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The Journal of Immunology, 2000, 165: 3260-3267.
Copyright © 00 by The American Association of Immunologists

Nonstandard Peptide Binding Revealed by Crystal Structures of HLA-B*5101 Complexed with HIV Immunodominant Epitopes1

Katsumi Maenaka2,*,{dagger}, Taeko Maenaka*, Hiroko Tomiyama{ddagger}, Masafumi Takiguchi{ddagger}, David I. Stuart*,§ and E. Yvonne Jones3,*,§

* Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Headington, Oxford, United Kingdom; {dagger} Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka, Japan; {ddagger} Division of Viral Immunology, Center for AIDS Research, Kumamoto University, Honjo, Kumamoto, Japan; and § Oxford Center for Molecular Sciences, New Chemistry Building, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the {alpha}1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Major histocompatibility complex class I is a glycoprotein expressed on the surface of normal nucleated cells, which binds a peptide Ag (normally an 8- to 10-mer) in the binding groove of its {alpha}1-{alpha}2 domain. Specific binding of TCR on CTL to MHC class I complexed with peptide on target cells initiates the cellular immune response (1). In contrast, MHC class I is also a target of other immunorelevant receptors, including CD8 and NK cell receptors (e.g., killer cell Ig-like receptor (KIR)4), which regulate various aspects of the immune response (Fig. 1GoA) (2).



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  		FIGURE 1. HLA-B*5101 structure. A, The crystal structure of the HLA-B*5101-KM1 (LPPVVAKEI) complex. The main chains of the MHC heavy chain and ß2-microglobulin are represented schematically and encased in a semitransparent surface (orange, heavy chain of HLA-B*5101; green, ß2-microglobulin, yellow ball-and-stick model, peptide KM1). The binding sites of MHC class I-specific receptors (TCR, CD8, and KIR) are indicated by circles. Surfaces were calculated in VOLUMES (R. Esnouf, unpublished), and this figure plus Figs. 2–5GoGoGoGo were produced by BOBSCRIPT (45 ) and Raster 3D (46 ). B, Final 2Fo-Fc electron density map (magenta mesh) of the KM2 (TAFTIPSI) peptide region of the HLA-B*5101-KM2 complex contoured at 1 {varsigma}. The bound peptide is shown in ball-and-stick representation.

 
Investigation of individuals who remained uninfected despite repeated exposure to HIV-1 (3, 4, 5) has strongly suggested that CTLs play an important role in the elimination of the virus. The human MHC class I molecule, HLA-B51, is one of a set of closely related classical HLA alleles (a serologically cross-reactive group (CREG)) including HLA-B35 and HLA-B53. While HLA-B*5101 itself has been identified to be the disease susceptibility gene for Behçet’s disease (6), it is also associated with a slow progression of AIDS. In a previous report, we identified, using reverse immunogenetics, several highly conserved immunodominant HIV-1 peptide epitopes displayed by HLA-B51, which play an essential role in the elimination of HIV-1 in most HIV-1-seropositive HLA-B51 individuals (7). HLA-B*5101 and HLA-B*3501 show a similar peptide-binding motif (Pro and Ala at position 2 (P2), and hydrophobic residues Leu, Ile, Val, and Met at the peptide C terminus (PC)), with the exception that HLA-B*3501 shows a strong preference for tyrosine at PC (8, 9, 10, 11, 12, 13). The latter allele is associated with a rapid progression to AIDS. HLA-B*5301 also shows the preference for Pro at P2 and a broad preference for Phe, Ile, Tyr, Leu, and Met at PC (14). Other differences between these closely related HLA-B molecules are: 1) HLA-B35 and HLA-B53 are fast assembly molecules for Ag presentation, while HLA-B51 is a slow assembly one (15), and 2) the affinity of peptides to HLA-B51 and HLA-B53 is generally lower than to HLA-B35 (7, 10, 14).

Members of the human KIR family are expressed on the surface of NK cells and some subsets of T cells, and regulate their immune response through specific binding to MHC class I (16). KIR family members belong to the Ig superfamily with extracellular regions containing two or three tandem Ig-like domains (denoted as KIR2D or KIR3D, respectively). HLA-B51 is a well-established ligand for a three Ig-domain example of the KIR family, KIR3DL1 (17). Like other KIR members, KIR3DL1 shows allele- and peptide-specific recognition, in particular discriminating between alleles with the Bw4/Bw6 serological epitopes (residues 77–83) in the C-terminal region of the {alpha}1 helix (18, 19, 20, 21).

Crystal structures for the other members of the CREG group, HLA-B35 and HLA-B53, have been reported previously (22, 23, 24). The HLA-B51 structure reported in this study finally allows a comparison of the structural differences among the full set of alleles in this CREG group. The aims of this work were to identify the structural basis for several immunologically interesting points: 1) the difference in peptide-binding motif and affinity of CREG HLA-B alleles; 2) the interactions of HIV-immunodominant peptides toward HLA-B*5101; 3) the nature of the TCR recognition surface presented by these HLA-B51-peptide complexes; 4) the structural characteristics of the putative KIR3D binding site (Bw4/Bw6 epitope). We selected two HIV-1 Pol peptides, the 9-mer KM1 (SF2-Pol-743-9; LPPVVAKEI) and the 8-mer KM2 (SF2-Pol-283-8; TAFTIPSI), both of which are immunodominant for HIV-seropositive patients and bind strongly to HLA-B51 (7). Detailed information on how these highly conserved immunodominant HIV-derived peptides are presented to T cells by HLA-B51 may be expected to be relevant to the study of CTL in the course of HIV-1 infection and the development of vaccines for the treatment and prevention of HIV infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Expression and purification of HLA-B51-KM1 and HLA-B51-KM2 complexes

rHLA-B51 heavy chain was produced as inclusion bodies by a conventional Escherichia coli expression system. It was solubilized with Urea-GuHCl solution and refolded with ß2-microglobulin and HIV-1-derived peptides (9-mer KM1 (SF2-Pol-743-9; LPPVVAKEI) and 8-mer KM2 (SF2-Pol-283-8; TAFTIPSI)) by dilution into the refolding solution, followed by purification using gel filtration and anion-exchange columns (25).

Crystallization and data collection

An initial crystallization trial was done by using the Crystal Screen I (Hampton Research, Riverside, CA). Very fine needle-type crystals were obtained by the sitting drop vapor diffusion method using microbridges (26) in Linbro tissue culture plates at 21°C. One microliter of a 10 mg/ml protein solution in 20 mM Tris, pH 8, was mixed in a 1:1 ratio with the crystallization reservoir solution (Crystal Screen I-41; 20% PEG4000, 0.1 M HEPES, 10% isopropanol, pH 7.5). Further optimization of these crystallization conditions failed. However, the macroscopic cross-seeding technique applied at these same crystallization conditions, using crystal seeds derived from HLA-B35, produced plate/rod shape crystals suitable for crystallographic analysis. X-ray diffraction data collections for the HLA-B51-KM1 and HLA-B51-KM2 complexes were conducted at station ID14 of the European Synchrotron Radiation Facility ESRF and at stations 9.6 and 7.2 of the Daresbury Synchrotron Radiation Source, respectively. The crystals of both complexes contained one molecule per asymmetric unit and belonged to the same space group, P212121 (see the details in Table IGo). The diffraction data were autoindexed, and integrated with the program DENZO, then scaled and merged in the program SCALEPACK (27) (Table IGo).


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  		Table I. Data collection and refinement statistics for the HLA-B51-KM1 and -KM2 complexes

 
Structure determination and refinement

Because the crystals of HLA-B51-KM1 and HLA-B51-KM2 are isomorphous with those of HLA-B53, whose structure has already been solved, the structures of HLA-B51-KM1 and HLA-B51-KM2 were first determined by using the crystal structure of the HLA-B53/HIV-Gag peptide complex without inclusion of the peptide (23). Three-domain ({alpha}1-{alpha}2 domains, {alpha}3 domain, ß2-microglobulin) rigid body adjustment with program CNS reduced the Rcryst to 29% (Rfree = 29.7%, resolution 25–3.5 Å) for KM-2 and to 30.1% (Rfree = 32.3%, resolution 25–2.8 Å) for KM-1. After one cycle of positional refinement (program CNS (28)), the Fo-Fc {phi}calc map clearly showed electron density for the bound peptide into which coordinates could easily be built. Further refinement was conducted using positional and individual B-factor refinement in program CNS and alternated with manual rebuilding in the interactive graphics program O (29). The final models include the whole molecule (heavy chain, ß2-microglobulin and peptide) and show Rcryst 19.9% (Rfree = 25.5%) between 25 and 2.2 Å for KM1 and Rcryst 19.4% (Rfree = 25.3%) between 25 and 3 Å for KM2. Clear electron density defines unambiguous conformations for the KM1 and KM2 peptide main chains and for most of the side chains. The exceptions are the P5 Ile side chain in KM2, which exhibits some flexibility, and the P7 Lys side chain in KM1, for which there is density indicative of multiple conformations, the major one of which was selected for refinement.

Coordinates have been deposited in the Protein Data Bank (accession codes 1e27 (HLA-B51-KM1) and 1e28 (HLA-B51-KM2)).

CTL assay of KM1- and KM2-restricted CTL clones and peptide-binding assay to HLA-B*5101 and HLA-B*5102 transfectant cells

Peptide-binding assay. RMA-S-B*5101 cells (7, 9), which are transfectants of the TAP-defective mouse cell RMA-S, express empty HLA-B*5101 on the cell surface when they are cultured at 26°C. Binding of the HIV-1 peptides to the HLA-B*5101 molecule was examined by HLA-B*5101 stabilization assays, as described previously (7). The BL50 value was calculated as the peptide concentration that yields the half-maximal levels of the mean fluorescence intensity.

CTL assay. Cytotoxicity was measured in a standard 51Cr release assay, as described previously (7). Recombinant vaccinia viruses containing gag/pol genes of HIV-1 SF2, as generated previously, were used to infect the HLA-B51 transfectant (7). The specific lysis = ((cpm exp - cpm spn)/(cpm max - cpm spn)) x 100, in which cpm exp and cpm spn are the cpm in the supernatant in wells containing both target and effector cells, and target cells only, respectively.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Overall structure

Soluble HLA-B51 molecules complexed with two different peptides derived from HIV-1 were produced by standard protocols (25); the heavy chain (residues 1–276) of HLA-B*5101 was overexpressed in E. coli and refolded by adding ß2-microglobulin and specific synthesized HIV-1 peptides (KM1, LPPVVAKEI and KM2, TAFTIPSI). The complexes crystallized in similar unit cells to other HLA-B alleles and their structures were determined by molecular replacement, using HLA-B53/Gag (23) as the search model. Coordinates were refined using x-ray diffraction data to 2.2 Å resolution for KM-1 and to 3 Å for KM-2 (see Table IGo). Electron density for the KM-2-bound peptide is shown in Fig. 1GoB.

The overall structures of the HLA in the HLA-B51-KM1 and HLA-B51-KM2 complexes are very similar to other MHC class I structures previously reported (Fig. 1GoA). The root-mean-square distances (r.m.s.d.) of some 180 C{alpha} positions in the {alpha}1 and {alpha}2 domains of HLA-B51 from equivalent atoms in the other CREG HLA-B alleles are less than 1 Å: 0.38–0.45 Å (HLA-B53 (23)) and 0.65–0.70 Å (HLA-B35, (22, 24)), with comparisons with other alleles yielding similar values, 0.45 Å (HLA-B8 (30)) and 0.61 Å (HLA-A2 (31)). The two HLA-B51 complexes are almost identical; r.m.s.d. = 0.37 Å.

Peptide-binding surface

Pool sequencing of eluted peptides and the binding analysis of individual peptides have shown that HLA-B*5101 has a peptide-binding motif with primary anchor residues, P2 and PC (8, 9, 10, 11). At P2, small amino acids (Gly, Ala, and Pro) are preferred. The HLA-B51 complex structures reveal P2 residues, Pro (KM1) and Ala (KM2), fitted into a small B pocket surrounded by Ile66, Phe67, and Tyr7 (Table IIGo). These B pocket residues are conserved in other alleles of the CREG, including HLA-B35 and HLA-B53. Structural comparisons with HLA-B35 and HLA-B53 reveal only slight adjustment in the HLA-B*5101-KM1 and HLA-B*5101-KM2 B pocket (22, 23, 24), reinforcing the notion that this pocket is a common feature of the CREG. This is in keeping with the common CREG anchor preference for Pro and small amino acids.


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  		Table II. Peptide hydrogen bond and van der Waals interactions to HLA-B51

 
In the F pocket (which accommodates the C-terminal peptide residue, PC), the hydrogen-bonding network between the N{zeta} of Lys146, O{gamma}1 of Thr143, and the C-terminal main chain carboxylate oxygens of the peptides differs only slightly from other B alleles (Table IIGo and Fig. 2Go). However, the F pocket peptide-binding motifs for the CREG B alleles differ in their PC preferences. HLA-B*5101 shows a preference for aliphatic side chains Leu, Ile, Val, and Met at PC. In both the HLA-B51 complex structures, the bulky hydrophobic amino acids, Ile80, Tyr84, and Trp95, form a relatively small F pocket making van der Waals interactions with the side chains of the peptide C terminus (Ile in both complexes). The binding motif is consistent with these structural data. HLA-B*3501 has a strong preference for tyrosine at the C terminus of the peptide. Crystal structures of HLA-B*3501 complexes have shown that the phenolic group of the C-terminal tyrosine pokes into the bottom of the groove with its OH group, forming a strong interaction with the side chain OH of Ser116. In HLA-B51 complexes, Ser is substituted by the much larger amino acid tyrosine at position 116; this directs the PC side chain toward the {alpha}1-helix rather than allowing access to the bottom of the central groove and hydrogen bond formation. While having serine at MHC residue 116, HLA-B*5301 has the same Bw4 epitope as HLA-B*5101, in particular a small Ala residue at position 81, which provides space in the F pocket. This F pocket allows the particularly broad specificity for both aliphatic and aromatic side chains at PC.



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  		FIGURE 2. Comparison of interactions in the C-terminal region of peptides bound to HLA-B*5101 and other CREG MHC class I molecules. A, HLA-B51-KM2; B, HLA-B51-KM1; C, HLA-B53-KPIVQYDNP (23 ); D, HLA-B35-VPLRPMTY (22 ). In Figs. 2Go and 4Go, the peptides are shown in ball-and-stick representation with orange bonds, while the amino acid residues of the MHC class I molecules involved in interactions to the peptide are shown primarily in green. Hydrogen bonds are shown as dotted yellow lines.

 
Center region

While P2 and PC are the primary anchors for HLA-B51 peptide binding, the complex structures reveal several other distinctive features of the B51 peptide-binding groove. In the D pocket (the P3 binding site), Pro in the KM1 peptide contacts the hydrophobic face of Tyr99, which is rotated by 140° around its C{alpha}-Cß bond relative to its side chain conformation in other MHC complexes to accommodate this interaction (Fig. 3Go). This rotation of the phenolic group is not observed in the HLA-B51-KM2 complex, which has Phe at P3. Therefore, this is a peptide-dependent conformational change and appears to depend on the size and shape of the side chain at P3. This conformation was also observed in the HLA-B*3501 9-mer complex, again in the presence of Pro at P3 (24), but in that case was one of two alternative conformations.



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  		FIGURE 3. Comparison of interactions in the central region (P3-P5) of peptides bound to HLA-B51-KM1 (orange) and HLA-B51-KM2 (green, gray) structures. The side chains of the bound peptides (P3-P6) and those of the amino acids of the MHC class I molecules involved in interactions are shown in ball-and-stick: blue, the difference of the P3 positioning; black, the rotation of Tyr99; and purple, the small hydrophobic pocket at the P5 site.

 
The overall main chain conformation of the peptide in the HLA-B*3501 9-mer complex, is quite similar to that of the HLA-B51-KM1 structure; however, in the center region of the HLA-B51 complexes, substitution of Lys97 with threonine makes more space, which results in the peptide being more deeply buried in the bottom of the groove. Despite having identical residues in the first three positions of the 9-mer peptides (KM1, LPPVVAKEI; B35 9-mer, LPPLDITPY), the P3 Pro is positioned deeper toward Tyr99 in HLA-B51, a positioning that appears correlated with the location of the central region (P5) of the peptide (Fig. 3Go). Despite the increased depth of the binding groove in HLA-B51, no strongly specificity conferring pocket is created, so that in the case of the KM2 peptide, the buried P5 Ile side chain shows some flexibility/multiple conformations (as assessed from the crystallographic electron density).

N-terminal nonstandard interaction

Tyr171 is conserved in most MHC class I alleles and CREG family members other than HLA-B*5101. As a tyrosine, this residue hydrogen bonds to the amide group of the N-terminal peptide residue P1 (Fig. 4Go). In HLA-B*5101, Tyr171 is replaced by histidine, necessitating an alternative hydrogen bond network to the peptide N terminus. The two B*5101 complexes achieve this by different mechanisms. In the HLA-B*5101-KM1 complex, a water molecule mediates the hydrogen-bonding network between N{epsilon}2 of His171 and the main chain nitrogen of the peptide amino terminus (Fig. 4Go). This water-mediated network raises the position of the N terminus of the bound peptide within the groove, similar to the unusual positioning previously reported for the B35 8-mer structure (Fig. 4Go) (22). In case of the 8-mer KM2 peptide with a threonine at P1, the O{delta} of its side chain makes new hydrogen bonds toward the side chain of Asn63, and the main chain carbonyl group interacts with the side chain OH of Tyr7 (Table IIGo). This rearrangement of the hydrogen-bonding network in the P1 pocket is novel and has not been found in any other MHC class I structures to date. Because nonstandard positioning of the N terminus is seen in both 8-mer and 9-mer complexes, this appears to be a peptide-independent property of HLA-B51, although the details of the structural configuration depend on the nature of the peptide P1 residue.



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  		FIGURE 4. Hydrogen-bonding networks in the P1 pocket of HLA-B-peptide complexes. A, HLA-B51-KM2; B, HLA-B51-KM1; C, HLA-B53-TPYDINQML (23 ); D, HLA-B35-VPLRPMTY (22 ). Structural representation and colorings are as in Fig. 2Go.

 
Peptide structure

The main chain conformation of the KM2 8-mer peptide bound to HLA-B51 is similar to that of the fully extended and deeply buried HLA-B8 8-mer (30), rather than that of the HLA-B35 8-mer (22) (Fig. 5Go). The central region of the HLA-B35 8-mer is highly exposed to solvent arching out of the binding groove in a manner typical of peptide-MHC class I structures (32). In many MHC class I alleles, there is limited space in this central region, with the large residue Lys97 filling the floor of the groove. In contrast, in HLA-B51 and HLA-B8, lysine at position 97 is replaced by Thr and Ser, respectively. These smaller residues open up the central portion of the groove to allow a more deeply buried peptide conformation.



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  		FIGURE 5. Comparison of peptide conformations bound in the various MHC class I structures. These superpositions are based on the {alpha}1-{alpha}2 domains of the MHC class I molecules. A, C{alpha} traces of 9-mer peptides: red (KM1), pink (Aw68 (33 )), yellow (B35 (24 )), light gray (A2, PDB code 1hhg (31 )), dark gray (B53 (23 )). B, C{alpha} traces: red (KM1) and cyan (KM2). C, C{alpha} traces of 8-mer peptides: cyan (KM2), blue (B8 (30 )), and gray (B35 (22 )).

 
The 9-mer, KM1 peptide in the HLA-B51 complex shows the "zig-zag" middle region conformation observed for a 10-mer peptide in a HLA-Aw68 complex (33), and in the HLA-B35 9-mer structure (24). In the case of the HLA-B35 9-mer structure, the C-terminal position was significantly closer to the P1 pocket than other MHC class I molecules. The zig-zag conformation is achieved in HLA-B51 by two different mechanisms (1): the N-terminal amino acid is lifted up to a nonstandard position by rearrangement of the hydrogen bond network due to the substitution of His for Tyr at position 171, and (2) as seen for the 8-mer peptide, the central region is deeply buried at the bottom of the groove.

Comparison of the KM1 and KM2 HLA-B51-peptide complexes reveals that the difference in number of residues for 8-mer and 9-mer peptides is accommodated in the region between P5 and PC. There is a striking conservation in the position of the deeply buried P5 residue, comparable with human (HLA-B8) and murine (H-2Db and H-2Kb) MHC class I structures for which P5 is a primary anchor. This is not apparent in the HLA-B51 peptide-binding motif, but as mentioned above, the comparison between KM1 and KM2 structures suggests cooperative effects between P3 and P5, which could fine-tune the location of the central region of the peptide and modulate the anchor-like specificity (Fig. 3Go). For example, in the KM1 structure, the deeply buried Pro at P3 makes the side chain of Tyr99 rotate toward the floor of the P5 pocket, which makes a small hydrophobic pocket suitable for Val at P5 (Fig. 3Go). In contrast, in the KM2 structure, the Phe at P3 faces toward the {alpha}2 helix, leaving Tyr99 in the standard position and instead prompting the Ile at P5 to be shifted toward the hydrophobic side chain at position 99.

CTL response, peptide-binding affinity, and assembly

Within the family of HLA-B51 molecules, the only difference between HLA-B*5101 and HLA-B*5102 is that HLA-B*5102 substitutes the standard tyrosine for the nonstandard His of HLA-B*5101. Therefore, HLA-B*5102 may be expected to have the standard hydrogen-bonding network to the peptide N terminus. The relationship between the structural change caused by the substitution of residue 171 and biological function, CTL response, can be assayed. CTL assays were performed using KM1- and KM2-restricted CTL clones to HLA-B*5101 or HLA-B*5102 C1R transfectants, which had the KM1 or KM2 peptide loaded, or were infected by the HIV recombinant vaccinia virus. For both peptide-loaded and vaccinia virus-infected transfectants, the CTL clones only killed the B*5101 transfectant, not the B*5102 one (Fig. 6Go). The KM1 and KM2 peptides bound to both HLA-B*5101 and HLA-B*5102 at broadly similar binding constants (data not shown); thus, the differences in CTL recognition are not due to differences in peptide-binding affinity. Superimposition into the context of known structures for TCR-MHC complexes (34, 35, 36) indicates that the N-terminal (P1) amino acid is likely to contact the CDR1 loop of the V{alpha}-chain for the TCRs of the KM1- and KM2-restricted CTL clones described in this work, and hence directly contribute to the functional recognition. Therefore, the data clearly suggest that not only the large conformational change resulting from novel interactions at the N-terminal position of KM2, but also the subtle rearrangement of the hydrogen bonding network in the KM1 complex affect the peptide-specific CTL response. The ability of the subtle change observed in the KM1 structure to have a dramatic effect on the CTL response is in good agreement with the previous observations that slight structural changes introduced by single residue mutations of a HIV-1 gag peptide in HLA-B8 complexes (30) or of the HTLV Tax peptide in HLA-A2 (37) could cause either antagonism or complete abolition of the CTL response.



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  		FIGURE 6. Recognition of KM1- and KM2-restricted CTL clones for C1R-B*5101 and C1R-B*5102 pulsed with each peptide or infected with recombinant vaccinia virus. The recognition of KM1 (top panel)- and KM2 (bottom panel)-restricted CTL clones for target cells prepulsed with each peptide () or target cells infected with vaccinia recombinant containing gag/pol gene of HIV-1 () were tested at an E:T ratio of 2:1. Relative specific lysis (%) was calculated as follows: 1) relative specific lysis of target cells pulsed with peptide = (% specific lysis of target cells pulsed with peptide) - (% specific lysis target cells without peptide), and 2) relative specific lysis of target cells infected with recombinant vaccinia virus = (% specific lysis of target cells infected with recombinant vaccinia virus) - (% specific lysis of target cells infected with wild-type vaccinia virus).

 
The peptide-binding affinity of HLA-B*5101 is similar to HLA-B*5301, but is generally lower than that of HLA-B*3501. Three related reports of peptide binding by HLA-B*5101 (9, 38, 39) point out that this is not due to the substitution at position 116 in the PC pocket or at position 171 in the P1 pocket (also confirmed by the above data on B*5101 vs B*5102 peptide-binding affinity). This is in contrast to the finding that the P1 site is important for high-affinity peptide binding for HLA-B*2703 (40). In contrast, Sobao et al. (14) have shown that the lower affinity of HLA-B*5301 is derived from the Bw4 epitope in the C-terminal portion of the {alpha}1 helix by comparing the binding affinity of various peptides toward the HLA-B*5301 (Bw4) and HLA-B*3501 (Bw6). Both HLA-B*5301 and HLA-B*5101 have the Bw4 epitope, as discussed above; the structures show that these residues result in more space near the {alpha}1 helix and do not show the shift of {alpha}2 helix that is observed in HLA-B*3501. Thus, the distinctive features resulting in the pronounced preference for Tyr at PC in HLA-B35 also appear to confer an enhanced binding affinity.

In contrast, the speed of assembly is fast for HLA-B35 and HLA-B53, but slow for HLA-B51 (12). Because the peptide-binding affinity of HLA-B35 is higher than that of HLA-B53 and HLA-B51, this result cannot be explained by the binding affinity of the peptides. The unique structural characteristics that appear plausible causative agents for the slow assembly are: 1) a nonstandard N-terminal position; 2) the central region around P5, which is deeply buried within the groove.

Binding site of the human killer cell Ig-like receptor KIR3DL1

Cytotoxic assay data suggest that one of the human killer cell Ig-like receptors, KIR3DL1, can recognize the Bw4 epitope (at residues 77–83) of MHC class I (18, 19). The Bw4 epitope coincides with the putative binding site on other MHC class I alleles of KIR family members such as the KIR2Ds (41, 42). KIR3DL1 differs from the KIR2Ds in having an extra membrane distal Ig-like domain (D0) in its extracellular region. Rojo et al. (17) have reported that all three Ig-like domains of the KIR3DL1 extracellular region are needed to mediate the binding to HLA-B*5101-transfected cells. Thus, KIR3DL1 may have another binding site on HLA-B*5101 for D0 (or this domain may simply be required for the structural stability of KIR3DL1). However, mAb 5.133, which can inhibit the cytotoxicity of KIR3DL1+NK cells, binds only the D1-D2 domain part of KIR3DL1. This observation plus the data on the specificity for the Bw4 epitope support the hypothesis that KIR3DL1 and KIR2Ds share a common MHC class I binding site. Given the known allele-based specificities of the KIRs, the structural differences of this putative KIR recognition surface on different MHC class I structures may usefully be assessed. Recently, Fan et al. (43) determined the crystal structure of HLA-Cw4, which is a ligand for the KIR2D molecule KIR2DL1, and pointed out an electrostatic complementarity of the putative binding site of HLA-Cw4 to that of KIR2DL1. Comparison of HLA-B51 with HLA-Cw4 clearly shows that the electrostatic distribution around the equivalent part of the Bw4 epitope is markedly different, as shown in Fig. 7Go. There are four charged-to-neutral or neutral-to-charged residue replacements: Glu76(B51)-Val(Cw4), Ile80(B51)-Lys(Cw4), Leu82(B51)-Arg(Cw4), and Arg83(B51)-Gly(Cw4). Comparison of the presumed recognition site for the MHC class I on KIR2DL1 and KIR3DL1 shows that the charged residues (highlighted by Fan et al. (43)) are replaced by the opposite-charged or neutral amino acids: Leu71(3DL1)-Gln (2D), Ala72(3DL1)-Asp(2D), and His183(3DL1)-Asp(2D). The exposed hydrophobic amino acid, Phe, at position 45, which is believed to be a major contributor to the binding affinity of KIR2DL1 (44), is replaced by Ser. Thus, the changes in electrostatic properties on the proposed binding surfaces appear broadly complementary. It is also possible that the Ser45 substitution in the three-domain KIR may weaken the interaction for the D1-D2 domains, in line with additional binding affinity being contributed by novel interactions involving D0.



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  		FIGURE 7. Distribution of electrostatic potential at the surface of KIRs (KIR2DL1 and KIR3DL1) and cognate ligand MHC class I molecules (HLA-Cw4 and HLA-B51-KM2). Left bottom, HLA-Cw0401-QYDDAVYKL (43 ). Right bottom, HLA-B51-KM2 (this study). Left top, Ectodomain of KIR2DL1 (47 ). Right top, The two membrane-proximal Ig domains of a KIR3DL1 homology model based on the KIR2DL3 structure (48 ). Key residues for KIR-MHC recognition are indicated. The electrostatic potential surface was produced by GRASP (49 ) and is colored red and blue for negative and positive potential, respectively.

 


   Acknowledgments
 
We thank T. Juji, K. Tadokoro, K. Tokunaga, I. Kumagai, M. Matsushima, T. Yabe, K. Tsumoto, A, Kikuchi, T. Nakayama, K. Harlos, S. Ikemizu, N. Zaccai, J. R. Wyer, B. Willcox, and G. F. Gao for their advice and discussion. We also thank J. Diprose and J. Grimes for data collection and T. Matsuda for peptide-binding assay.


   Footnotes
 
1 K.M. has received support from a long-term fellowship of the Human Frontier Science Programme. This work is supported in part by the Japan Health Science Foundation and a Grant-in-Aid for Scientific Research (10557034 and 10167217) from Ministry of Education, Science, Sport, and Culture, Japan. The Oxford Center for Molecular Sciences is supported by the Biotechnical and Biological Sciences Research Council, Medical Research Council, and the Engineering and Physical Sciences Research Council. D.I.S. and E.Y.J. are supported by the Medical Research Council and Royal Society, respectively. Back

2 Address correspondence and reprint requests to Dr. Katsumi Maenaka, Structural Biology Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan. Back

3 Address correspondence and reprint requests to Dr. E. Yvonne Jones, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN U.K. Back

4 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; CREG, cross-reactive group, r.m.s.d., root-mean-square distance. Back

Received for publication April 12, 2000. Accepted for publication June 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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