the side chains of peptide antigens in HLA-Aw68.
Nature
342
:
692
18
Guo, H.-C., D. R. Madden, M. L. Silver, T. S. Jardetzky, J. C. Gorga, J. L. Strominger, D. C. Wiley.
1993
. Comparison of the P2 specificity pocket in three human histocompatibility antigens, HLA-A*6801, HLA-A*0201, and HLA-B*2705.
Proc. Natl. Acad. Sci. USA
90
:
8053
19
Madden, D. R., J. C. Gorga, J. L. Strominger, D. C. Wiley.
1991
. The structure of HLA-B27 reveals nomamer “self-peptides” bound in an extended conformation.
Nature
353
:
321
20
Madden, D. R., J. C. Gorga, J. L. Strominger, D. C. Wiley.
1992
. The three-dimensional structure of HLA B-27 at 2.1 Å resolution suggests a general mechanism for tight peptide binding to MHC.
Cell
70
:
1035
21
Fremont, D. H., M. Matsumura, E. A. Stura, P. A. Peterson, I. A. Wilson.
1992
. Crystal structures of two viral peptides in complex with murine MHC class I H2-Kb.
Science
257
:
919
22
Silver, M. L., H.-C. Guo, J. L. Strominger, D. C. Wiley.
1992
. Atomic structure of a human MHC molecule presenting an influenza virus peptide.
Nature
360
:
367
23
Guo, H.-C., T. S. Jardetzky, T. P. J. Garrett, W. S. Lane, J. L. Strominger, D. C. Wiley.
1992
. Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle.
Nature
360
:
364
24
Young, A. C. M., W. Zhang, J. C. Sacchettini, S. G. Nathenson.
1994
. The three-dimensional structure of H-2Db at 2.4 Å resolution: implications for antigen-determinant selection.
Cell
76
:
39
25
Parker, K., M. DiBrino, L. Hull, J. E. Coligan.
1992
. The β2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound.
J. Immunol.
149
:
1896
26
Silver, M. L., K. C. Parker, D. C. Wiley.
1991
. Reconstitution by MHC-restricted peptides of HLA-A2 heavy chain with β2-microglobulin, in vitro.
Nature
350
:
619
27
Fahnestock, M. L., I. Tamir, L. Nahri, P. J. Bjorkman.
1992
. Thermal stability comparison purified empty and peptide-filled forms of a class I MHC molecule.
Science
258
:
1658
28
Bouvier, M., H. C. Guo, K. J. Smith, D. C. Wiley.
1998
. Crystal structures of HLA-0201 complexed with antigenic peptides with either the amino- or carboxy-terminal group substituted by a methyl group.
Proteins Struct. Funct. Genet.
33
:
97
29
Gakamsky, D. M., P. J. Bjorkman, I. Pecht.
1996
. Peptide interaction with a class I major histocompatibility complex-encoded molecule: allosteric control of the ternary complex stability.
Biochemistry
35
:
14841
30
Pamer, E., P. Cresswell.
1998
. Mechanisms of MHC class I-restricted antigen processing.
Annu. Rev. Immunol.
16
:
323
31
Fruh, K., A. Gruhler, R. M. Krishna, G. J. Schoenhals.
1999
. A comparison of viral immune strategies targeting the MHC class I assembly pathway.
Immunol. Rev.
168
:
157
32
Bouvier, M. N., D. C. Wiley.
1994
. Importance of peptide amino and carboxyl termini to the stability of MHC class I molecules.
Science
265
:
398
33
Elovaara, I., S. Koenig, A. Y. Brewah, R. M. Woods, T. Lehky, S. Jacobson.
1993
. High human T-cell lymphotropic virus type 1 (HTLV-1)-specific precursor cytotoxic T lymphocyte frequencies in patients with HTLV-1-associated neurological disease.
J. Exp. Med.
177
:
1567
34
Madden, D. R., D. N. Garboczi, D. C. Wiley.
1993
. The antigenic identity of peptide-MHC complexes: a comparison of the conformations of five viral peptides presented by HLA-A2.
Cell
75
:
693
35
Garboczi, D. N., P. Ghosh, U. Utz., Q. R. Fan, W. E. Biddison, D. C. Wiley.
1996
. Structure of the complex between human T cell receptor, viral peptide, and HLA-A2.
Nature
384
:
134
36
Ding, Y.-H., K. J. Smith, D. N. Garboczi, U. Utz, W. E. Biddison, D. C. Wiley.
1998
. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids.
Immunity
8
:
403
37
Ding, Y. H., B. M. Baker, D. N. Garboczi, W. E. Biddison.
1999
. Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical.
Immunity
11
:
45
38
Utz, U., S. Koenig, J. E. Coligan, W. E. Biddison.
1992
. Presentation of three different viral peptides, HTLV-1 Tax, HCMV gB, and influenza virus M1, is determined by common structural features of the HLA-A2.1 molecule.
J. Immunol.
149
:
214
39
Garboczi, D. N., D. T. Hung, D. C. Wiley.
1992
. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides.
Proc. Natl. Acad. Sci. USA
89
:
3429
40
Brünger, A. T., P. D. Adams, G. M. Clore, W. L. Delano, P. Gros, R. W. Grosse-Kunstleve, J.-S. Jiang, J. Kuszewski, M. Nilges, N. S. Pannu, et al
1998
. Crystallography and NMR system: a new software system for macromolecular structure determination.
Acta Crystallogr. D
54
:
901
41
Jones, T. A., J.-Y. Zou, S. W. Cowan, M. Kjeldgaard.
1991
. Improved methods for building protein models in electron density maps and the location of errors in these models.
Acta Crystallogr. A
47
:
110
42
Baker, B. M., Y. H. Ding, D. N. Garboczi, W. E. Biddison, D. C. Wiley.
1999
. Structural, biochemical, and biophysical studies of HLA-A2 altered peptide ligand binding to viral-peptide specific human T-cell receptors.
Cold Spring Harbor Symp. Quant. Biol.
64
:
235
43
Utz, U., D. Banks, S. Jacobson, W. E. Biddison.
1996
. Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.
J. Virol.
70
:
843
44
Morgan, C. S., J. M. Holton, B. D. Olafson, P. J. Bjorkman, S. L. Mayo.
1997
. Circular dichroism determination of class I MHC-peptide equilibrium dissociation constants.
Protein Sci.
6
:
1771
45
Fahnestock, M. L., J. L. Johnson, R. M. R. Feldman, T. J. Tsomides, J. Mayer, L. O. Narhi, P. J. Bjorkman.
1994
. Effects of peptide length and composition on binding to an empty class I MHC heterodimer.
Biochemistry
33
:
8149
46
Smith, K. J., S. W. Reid, D. I. Stuart, A. J. McMichael, E. Y. Jones, J. I. Bell.
1996
. An altered position of the α2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501.
Immunity
4
:
203
47
Fremont, D. H., E. A. Stura, M. Matsumura, P. A. Peterson, I. A. Wilson.
1995
. Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.
Proc. Natl. Acad. Sci. USA
92
:
2479
48
Davis, M. M., J. J. Boniface, Z. Reich, D. Lyons, J. Hampl, B. Arden, Y. Chien.
1998
. Ligand recognition of αβ T cell receptors.
Annu. Rev. Immunol.
16
:
523
49
Alam, S. M., P. J. Travers, J. L. Wung, W. Nasholds, S. Redpath, S. C. Jameson, N. R. J. Gascoigne.
1996
. T-cell receptor affinity and thymocyte positive selection.
Nature
381
:
616
50
Lyons, D. S., S. A. Lieberman, J. Hampl, J. J. Boniface, Y.-h. Chien, L. J. Berg, M. M. Davis.
1996
. A TCR binds to antagonist ligands with lower affinities and faster dissociation rates than to agonists.
Immunity
5
:
53
51
Luzzati, V..
1952
. Traitement statistique des erreurs dans la determination des structures cristallines.
Acta Crystallogr.
5
:
802
52
Laskowski, R. A., M. W. MacArthur, D. S. Moss, J. M. Thornton.
1993
. PROCHECK: a program to check the stereochemical quality of protein structures.
J. Appl. Crystallogr.
26
:
283
53
Kraulis, J. P..
1991
. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures.
J. Appl. Crystallogr.
24
:
946