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The Journal of Immunology, 2004, 172: 6175-6184.
Copyright © 2004 by The American Association of Immunologists

Structures of HLA-A*1101 Complexed with Immunodominant Nonamer and Decamer HIV-1 Epitopes Clearly Reveal the Presence of a Middle, Secondary Anchor Residue1

Lenong Li and Marlene Bouvier2

School of Pharmacy, University of Connecticut, Storrs, CT 06269


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HLA-A*1101 is one of the most common human class I alleles worldwide. An increased frequency of HLA-A*1101 has been observed in cohorts of female sex workers from Northern Thailand who are highly exposed to HIV-1 and yet have remained persistently seronegative. In view of this apparent association of HLA-A*1101 with resistance to acquisition of HIV-1 infection, and given the importance of eliciting strong CTL responses to control and eliminate HIV-1, we have determined the crystal structure of HLA-A*1101 complexed with two immunodominant HIV-1 CTL epitopes: the nonamer reverse transcriptase(313–321) (AIFQSSMTK) and decamer Nef(73–82) (QVPLRPMTYK) peptides. The structures confirm the presence of primary anchor residues P2-Ile/-Val and P9-/P10-Lys, and also clearly reveal the presence of secondary anchor residues P6-Ser for reverse transcriptase and P7-Met for Nef. The overall backbone conformation of both peptides is defined as two bulges that are separated by a more buried middle residue. In this study, we discuss how this topology may offer functional advantages in the selection and presentation of HIV-1 CTL epitopes by HLA-A*1101. Overall, this structural analysis permits a more accurate definition of the peptide-binding motif of HLA-A*1101, the characterization of its antigenic surface, and the correlation of molecular determinants with resistance to HIV-1 infection. These studies are relevant for the rational design of HLA-A*1101-restricted CTL epitopes with improved binding and immunological properties for the development of HIV-1 vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several lines of evidence suggest that CD8+ CTL responses represent an important cellular immune defense during the course of HIV-1 infection. HIV-1-specific CD8+ CTLs are associated with the control of viremia during the early acute phase of the infection and decline significantly during progression to AIDS (1, 2, 3, 4). Strong HIV-1-specific CD8+ CTL responses have also been detected in individuals who remain clinically asymptomatic for long periods of time (1, 3, 4). Perhaps more importantly, CD8+ CTL responses to HIV-1 peptides have been detected in HIV-1-seronegative children born from seropositive mothers (5, 6, 7), in HIV-1-seronegative sex workers at high risk of infection (8, 9), and in HIV-1-seronegative health care workers with repeated exposures to contaminated blood (10, 11). Because CD8+ CTL responses are restricted by polymorphic gene products of the MHC region, it is not surprising that certain class I HLA alleles have been associated, at levels that are statistically significant, with resistance to HIV-1 infection in several cohorts of highly exposed, persistently seronegative (HEPS)3 individuals (9, 12, 13, 14). For example, a significant increase of the class I HLA-A*1101 allele has been observed in cohorts of highly exposed female sex workers from Northern Thailand who have been persistently HIV-1 seronegative, but are seropositive for other sexually transmitted diseases (9, 15, 16). Although the ability of the host to resist HIV-1 infection is likely to depend on multiple factors, these cases of apparent resistance to HIV-1 infection suggest that HLA-A*1101 may play a role in these processes.

HLA-A*11 is one of the most common class I alleles in worldwide populations, ranging from 4 to 33% depending on the particular ethnic background (17), and is highly prevalent in Southeast Asia. Fifteen natural variants (-A*1101 to -A*1115) have been reported to date, with HLA-A*1101 being most prevalent. Although HLA-A*1101 recognizes predominantly peptides of 9 or 10 residues in length, binders of 8, 11, or 12 residues have been identified (18, 19, 20, 21). The peptide-binding motif of HLA-A*1101 is defined by a preference for residues with small or aliphatic side chains (Ala, Ile, Leu, Met, Ser, Thr, or Val) at position (P) 2, and with positively charged side chains (Arg or Lys) at the C terminus position (18, 19, 22). HLA-A*1101 belongs to the HLA-A3 supertype that also includes among its common members HLA-A*0301, -A*3101, -A*3301, and -Aw*6801 as defined on the basis of their shared peptide-binding motifs (17, 19). To date, x-ray crystal structures of only HLA-Aw*6801 have been reported (23, 24, 25).

In this study, we characterized molecular interactions between HLA-A*1101 and two HIV-1 CTL epitopes. We have determined the first x-ray crystal structure of HLA-A*1101 in complex with HIV-1 peptides derived from reverse transcriptase (RT) and Nef proteins: the nonamer RT(313–321) (AIFQSSMTK) and the decamer Nef(73–82) (QVPLRPMTYK). These HLA-A*1101-restricted CTL epitopes have been shown to elicit immunodominant CTL responses in Northern Thai HEPS and HIV-1-infected sex workers (9). The RT(313–321) and Nef(73–82) peptides are also conserved between the predominant North American clade B and the circulating recombinant form 01_AE, the most prevalent HIV-1 strain in Southeast Asia. The goal of our studies is to examine the detailed interactions between HLA-A*1101 and highly conserved immunodominant HIV-1 epitopes as a necessary step in understanding how the molecular properties of HLA-A*1101/peptide complexes may provide a functional advantage in resistance to HIV-1 infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptide synthesis

RT(313–321) (AIFQSSMTK) and Nef(73–82) (QVPLRPMTYK) peptides (refer thereafter as RT and Nef, respectively) and their Ala mutants at P6 and P7, respectively, were synthesized on an ABI 431A peptide synthesizer (Applied Biosystems, Foster City, CA) and purified by HPLC reverse-phase chromatography (Tufts University, Boston, MA).

Reconstitution of HLA-A*1101/peptide complexes

Class I H chain HLA-A*1101 (residues 1–275) was cloned into the PLM1 vector and expressed in the Escherichia coli strain BL21(DE3)pLysS (Stratagene, La Jolla, CA), as described previously (26). The expression plasmid for {beta}2-microglobulin was a gift of D.C. Wiley (deceased). HLA-A*1101/peptide complexes were reconstituted and purified, as described previously (26). Stock solutions of complexes (20–40 mg/ml) in 10 mM MOPS, pH 7.4, were kept frozen at –70°C.

Thermal stability

The thermal stability of HLA-A*1101/peptide complexes was determined by circular dichroism using a Jasco J-810 spectropolarimeter equipped with a thermoelectric temperature controller. Denaturation curves were obtained in duplicate by monitoring the change in ellipticity at 218 nm in the temperature range 10–90°C using a scan rate of 40°C/h and a 1-mm path length cuvette. The midpoint temperatures (Tm) of averaged denaturation curves were determined, as described previously (27). The concentration was 0.20 mg/ml in 10 mM MOPS, pH 7.4.

Crystallization

Crystallization conditions of the HLA-A*1101/RT complex were established using the Crystal Screen I (Hampton Research, Riverside, CA) and the hanging-drop vapor diffusion technique at room temperature. After several days, microcrystals were obtained with solution No. 9 of Screen I and were used to produce a seeding solution in 16% polyethylene glycol (PEG) 6000, 25 mM MES, pH 6.5, and 0.1% sodium azide. Crystals used for data collection were grown by mixing 3.5 µl of an 11 mg/ml protein solution with 28% PEG 4000, 0.3 M sodium acetate, 0.2 M sodium citrate, pH 5.6, and 0.5 µl of seeding solution. Drops were placed over a reservoir solution containing 14.5% PEG 4000, 0.16 M sodium acetate, and 0.1 M sodium citrate, pH 5.6. Crystals appeared within 5–10 days as plates with maximum dimensions of 150 x 80 x 40 µm. Crystals of the HLA-A*1101/Nef complex were obtained, as described previously (26).

Data collection and processing

Single crystals of the HLA-A*1101/RT complex were soaked for several minutes in the reservoir solution supplemented with 20% 2-methyl-2,4-pentanediol as a cryoprotectant and then flash cooled directly in liquid nitrogen. X-ray diffraction data were collected on the beamline F1 (wavelength 0.9474 Å) at Cornell High Energy Synchrotron Source (Ithaca, NY) using a Quantum 4 charge-coupled device detector (Area Detector Systems, Poway, CA). Data were autoindexed and integrated with the program DENZO (28), then scaled and merged with the program SCALEPACK (28). Data collection for the HLA-A*1101/Nef complex has been described previously (26). Crystallographic data are presented in Table I.


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  		Table I. Crystallographic data and refinement statistics

 
Structure determination and refinement

The structure of HLA-A*1101/Nef complex was determined by molecular replacement using HLA-A*0201/Tax8 complex (Protein Data Bank code: 1DUZ (30)) (peptide excluded) as a search model in AMoRe (31). Two clear solutions for both the rotation and translation functions indicated the presence of two molecules (molecules 1 and 2) in the asymmetric unit. Rigid body fitting in AMoRe yielded a correlation coefficient of 40.7% and an Rfactor of 44.0% on data from 8.0–4.0 Å resolution. Residues that differ between -A*0201 and -A*1101 were replaced, and geometric adjustments were made in program O (32) under the guidance of FO-FC and 2FO-FC electron density maps. Refinement of the model was done using simulated annealing, energy minimization, and restrained individual B factor in program crystallography and nuclear magnetic resonance systems (33). In the first cycles, noncrystallographic symmetry (NCS) restraints of 300 Kcal mol–1 Å2 were applied to main chain atoms (loops and peptides excluded) using data to 2.8 Å, resulting in Rfree and Rcryst of 0.34 and 0.29, respectively. Further refinement with NCS restraints did not yield significantly improved R factors using data to higher resolution. Releasing NCS restraints gave the best refinement results based on R factors (Rfree and Rcryst of 0.27 and 0.24, respectively) and quality of electron density after some cycles of refinement using data to 2.22 Å. In the final stages of refinement, a composite omit map was calculated to eliminate model bias. Water molecules were gradually introduced during model building and were selected from >3.0{varsigma} peaks in the FO-FC electron density map at 3.5 Å or less from hydrogen bond donors or acceptors. A total of 427 water molecules was added to the model. Throughout refinement, the agreement between the model and the observed data was monitored by calculating Rfree based on 5% of the reflections. The final Rfree and Rcryst values (with a bulk solvent correction) are 25.5 and 21.2%, respectively, for all reflections between 49.3 and 2.22 Å (Table I). In the final model, electron density for the entire peptides 1 and 2 was continuous and clearly visible. Although backbone conformations of molecules 1 and 2 are essentially identical, side chains at the most solvent-exposed positions, P4, P5, and P8, show slightly different orientations, as do downward-pointing P7-Met side chains.

For the HLA-A*1101/RT complex, two molecular replacement solutions corresponding to two molecules (molecules 1' and 2') in the asymmetric unit were readily obtained in AMoRe using the refined coordinates of the HLA-A*1101/Nef complex (peptide excluded). The structure was then refined using similar protocols as those described for HLA-A*1101/Nef complex. The final Rfree and Rcryst values (with a bulk solvent correction) are 27.2 and 23.2%, respectively, for all reflections between 23.7 and 2.40 Å (Table I). In the final model, electron density for the entire peptides 1' and 2' was well resolved. Backbone conformations of molecules 1' and 2' are essentially identical, but the small size of the P1-Ala side chain caused a rotation around its {psi} angle (34), resulting in different hydrogen bonds at the N terminus of molecules 1' and 2'. Side chains at the most solvent-exposed positions P4 and P7 and at the secondary anchor position P6 adopt slightly different orientations in molecules 1' and 2'.

Molecules 2' and 2 of HLA-A*1101/RT and HLA-A*1101/Nef complexes, respectively, have been selected for characterization in this study. The coordinates of both complexes have been deposited in the Protein Data Bank and will be released upon publication (codes: 1Q94 for RT and 1QVO for Nef).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overall structures of HLA-A*1101/peptide complexes

The two structures of HLA-A*1101 are very similar to those of other human class I HLA molecules. The root-mean-square (r.m.s.) deviation between HLA-A*1101/RT and HLA-Aw*6801 (25) is 0.9 Å for superposition over all C{alpha} atoms. Similar comparison with a different allele of the HLA-A locus, HLA-A*0201 (30, 35, 36), revealed only small structural differences and r.m.s. deviations in the range 0.7–0.9 Å for superposition over all C{alpha} atoms. Although the two HLA-A*1101/peptide complexes show r.m.s. deviation of 1.2 Å for superposition over all C{alpha} atoms, independent superposition of individual domains yielded smaller values: 0.46 Å, 0.80 Å, and 0.68 Å for {alpha}1/{alpha}2, {alpha}3, and {beta}2-microglobulin domains, respectively. Regions of major conformational differences (r.m.s. deviations greater than ~1 Å) between the two HLA-A*1101 H chains are rather localized: {alpha}1/{alpha}2 domains (Fig. 1), residues 15–19, 41, 89–90, 146, and 149–150; and {alpha}3 domain, residues 195, 196, 218–228, 252–259, 266–268, and 271–275. As discussed below, although some of these differences in {alpha}1/{alpha}2 domains arise from crystal packing contacts (e.g., residue 146 in HLA-A*1101/RT complex), or are localized in regions of conformational flexibility, distinct conformations in the two HLA-A*1101 H chains appear to be directly induced by bound peptides.



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  		FIGURE 1. Independent superposition of {alpha}1/{alpha}2 domains of HLA-A*1101/RT (blue) and HLA-A*1101/Nef (red) complexes showing regions (yellow) of major conformational differences (r.m.s. deviations greater than ~1 Å) between the two HLA-A*1101 H chains. Peptides are omitted.

 
Overall conformations of bound RT and Nef peptides

Clear electron density for RT and Nef peptides within the binding groove of HLA-A*1101 is displayed in Fig. 2, respectively. Both peptides adopt an extended conformation that is stably maintained by a large number of polar and nonpolar interactions between HLA-A*1101 residues lining the binding cleft and peptides (Table II). Crystal contacts between any symmetry-related molecules in close proximity to the peptide-binding groove of HLA-A*1101/RT and /Nef complexes appear to induce no conformational changes in RT peptide 2' or Nef peptide 2, except for minor reorientation of exposed side chains at P7 for RT and at P5 and P8 for Nef.



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  		FIGURE 2. Final 2FO-FC electron density maps of HIV-1 peptides bound in the groove of HLA-A*1101 contoured at 1.5 {varsigma}. Peptide side chains are labeled from the N (P1)- to the C (P9 or P10)-terminal end of the binding groove. Only the {alpha}1 helix and {beta}-sheet floor are shown. A, RT peptide; B, Nef peptide. Figure produced using program SETOR (37 ).

 

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  		Table II. Hydrogen bond and Van der Waals interactions between HIV-1 peptides and HLA-A*1101 residues

 
Peptide side chains at P2, P6 (P7 for Nef), and P9 (P10 for Nef) are buried within the groove and display varying degrees of solvent exposure (areas calculated with GETAREA (38) using a 1.4 Å radius probe): RT, P2, 0.0 Å2; P6, 6.7 Å2; and P9, 10.3 Å2; Nef, P2, 1.4 Å2; P7, 6.5 Å2; and P10, 4.0 Å2. The P3 side chain of both peptides faces the {alpha}2 helix and resides within the interior of the cleft (solvent exposure: RT, 8.0 Å2 and Nef, 22.0 Å2). In the central region, from P4 to P8 (P9 for Nef), the backbone of both peptides arches out of the binding groove twice as a direct consequence of the P6-C{alpha} atom (P7-C{alpha} atom for Nef) being considerably more buried within the binding groove. For RT (Fig. 2A), side chains at P4, P5, P7, and P8 point up and show the highest degree of solvent exposure (P4, 56.7 Å2; P5, 29.1 Å2; P7, 39.5 Å2; and P8, 53.0 Å2). Similarly, for Nef (Fig. 2B), side chains at P4, P5, P8, and P9 are oriented toward the exterior of the binding cleft and display the highest solvent exposure values (P4, 73.6 Å2; P5, 117.9 Å2; P8, 32.0 Å2; and P9, 95.3 Å2). Consequently, solvent-exposed peptide side chains from the central region of RT and Nef make fewer contacts with HLA-A*1101 residues along the binding groove (Table II).

Overall, when the {alpha}1/{alpha}2 domains of both complexes are superimposed (Fig. 3), the RT and Nef peptides adopt similar conformations with nearly identical positioning of their N (P1)- and C (P9 or P10)-terminal ends within the groove. Perhaps most notably, the RT and Nef peptides have a middle residue, P6-Ser for RT or P7-Met for Nef, buried within the groove (Fig. 3A). The additional length of Nef is accommodated by a prominent vertical bulge out of the binding groove involving residues P4-Leu and P5-Arg, followed by a small zigzag across the width of the groove between residues P6-Pro and P7-Met (Fig. 3). At position P7, the backbone of both peptides reunite such that P6, P7, P8, and P9 of the nonamer peptide align with P7, P8, P9, and P10 of the decamer peptide (Fig. 3). The ability of RT and Nef to zigzag vertically and arch out of the binding groove, in contrast to zigzag horizontally across the width of the groove, is a feature that has been observed in other structures of human class I HLA molecules, including HLA-Aw*6801, -B8, -B*3501, and -B*5101 (23, 24, 25, 39, 40, 41), and of mouse and rat class I HLA molecules, including H-2Kb, -2Db, and RT1-Aa (42, 43, 44).



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  		FIGURE 3. Superposition of the bound nonamer RT (blue) and decamer Nef (red) peptides. A, Side view through the {alpha}2 helix. Side chain positions for RT and Nef are labeled in blue and red, respectively; B, top view with peptides oriented as in A. Only C{alpha} atoms for RT and Nef, labeled in blue and red, respectively, are shown. The P1-C{alpha} atom of both peptides is labeled in black.

 
Interactions at the N- and C-terminal end of the binding groove

In the HLA-A*1101/RT complex structure (Fig. 4A and Table II), the positively charged N-terminal amino group of RT forms a water-mediated network of hydrogen bonds with residues Tyr7, Tyr59, Glu63, and Tyr171. The main chain carbonyl group of P1 makes a hydrogen bond with Tyr159, while the small methyl side chain is oriented toward the interior of the A pocket and packs against Met5, Tyr7, Tyr167, and Tyr171 (Fig. 4A and Table II). In the HLA-A*1101/Nef complex structure (Fig. 4B), the larger size of P1-Gln side chain causes the N-terminal ammonium group of Nef to rotate around its {psi} angle relative to its conformation in the structure of HLA-A*1101/RT complex. This residue change leads to a different network of hydrogen bonds: the N-terminal ammonium group of Nef forms a pentagonal hydrogen-bond network with conserved residues Tyr7, Tyr59, Tyr171, and bound water molecule w20 (Fig. 4B and Table II). The main chain carbonyl group of P1 makes a hydrogen bond with Tyr159 (Fig. 4B and Table II), while the Gln side chain interacts with the guanidinium group of Arg163 and bound water molecule w426 (Table II). Overall, the two structures of HLA-A*1101 complexes show that different hydrogen-bond networks are possible at the N-terminal end of the binding groove depending on the size of the peptide side chain at P1.



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  		FIGURE 4. Network of hydrogen bonds at the N- and C-terminal ends of the binding groove. A, A pocket for RT. Only positions P1-Ala and P2-Ile are shown; B, a pocket for Nef. Only positions P1-Gln and P2-Val are shown; C, F pocket for RT. Only positions P8-Thr and P9-Lys are shown; D, F pocket for Nef. Only positions P9-Tyr and P10-Lys are shown. In A–D, peptides are shown primarily in yellow, HLA-A*1101 residues are shown primarily in green, while bound water molecules are shown in orange. Hydrogen bonds are illustrated as dotted lines.

 
At the C-terminal end of the binding groove, the C-terminal carboxylate group of RT makes direct hydrogen bonds to Tyr84 and Thr143, and via a bound water molecule (w2) to Asp77 (Fig. 4C and Table II). The main chain amino group of P9 interacts with Asp77. A nonstandard positioning of conserved Lys146 is observed in the F pocket of the HLA-A*1101/RT complex structure (Fig. 4C) due to crystal packing contacts to Tyr84 of molecule 1': the side chain of Lys146 is flipped away and forms hydrogen bonds with P8-Thr of RT (Fig. 4C and Table II). This rather unusual orientation of Lys146 has been previously observed in the structure of rat class I RT1-Aa (44). The HLA-A*1101/Nef complex structure reveals more standard interactions (Fig. 4D and Table II) in which the C-terminal carboxylate group of Nef makes direct hydrogen bonds to conserved residues Tyr84, Thr143, and Lys146, and via a bound water molecule (w5) to both Asp77 and Thr80. The main chain amino group of P10 interacts with Asp77 (Fig. 4D and Table II). Overall, although different networks of hydrogen bonds are made by the RT and Nef peptides at the C-terminal end of the groove, the observed interactions satisfy the hydrogen-bonding potential of conserved Tyr84, Thr143, Lys146, and Trp147.

P2 and C terminus anchor residues

The HLA-A*1101/RT complex structure shows that polymorphic residues lining the B pocket (Fig. 5A) provide a well-defined hydrophobic cavity for making Van der Waals interactions with the P2-Ile side chain of RT (Table II). Positioning of the smaller aliphatic P2-Val side chain of Nef within the corresponding B pocket of HLA-A*1101 is essentially similar (Table II). The main chain amino group of both peptides at P2 is tethered by a hydrogen bond to Glu63 (Fig. 5A and Table II). The main chain carbonyl group of RT at P2 makes a hydrogen bond to Asn66 via a bound water molecule (w8) (Fig. 5A and Table II), while that of Nef makes hydrogen bonds to bound water molecules w23 and w46 (Table II). Comparisons of the two HLA-A*1101 H chains (Fig. 1) reveal that conformational differences in the region of the B pocket are most notable at residue 41 located on a flexible, solvent-exposed loop.



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  		FIGURE 5. A, Interactions within the B pocket between HLA-A*1101 residues and RT. Only positions P1-Ala, P2-Ile, and P3-Phe are shown; B, interactions within the F pocket between HLA-A*1101 residues and P9-Lys side chain of RT. Only positions P8-Thr and P9-Lys are shown.

 
The F pocket of HLA-A*1101 is defined by a triad of negatively charged residues (Fig. 5B): Asp74 and Asp77, which poke in from the wall of the pocket, and Asp116, which lines the base of the pocket. The P9-Lys side chain of RT extends deep down into the F pocket where the N{epsilon}-ammonium headgroup makes a water-mediated hydrogen bond to the carboxylate groups of Asp74 and Asp77, and a direct hydrogen bond to Asp116 side chain as well as to bound water molecule w124 (Table II). The clustering of three Asp residues within the F pocket has been observed in HLA-Aw*6801, -B*2705, and rat RT1-Aa and is consistent with these alleles selecting positively charged residues at their C-terminal anchor positions (23, 24, 25, 44, 45). The aliphatic moiety of P9-Lys side chain of RT makes Van der Waals interactions with Asp77, Leu81, Tyr123, Thr143, and Trp147 (Table II). In comparison with the HLA-A*1101/RT complex structure, the P10-Lys side chain of Nef is positioned slightly different with a reorientation of its N{epsilon}-ammonium headgroup causing Asp116 to adopt a different rotamer conformation to maintain a critical salt-bridged interaction with P10-Lys side chain. Comparisons of the two HLA-A*1101 structures (Fig. 1) show that major conformational changes in the region of the F pocket are localized at residues 146, 149, and 150. It is most likely that these structural differences are induced by bound peptides as well as by crystal contacts involving Lys146.

In conclusion, the chemical and stereochemical nature of the B pocket is consistent with the preference of HLA-A*1101 to accommodate small or aliphatic side chains at anchor position P2. In contrast, the strong negatively charged environment provided by the presence of three Asp residues within the F pocket of HLA-A*1101 accounts for the ability of this allele to select antigenic peptides with positively charged residues at the C terminus anchor position.

Middle anchor residues

Although P2 and P9 represent the primary anchor residues for HLA-A*1101, electron density of RT and Nef (Fig. 2) unambiguously reveals the presence of secondary anchor residues at P6 and P7, respectively. These results extend biochemical analysis of HLA-A*1101/peptide complexes in which a preference for binding peptides with a small or aliphatic side chain at a "middle position" was demonstrated (17, 18, 20, 22).

The small P6-Ser side chain of RT is accommodated within the C pocket (Fig. 6A) and makes hydrogen bonds to Gln70 and a bound water molecule (w67) (Table II). The main chain amino group of P6 interacts with water molecule w67, while its carbonyl group makes no hydrogen bonds (Table II). In comparison with the structures of other class I HLA/nonamer peptide complexes, the presence of an Ile at position 97, in contrast to a bulkier residue such as Arg97 (in HLA-A*0201 and HLA-B53) or Met97 (in HLA-Aw*6801), provides a more open space on the floor of the groove in HLA-A*1101, allowing nonamer peptides to adopt a more buried backbone conformation with a downward-pointing side chain at P6. An examination of the C pocket in HLA-A*1101 suggests that nonamer peptides carrying small or aliphatic side chains at P6, such as Ala, Ile, Leu, Met, Ser, Thr, and Val, can be accommodated within this pocket. Although HLA-A*1101 is the first example of a human class I allele to use P6 as a secondary anchor residue, the structure of SEV-9 peptide bound to H-2Kb revealed that position P6 serves as a primary anchoring point in this mouse system (42). Position P6 is the point at which the backbone of RT and Nef reunites (Fig. 3) such that this position aligns with P7 of Nef. The P7-Met side chain points downward into the corresponding E pocket (Fig. 6B) and makes Van der Waals interactions with the aliphatic moieties of Arg114, Trp133, Ala152, and Gln156 (Table II). The P7-Met side chain shows slightly dissimilar orientations between molecules 1 and 2 of the asymmetric unit due to a change in the conformation of HLA residue 156 within the corresponding E pockets. An examination of the E pocket in HLA-A*1101 suggests that decamer peptides carrying small or aliphatic side chains at P7 such as Ala, Ile, Leu, Met, Ser, Thr, and Val can more favorably fit within this pocket than a large Tyr, Phe, or Trp side chain.



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  		FIGURE 6. A, Interactions within the C pocket between HLA-A*1101 residues and P6-Ser side chain of RT. Only positions P5-Ser, P6-Ser, and P7-Met are shown; B, interactions within the E pocket between HLA-A*1101 residues and P7-Met side chain of Nef. Only positions P6-Pro, P7-Met, and P8-Thr are shown.

 
We have assessed the energetic contribution of middle, secondary anchor residues P6-Ser of RT and P7-Met of Nef to the stability of the HLA-A*1101/peptide complexes by substituting these anchors with Ala. The stability of HLA-A*1101 complexes assembled with cognate and Ala-mutant peptides was determined from thermal denaturation curves (27). The midpoint of the denaturation curves, Tm, has been shown previously to correlate with the binding affinity of peptides for class I HLA molecules (27). Results showed that Tm(RT) = 63.7°C and Tm(Nef) = 61.2°C, while Tm(RT-Ala) = 62.5°C and Tm(Nef-Ala) = 59.4°C. This indicates that P6-Ser of RT and P7-Met of Nef contribute 1.2°C and 1.6°C, respectively, to the overall thermal stability of these complexes. It is possible that in addition to reflecting a loss of hydrogen bonds involving P6-Ser side chain (Fig. 6A) or hydrophobic interactions involving P7-Met side chain (Fig. 6B), these differences in stability also suggest that Met is only slightly more optimal as secondary anchor for the E pocket than Ser is for the C pocket.

TCR recognition surface

In the HLA-A*1101/RT complex structure (Figs. 2A and 3A), bulging residues at P4, P5, P7, and P8 show the highest degree of solvent exposure and as such represent potential TCR contact sites. Similarly, bulging residues at P4, P5, P8, and P9 of Nef (Figs. 2B and 3A) also display the highest degree of solvent exposure and are likely to be recognition sites by TCRs.

Peptide titration experiments using an HLA-A*11-restricted CTL clone specific for RT and derived from an HIV-1-infected patient have shown that an Ala substitution at either P7 or P8 yielded mutants that poorly activated the RT-specific CTL clone, while binding affinity for HLA-A*11 was essentially unchanged (46). This is consistent with P7-Met and P8-Thr side chains of RT pointing toward the solvent (Figs. 2A and 3A). In marked contrast, mutants of RT carrying single Ala substitutions at either bulging position P4 or P5 were both efficient at activating the identical TCR (46). Although this is somewhat surprising in the context of our structure, these results may reflect some degrees of flexibility in the recognition of antigenic surfaces by the RT-specific TCR isolated in the HIV-1-seropositive donor. The cross-reactive nature of this HLA-A*11-restricted RT-specific TCR was indeed demonstrated by its ability to be activated in vitro, albeit with lower efficiency, upon recognition of RT in the context of a different HLA-A3-like molecule (46). Although no systematic Ala scanning mutagenesis studies of HLA-A*11-restricted CTL clones specific for Nef have been reported, it was nonetheless shown that naturally occurring mutants of Nef with substitutions at P2 and P9 elicit considerably reduced CTL responses in vitro (47). This impaired CTL recognition was, however, associated with a failure of Nef mutants to bind to HLA-A*1101, consistent with positions P2 and P9 acting as primary anchor residues rather than TCR recognition sites.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The structures of HLA-A*1101/peptide complexes have demonstrated how two distinct HIV-1 CTL epitopes are bound within the groove of HLA-A*1101 and are presented to TCRs. This knowledge is important for improving computer-based predictions of high-affinity HLA-A*1101-restricted CTL epitopes in viral- and tumor-associated proteins (48, 49) and for understanding presentation of CTL epitopes by HLA-A*1101.

The structural features of the HLA-A*1101-binding groove that permit peptides to adopt a backbone conformation with two bulges separated by a secondary anchor residue may offer various advantages in the selection and presentation of CTL epitopes by HLA-A*1101 with functional implications on the ability of this allele to eliminate HIV-1: 1) a bulging backbone conformation allows longer peptides to bind without altering conserved interactions at the N- and C-terminal ends of the groove. This is supported by the large number of HLA-A*1101-restricted peptides of 10–12 residues in length that have been reported in the literature (18, 19, 20, 21). An unrestricted specificity for peptides of various lengths is likely to increase the potential of HLA-A*1101 to acquire stabilizing peptides during its maturation process in the endoplasmic reticulum (ER). 2) Given that mutants of RT and Nef peptides carrying conserved residue mutations at solvent-exposed positions P4, P5, and P8 have been shown to retain binding affinity for HLA-A*1101 (46, 50), these findings suggest a mechanism to substantially expand the repertoire of CTL epitopes presented by HLA-A*1101. This is functionally relevant because peptide-specific CTL clones are known to exhibit a certain degree of structural and functional flexibility in the recognition of cognate peptide mutants (51). Thus, in the context of viruses with high genetic variability such as HIV-1, HLA-A*1101 may have a functional advantage by counteracting immune escape mechanisms based on mutations of CTL epitopes. 3) Given that increased surface contacts between bound peptides and TCRs have been suggested as favorable to enhance the immunogenicity of class I HLA/peptide/TCR complexes (52, 53), the observed bulging backbone conformation of HLA-A*1101-restricted peptides may thus promote interactions with TCRs that lead to productive signaling. 4) Finally, in addition to contributing specificity to the peptide-binding motif of HLA-A*1101, secondary anchor residues P6-Ser of RT and P7-Met of Nef also make an energetic contribution to the specificity of the C and E pockets, respectively. The more buried backbone conformation of middle residues P6 and P7 thus provides a mechanism to stably anchor the central region of peptides within the binding groove of HLA-A*1101.

Recent crystallographic studies have demonstrated a general diagonal orientation of TCRs over class I HLA/peptide complexes (52, 54, 55). To date, no crystal structures of a soluble HLA-A*1101-restricted TCR have been reported. Nonetheless, to obtain insights into the recognition of HLA-A*1101/peptide complexes by TCRs, we have docked the A6 TCR restricted to HLA-A2/Tax complex (54) onto the structures of HLA-A*1101/peptide complexes (data not shown). In this model, bulging residues at P4 and P5 of both RT and Nef are positioned directly under the hypervariable complementarity-determining region (CDR) 3{alpha} and 3{beta} loops. Indeed, residues P4 and P5 of both peptides form numerous interactions with CDR3{alpha}, 3{beta}, and 1{alpha} loops. Interestingly, in this model, residue P4 of RT and Nef also form a small number of rather close contacts with CDR3{alpha} and 1{alpha} loops. The putative binding interface between the A6 TCR and HLA-A*1101 also includes substantial interactions between residues P7 (P8 for Nef) and P8 (P9 for Nef) and the CDR3{beta} loop. Longer peptides of 11 and 12 residues in length with more prominent bulges are likely to clash with the A6 CDR3 loops unless conformational changes at the binding interface occur upon complex formation, or TCRs capable of accommodating highly protruding peptide backbone are selected (56). The availability of HLA-A*1101-restricted T cell clones specific for RT and Nef will permit to assemble soluble HLA-A*1101/peptide/TCR complexes and to examine molecular details of the binding interface.

Evidence has been provided that HLA-A*11 is characterized by an unusual ability to mature efficiently in the ER and to be expressed normally at the cell surface when proteasome function is severely inhibited by the specific inhibitor lactacystin (57). Perhaps most significant, it was shown that HLA-A*3, but neither HLA-A*31, -A*33, nor -Aw*68, exhibits the same anomalous maturation and intracellular transport as HLA-A*11 upon inhibition of proteasome activity (57). These functional differences are quite interesting given that all five of these alleles belong to the HLA-A3 superfamily on the basis of their shared peptide-binding motifs. In this context, it was recently shown that cells have evolved a proteasome-independent proteolytic system in the form of the cytosolic tripeptidyl peptidase II complex that can generate the Nef decamer peptide used in this study (58). Given that HIV-1 can interfere with proteasome activity (59), these findings suggest that a similar mechanism may occur in vivo in the context of HIV-1, highlighting the apparent ability of HLA-A*1101 to mature normally in the ER under conditions in which the physiological pool of CTL epitopes is altered either qualitatively or quantitatively, or at the level of the source of peptides.

An examination of the primary sequence of HLA-A3-like molecules reveals that residue 97 is the only residue within the binding groove that is exclusively common in both HLA-A*0301 and -A*1101; indeed, while HLA-A*0301 and -A*1101 carry Ile97, HLA-A*3101, -A*3301, and -Aw*6801 have Met97. A preliminary comparison of HLA-A*1101 and -Aw*6801 structures (L. Li and M. Bouvier, unpublished data) reveals that residue 97, positioned in the central region of the groove (Fig. 6), modulates two coupled processes: the preference for binding either a Lys or a bulkier Arg C-terminal anchor residue within the F pocket, and the presence of a middle, secondary anchor residue with a downward-pointing side chain. Thus, residue Ile97 appears to play an important role in the fine selection and presentation of antigenic peptides by HLA-A*0301 and -A*1101 relative to other HLA-A3-like molecules. This structural property may have implications on how HLA-A*0301 and -A*1101 function relative to other alleles of the HLA-A3 superfamily in the context of disease states.

Taken together, the apparent association of HLA-A*1101 with resistance to HIV-1 infection may reflect, at least in part, an interplay of functional advantages that particular structural features of the peptide-binding groove may confer to HLA-A*1101: an enhanced ability for selecting HIV-1 CTL epitopes that can be supplied independently of proteasome function and for presenting them efficiently to T cells. An increased understanding of immune responses and molecular mechanisms responsible for resistance to HIV-1 infection in HLA-A*1101-positive HEPS subjects is important for efforts in developing HIV-1 vaccines.


   Acknowledgments
 
We thank Shu Wang for circular dichroism experiments, Dr. James Knox and members of his laboratory for their assistance with x-ray data collection, Drs. Mike Silver and Don C. Wiley for providing the coordinates of HLA-Aw*6801/Np(91–99) complex (see Ref. 23 ), and Dr. Janet M. McNicholl for discussion and for carefully reading the manuscript.


   Footnotes
 
1 This project was supported by the National Institutes of Health/National Institute of Allergy and Infectious Diseases (Grant AI46309) (to M.B.) and is now supported by the Robert Leet and Clara Guthrie Patterson Trust (to M.B.). Part of this work is based upon research conducted at the Cornell High Energy Synchrotron Source, which is supported by the National Science Foundation and the National Institutes of Health/National Institute of General Medical Sciences under Award DMR 0225180. Back

2 Address correspondence and reprint requests to Dr. Marlene Bouvier, School of Pharmacy, 372 Fairfield Road U-92, Storrs, CT 06269. E-mail address: bouvier{at}uconnvm.uconn.edu Back

3 Abbreviations used in this paper: HEPS, highly exposed, persistently seronegative; CDR, complementarity-determining region; ER, endoplasmic reticulum; NCS, noncrystallographic symmetry; P, position; PEG, polyethylene glycol; r.m.s., root-mean-square; RT, reverse transcriptase; Tm, midpoint temperature. Back

Received for publication October 21, 2003. Accepted for publication March 2, 2004.


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 Introduction
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 Discussion
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G. B. E. Stewart-Jones, G. Gillespie, I. M. Overton, R. Kaul, P. Roche, A. J. McMichael, S. Rowland-Jones, and E. Y. Jones
Structures of Three HIV-1 HLA-B*5703-Peptide Complexes and Identification of Related HLAs Potentially Associated with Long-Term Nonprogression
J. Immunol., August 15, 2005; 175(4): 2459 - 2468.
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