Dimorphism at position 283 in the D2 domain changes KIR3DL1 conformation
KIR3DL1*015 is the most prevalent form of 3DL1 and has the consensus sequence (10
). Using the 3DL1*015-EGFP construct as template, 42 different point mutants were made, each corresponding to a natural substitution present in another form of 3DL1 or in 3DS1, the allotypic activating receptor. Mutant cDNA were transiently transfected into Jurkat T cells, which express neither KIR nor any 3DL1-binding HLA class I allotype. The transfected cells were assessed for binding to three phycoerythrin-conjugated monoclonal antibodies: DX9, specific for 3DL1 (25
); Z27 that binds strongly to 3DL1 and weakly to 3DS1 (26
); and 177407 that was raised against 3DL1, but also cross-reacts with KIR3DL2 (data not shown). The binding data obtained with these antibodies was normalized to the abundance of KIR3DL1, as measured by EGFP fluorescence (12
Interaction of monoclonal antibodies with KIR3DL1*015 point mutants
Twenty mutations affected binding to one or more of the anti-KIR3DL1 antibodies; all were in the extracellular domains, with greater abundance in D0 and D2, than D1 (). Mutation at three positions greatly reduced binding to all three antibodies (), indicating that threonine 203 in 3DL1*037 and proline 290 in 3DS1*045 likely reduce cell surface expression of these rare variants, as previously shown for serine 86 in 3DL1*004 (12
). Overall, DX9 and Z27 exhibited similar reaction patterns with the panel of mutants, although clear differences were observed for the position 30, 32 and 166 mutants. In contrast, antibody 177407 exhibited a very different specificity: only eight mutations reduced 177407 binding, compared to 15 for DX9 and 16 for Z27, and the mutations affecting 177407 binding are largely non-overlapping with those that affect DX9 and Z27 binding (). These results point to considerable overlap between the epitopes recognized by DX9 and Z27, but little overlap between them and the epitope recognized by 177407.
Mutation at position 283 in D2 uniquely changes the capacity of KIR3DL1*015 to bind anti-KIR3DL1 monoclonal antibodies
Mutation at position 283 in the D2 domain gave a unique pattern of antibody binding: substitution of leucine for tryptophan increased the binding to 177407 while reducing the binding to DX9 and Z27 ( and ). Thus tryptophan 283 favors binding to DX9 and Z27, whereas leucine 283 favors binding to 177407. Position 283 in 3DL1 corresponds to position 188 in KIR2DL, an invariant tryptophan in all KIR2DL. Crystallographic structures of KIR2DL1, 2 and 3 showed tryptophan 188 is one of eleven hydrophobic residues forming an inter-domain core that affects the hinge angle between the D1 and D2 domains (1
). These residues are conserved in all KIR2DL and KIR3DL1, the only exception being position 283 in 3DL1 where leucine is the only alternative to the otherwise constant tryptophan.
That the constituent residues of the hydrophobic core are conserved in 3DL1*015 predict it has a core like that of KIR2DL. In the 3DL1*015-W283L mutant, replacement of the bulky, aromatic tryptophan at position 283 by the smaller, aliphatic leucine is predicted to alter the hinge angle and change the relative orientation of the D1 and D2 domains. Such a conformational difference could account for the observed differences in antibody binding between *015 and *015-W283L. Because residue 283 is buried and surface inaccessible in the 2DL2 structure, the tryptophan/leucine polymorphism in 3DL1 is more likely to exert its effects on antibody binding in an indirect manner rather than contributing directly to antibody contact.
Dimorphism at position 283 distinguishes the two KIR3DL1 lineages
Tryptophan and leucine at position 283, are both at high frequency in human populations, and comprise one of seven dimorphisms that distinguish the 015- and 005-lineages of 3DLl allotypes (10
). These positions are distributed throughout the extracellular domains, but only substitution at position 182 in D1 and 283 in D2 had significant effects upon antibody binding (). Whereas substitution of tryptophan (015-lineage) to leucine (005-lineage) increased 177407 binding and decreased DX9 and Z27 binding, substitution of proline (015-lineage) to serine (005-lineage) at position 182 reduced DX9 and Z27 binding but had little effect on 177407 (). Thus both the serine 182 and leucine 283 residues present in 3DL1*005 have the effect of reducing interaction with DX9 and Z27 while preserving or increasing the interaction with 177407. In each case the effects are quantitative and for neither position 182 or 283 did the lineage-specific substitution abrogate binding of any antibody.
Mutation at lineage-specific sites and comparison of anti-KIR3DL1 antibodies
At the seven positions which distinguish the two 3DL1 lineages, 3DS1 has four identical to 3DL1*015 (-20, 47, 182 and 283) and three identical to 3DL1*005 (-9, 2 and 54). That 3DS1 is not significantly more like one 3DL1 lineage than the other, is consistent with 3DS1 and 3DL1 having diverged before 3DL1 split into the 005- and the 015-lineages (10
). Also consistent with this evolutionary model, 3DS1 is distinguished from both 3DL1 lineages at six positions (58, 92, 138, 163, 166 and 199) that have no overlap with those distinguishing the two 3DL1 lineages. When the 3DS1 residues at these positions were individually introduced into 3DL1*015, the resulting mutants retained substantial reactivity with all three antibodies, which bound to levels >50% of that for 3DL1*015, except for mutation at position 166 which reduced DX9 binding by ~60% (). Thus the very low Z27 binding and the lack of DX9 binding that characterize 3DS1 (26
) cannot be attributed to the effect of a single substitution. Consequently, these properties likely arise from the concerted effect of two or more of the six 3DS1-specific substitutions.
We also assessed five additional anti-KIR3DL1 monoclonal antibodies for binding to 3DL1*015, *015-W283L, *005 and *004. This comparison revealed no novel specificity: three antibodies were shown to be similar to DX9, two were similar 177407, and none resembled Z27 ().
Conformational change caused by position 283 dimorphism does not determine the high- and low-binding phenotypes of 3DL1 allotypes
Our previous studies have shown that peripheral blood NK cells expressing particular KIR3DL1 allotypes can be distinguished by the amount of DX9 or Z27 antibody they bind (11
). For example, 3DL1*015 binds these antibodies to high level, 3DL1*005 to low level. To see if these differences correlated with the conformational change conferred by polymorphism at position 283, we examined the binding of 177407 to peripheral blood NK cells of defined KIR3DL1/S1
genotype and compared the results to those obtained with DX9 and Z27 (). If conformational change was the basis for the difference, the expectation was that 3DL1*005-expressing NK cells would bind more 177407 than 3DL1*015-expressing NK cells. That was not the case: all three antibodies distinguished high binding (3DL1*015 and *020) from low binding (*005 and *007) allotypes. Although antibody 177407 is different from DX9 and Z27 in conformational sensitivity, it too can distinguish between the high- and low-expressing 3DL1 allotypes. Thus this major distinction, between high- and low-binding allotypes, is not correlated with either the different epitope specificity of the antibodies or the presence at position 283 of either tryptophan (*015, *020 and *007) or leucine (*005). It can therefore be attributed to varying levels of cell-surface expression of KIR3DL1, as was previously concluded (11
Like DX9 and Z27, the 177407 antibody distinguishes KIR3DL1 allotypes expressed on the surface of peripheral blood NK cells
One consistent difference was that *005 bound more 177407, but less DX9 and Z27, than *007 (). Thus *005, which has leucine 283, binds better to 177407, whereas *007, which has tryptophan 283, bound better to DX9 and Z27, an observation consistent with the comparison of *015 and *015-W283L (). Thus this difference in antibody binding could reflect conformational change between *005 and *007, rather than their relative abundance at the cell surface. However, the effect is small and elevates neither *005 nor *007 into the range observed for high-binding allotypes such as *015.
Dimorphisms in D0, D1 and D2 combine to alter avidity of KIR3DL1 for A24nef
3DL1*015 and *005 differ by five substitutions: three in D0 (positions 2, 47 and 54), one in D1 (position 182) and one in D2 (position 283). These substitutions are recombined in the common 3DL1*001 allotype, which has D0 derived from *005 and D1+D2 derived from *015. Previous qualitative analysis showed that the A24nef tetramer, comprising HLA-A*2402 and a peptide derived from the nef2 protein of HIV, binds to 3DL1*001, *005, and *015 (14
). Here, we quantified the binding of A24nef to Jurkat cells expressing 3DL1*001, *005, *015 and selected mutants (). The results are summarized in and representative examples of the flow cytometric analysis are shown in . As controls, the levels of 3DL1 cell-surface expression were assessed using the anti-KIR3DL1 antibodies (). The observed binding was highly specific for the A24nef tetramer, as no binding was seen with five other tetramers tested (see Materials and Methods).
Interactions between D0 polymorphisms and D1+D2 polymorphisms determine the binding of 3DL1 to the A24nef tetramer
Jurkat cells transfected with 3DL1*001 bound A24nef to a higher level (mfi:12.9) than cells transfected with either 3DL1*005 (9.8) or *015 (8.6). This hierarchy of binding did not correspond to that observed with the anti-KIR3DL1 antibodies, all of which bound less to 3DL1*001 than *015; and 177407 bound less to 3DL1*001 than *005 (). Thus the elevated binding of A24nef to 3DL1*001 compared to*005 and *015 reflects superior avidity for A24nef rather than greater abundance at the cell surface.
These results demonstrate that in 3DL1*001, the combination of the 3DL1*005 D0 with the D1+D2 of 3DL1*015 binds more effectively to A24nef than the ‘parental’ domain combinations in either 3DL1*015 or 3DL1*005. The reciprocal recombinant to 3DL1*001 combines the D0 of 3DL1*015 with D1+D2 of 3DL1*005. Such an allotype has not been identified in any human population (10
), but is identical in structure to the *015-P182S-W283L mutant. Although we found this mutant was well expressed at the cell surface as detected by antibodies, it was unable to bind A24nef (). Thus the combination of the 3DL1*015 D0 and the 3DL1*005 D1+D2 is far inferior to either 3DL1*001, *005 or *015 in binding A24nef. These results clearly demonstrate the important role of complementary polymorphisms in D0 and D1+D2 for the functional interaction of KIR3DL1 with HLA class I ligands. In particular, the inter-lineage recombination that produced 3DL1*001 combined features of 3DL1*015 and *005 that synergize to increase the avidity of 3DL1*001 for the A24nef tetramer. Since its origin in one individual, 3DL1*001 has spread throughout the world, being now present in all major population groups except Amerindians (10
). These results also demonstrate that the ‘enhancing’ effect D0 has on the binding of HLA class I to KIR3DL1 does not depend on interaction between two cell membranes and formation of an immunological synapse, one of several models we considered previously (9
), as the effect is clearly seen here for the interaction of tetrameric HLA class I ligand with cell-surface associated receptor.
Comparing the double mutant at positions 182 and 283 with the single mutants showed that the inability of *015-P182S-W283L to bind A24nef was principally due to leucine 283, with a minor contribution from serine 182 (). The loss of A24nef binding by *015-W283L is paralleled by increased binding to antibody 177407. Thus the conformation of 3DL1 that favors binding to 177407 appears less permissive to A24nef binding. Although the introduction of serine 182 and leucine 283 into 3DL1*015 abrogates binding to A24nef, the presence of these residues in *005 is permissive for A24nef binding. Because residues 182 and 283 are the only differences in D1+D2 between 3DL1*015 and *005, these observations imply that the substitutions distinguishing 3DL1*005 from *015 in D0 affect the conformation of D1+D2 to make 3DL1*005 permissive for A24nef binding. Highest binding is, however, achieved by the combination present in 3DL1*001 of D0 from *005 with D1+D2 from *015 (). As 3DL1*001 only differs from *015 at positions 2, 47 and 54 in D0, its superiorA24nef binding must arise from one or more of these substitutions. Single mutations at each of these positions bound A24nef like 3DL1*015, as did a double mutant at positions 47 and 54. Either all three substitutions are necessary for superior binding, or it is the combination of methionine 2 with either isoleucine 47 or isoleucine 54. In 3DL1*005 these same substitutions compensate for the presence of serine 182 and leucine 283, permitting this allotype to bind A24nef at a level comparable to that of 3DL1*015.
Model for the structure of KIR3DL1*015 and its interaction with A*2402
Previously, alanine substitutions at positions 49, 50, 51 and 52 in D0 increased Bw4+
HLA-B binding by 3DL1, but not at twelve other positions examined (9
). Alanine at either position 50 or 51 could also compensate for the reduced binding to 3DL1 caused by mutating residue 76 in HLA-B from glutamate to alanine (9
). Residues 49-52 are invariant in 3DL1, but they are flanked by the dimorphisms at residues 47 and 54 shown here to modulate D0 function. Positions 5, 20, 31, 32 and 51 in D0 have also been identified as sites of positive natural selection (10
). Of these, 5, 31, 32 and 51 are predicted to cluster on the D0 surface, whereas 20 lies apart, but also on the surface. Together these functional and phylogenetic analyses suggest that residues 47-54 mark a functional site of interaction between the D0 domain of 3DL1 and bound Bw4+
HLA class I ligand. In the absence of a crystallographic structure for 3DL1, we explored this possibility by using multiple sequence alignment, homology modeling, and the functional data to construct structural models for 3DL1*015 and its interaction with A24nef.
Model of the 3DL1*015 structure
D1+D2 of 3DL1*015 were modeled on D1+D2 in the crystallographic structure of 2DL1 bound to HLA-C*04 (PDB file 1IM9) (19
). Based upon similarities observed in the multiple sequence alignment (), the D0 domain of 3DL1*015 was modeled on D1 in the crystallographic structure of 2DL2 bound to HLA-C*03 (see Materials and Methods). The models can be viewed at http://csb.stanford.edu/karine/3DL1
. The high sequence identity between D1 and D2 of KIR3DL1 and their KIR2DL counterparts (~77% and ~88%, respectively), gives confidence in the validity of the structural models for 3DL1 D1 and D2. In contrast, D0 of 3DL1 has only ~38% sequence identity with D2 of 2DL2. However, the structure of a protein with >30% sequence identity to a known structure can often be predicted with accuracy equivalent to a low-resolution X-ray structure (32
). Consequently, detection of secondary structures elements like α-helices and β-sheets is more reliable, whereas attribution of length and conformation for loops are less accurate. Similarly for the position of D0 relative to D1/D2 which was inferred on the basis of functional properties and structural constraints. Shifts of D0 with respect to D1-D2 might occur, because D0 is not predicted to interact strongly with D1, and the linker between them appears flexible. We should stress that the model of 3DL1*015 presented here has limitations in its structural foundation, but is consistent with current knowledge of the function and polymorphism of KIR3DL1/S1.
Multiple sequence alignment for the extracellular ligand-binding domains domains (D0, D1 and D2) of human KIR
Whereas the interaction of D1 with D2 in the model of 3DL1*015 appears to be dominated by a core of eleven hydrophobic residues, sequence comparison revealed no equivalent core of hydrophobic residues between D0 and D1 (): only one hydrophobic interaction, between tryptophan 13 of D0 and glycine 138 of D1, being predicted. Constraining the position of D0 with respect to D1+D2 is the length of the linker (residues 96-103) between D0 and D1. Consequently, the only possible position for D0 that permitted contact with HLA class I ligand bound to D1+D2, as deduced from previous (9
) and present studies in mutagenesis, was in the space between D1 and D2.
In this orientation three flexible loops of D0 (residues R48-L54, Y30-N35, and H78-V92) and the N-terminal region (H1-P9) are positioned close to the ligand-binding site. To model the rotational orientation of D0 with respect to D1+D2 we applied four additional constraints. Firstly, the N-terminal region could not be buried through interdomain interactions, because of its hydrophilicity and potential flexibility. Secondly, our functional analysis implicated residues 2, 47 and/or 54 in the enhanced binding of ligand. The positioning of residue 2 in the N-terminal region was limited by the constraint to keep this region accessible; residue 47 is located in a β-sheet and could not be placed near the binding site without stretching the D0-D1 linker; and so residue 54 was placed as close to the ligand-binding site as possible. Thirdly, residue 86 was made surface accessible because it is implicated in chaperone interactions (12
). Fourthly, we required that the interaction of D0 with the other two domains be stabilized by hydrophobic interaction. Of the possible hydrophobic contacts examined, only those involving tryptophan 13 of D0 were compatible with the other three constraints. Moreover, tryptophan 13 provided an excellent candidate for fitting into the hydrophobic core of D1+D2, notably by making stacking interactions with tyrosine 281 of D2. In the deduced model, the D0 domain is seen to interact intimately with both D1 and D2, forming a compact, rather than an elongated, structure ().
Model for the interaction of the D0, D1 and D2 domains in KIR3DL1
Of the non-covalent interactions between domains, those between D0 and D1 are fewest in number in the model. Hydrophobic interactions are made by residues 187, 188 and 191, part of a stretched loop maintained by the functionally influential proline 182 (). Arginine 20, which defines the smaller of the two sites of positive natural selection on the D0 domain surface (10
), interacts with residues 96-103 in the hinge between D0 and D1. Substitution of arginine 20 for glutamine may alter conformation, as detected by reduced 177407 antibody binding to *015-R20Q ( and ), by affecting the angle between the two domains. Although D0 and D2 are not directly connected in the polypeptide chain, the model predicts they interact strongly through hydrophobic contacts, a salt bridge and polar interactions. As well as the hydrophobic interaction between tyrosine 281 in D2 and tryptophan 13 in D0, arginine 277 in D2 is surrounded by four residues of D0: isoleucine 49, phenylalanine 50, histidine 51 and glycine 52 and interacts with phenylalanine 50 and arginine 53.
Model for the interaction of KIR3DL1*015 with HLA-A*2402
The crystallographic structure of A*2402 (PDB file 2BCK) (22
) was first docked onto the D1+D2 domains of 3DL1*015 using the complex of HLA-C*04 bound to KIR2DL1 as the model (19
). The configurations of certain side chains were then changed manually, either to avoid steric incompatibility between the two proteins or to optimize polar or salt bridge interactions. Finally, the energy of the system was minimized to optimize the contacts between 3DL1*015 and A*2402. Clearly, the limitations to the model of 3DL1*015 will extend to this model of the complex formed by 3DL1*015 and A*2402, which has additional potential for inaccuracy that derives from using the complex of HLA-C*04 and KIR2DL1 as template for docking HLA-A*2402 on KIR3DL1.
In the model the D0, D1 and D2 domains of 3DL1*015 come together to form a flat surface predicted to contact A*2402 using eight loops: two in D0 (residues 30-34 and 51-55); three in D1 (residues 115-118, 138-142 and 162-169); and three in D2 (198-202, 225-230 and 275-282) (). Consistent with this contribution of D0, the larger site of positive natural selection on the D0 domain surface (10
) comprises residues from both the loops (31, 32 and 51) as well as position 5 from the amino-terminal region (which is near the binding site). Importantly, leucine 54 in D0 appears to contact the essential arginine 83 in the Bw4 motif, and arginine 31 in D0 contacts asparagine 86, also in the helix of the A*2402 α1
domain. As in the complexes of KIR2DL with HLA-C (19
), the binding loops of the D1 and D2 domains of 3DL1*015 are predicted to interact with the α1
helices of A*2402, respectively.
Model for the binding of HLA-A*2402 to 3DL1*015
A striking feature of the model is the predominance of hydrophobic interactions between 3DL1*015 and A*2402, and loss of the conserved electrostatic bonds that characterize the binding of KIR2DL to HLA-C (19
). For example, the two salt bridges common to the interaction of KIR2DL with HLA-C and involving the D1 domain (D135-R145 and D183-K146) are absent from the modeled complex of 3DL1*015 bound to A*2402. Instead, there are two salt bridges involving D2 (E282-R83 and E201-R145), which make a greater contribution to interactions with HLA class I than observed for KIR2DL. However, the majority of the binding is mediated by hydrophobic contacts with D1 (L54-R83, I139-R83, K141-R83, L166-E76/ pCys8, M165-E76, F276-I142 and H278-I142) including one between leucine 166 and the cysteine at position 8 in the peptide bound to A*2402.
Arginine 83 of A*2402 is predicted to bind to a specific pocket of 3DL1*015
Interaction of KIR2D with HLA-C involves specificity-determining residues at position 44 in the D1 domain of the KIR and at position 80 of HLA-C. For example, the lysine 80 of HLA-C*04 is bound in a pocket by 2DL1, making hydrophobic interaction with methionine 44 of 2DL1, as well as a hydrogen bond to serine 184 and a salt bridge with glutamate 187. Whereas the specificity-determining residue of the HLA-C is position 80, the residue essential for Bw4 binding to 3DL1 is arginine 83 (7
). In the model, arginine 83 can make an analogous set of interactions to those observed for lysine 80 in the structure of 2DL1 bound to HLA-C*04 (18
). Thus it forms a hydrophobic interaction with isoleucine 139 (equivalent to methionine 44 in 2DL1), a hydrogen bond with serine 279 (equivalent to serine 184 in 2DL1) and a salt bridge with glutamate 282 (equivalent to glutamate 187 in 2DL1).
The arginine 83 binding pocket of 3DL1*015 is formed at the junction of the D0, D1 and D2 domains and can be divided into three strata: upper, intermediate and lower. Upper residues are hydrophobic (L54, I139 and L166) or positively charged (K141 and H278), intermediate residues are polar (S11, H29, Q56, G138, R277, S279 and E282), and lower residues are components of the hydrophobic core (W13, A169, G170, P280 and Y281). The salt bridge that arginine 83 makes with glutamate 282 is part of a rigid loop structured by tyrosine 281 and tryptophan 283, residues deeply anchored in the hydrophobic core of 3DL1. Substitution of leucine for tryptophan at position 283 of 3DL1*015 is predicted to change the position of glutamate 282 in the binding pocket, consistent with the differential antibody reactivity and loss of A24nef binding by mutant 3DL1*015-W283L. Inability to form the salt bridge between arginine 83 of A*2402 and glutamate 282 of 3DL1*015 could account for the complete loss of A24nef binding.
Functional tests of the structural model
To test key features of the modeled interaction between 3DL1*015 and A24nef, additional mutants were made and, in combination with selected mutants from the first set, examined for binding to A24nef (). The combination of serine 182 and leucine 283 is permissive for A24nef binding in the presence of one D0 motif (M2, I47, I54 – as in *005) but not the other (V2, V47, L54 – as in *015-P182S-W283L). The model suggested this difference was caused by altered flexibility of the N-terminal region (residues 1-8). With the non-permissive motif, this region was predicted to be flexible, whereas with the permissive motif it was predicted to be tethered, with methionine 2 being buried between phenylalanine 9 and tyrosine 30. There the juxtaposition of methionine 2 and leucine 54 reinforced the hydrophobicity and strength of the interaction between D0 and A*2402. Consistent with this model, mutant *005-Y30C, in which tyrosine 30 was substituted for the smaller cysteine, failed to bind A24nef ().
Binding of A24nef to mutants of 3DL1*015 and 3DL1*005
The model predicts that a cluster of electropositive residues on the D0 surface, comprising arginine 31, histidine 32, arginine 33 and arginine 53, is important for ligand binding. Consistent with this hypothesis, increasing the electropositivity by substituting arginine for histidine at position 32, gave *005-H32R stronger avidity for A24nef than *005, *015 and *001. The only hydrophobic interaction predicted between D0 and D1 involves tryptophan 13 (D0) and glycine 138 (D1). Glycine 138 is also predicted to form hydrophobic interactions with tyrosine 281 of D2 and arginine 83 of A*2402. Both tryptophan 13 and glycine 138 are conserved in 3DL1, but glycine 138 is replaced by tryptophan in 3DS1 (10
). Although mutant *015-G183W showed no perturbation in antibody binding (), binding to A24nef was severely reduced ().
The model predicts that arginine 277 in D2 has hydrophobic interactions with the loop comprising residues 47-56 of D0 and electrostatic interactions with aspartate 230 of D2. Proximal to arginine 277 is a flexible loop containing phenylalanine 276, histidine 278 and serine 279 of KIR3DL1 that are seen to interact with residues isoleucine 142, lysine 146 and isoleucine 80 of A*2402. Thus residue 277 has a network of interactions with residues in D0, D2 and A*2402. In four natural allotypes (3DL1*006, *022, *034 and *035) arginine 277 is replaced by cysteine. Introducing cysteine 277 into 3DL1*015 abrogated A24nef binding () and caused some reduction in antibody binding. In contrast, replacement of arginine 277 with histidine, the residue distinguishing the rare 3DS1*048 allotype (10
), preserved the capacity to bind A24nef. This indicates the importance of the network of electrostatic interactions around arginine 277 for ligand binding, as predicted by the model.
Three 3DS1-specific residues abrogate A24nef binding when introduced into 3DL1
We examined the capacity of KIR3DS1, the activating counterpart of KIR3DL1, to bind A24nef. Jurkat cells tranfected with 3DS1*013 failed to bind A24nef (), although cell-surface 3DS1 was detected with the Z27 antibody. A similarly negative result was obtained with 3DL1*004, which is sequestered inside cells and does not reach the cell surface (12
). That KIR3DS1 fails to bind A24nef, is consistent with previous failure to detect interaction of 3DS1 with Bw4+
HLA class I (34
). Mutant *015-G183W is an example where the substitution is to a 3DS1-specific residue and this led to reduction in A24nef binding (). Three other mutants containing a single 3DS1-specific residue at either position 163, 166 and 199, were shown to be incapable of binding A24nef.
In the model, proline 163 and leucine 166 are part of a flexible HLA-binding loop comprising residues 161-166 that connects strands E and F in the D1 domain. Proline 163 is predicted to structure the conformation of this loop while leucine 166 forms hydrophobic contacts with glutamate 76 and isoleucine 80 of A*2402, and also with the cysteine at position 8 of the bound nef peptide. Proline 163 and leucine 166 are completely conserved in 3DL1, but in 3DS1are replaced by serine 163 and arginine 166. Mutants *015-P163S and *015-L166R were b oth expressed at the cell surface, the former giving reduced binding with all three antibodies, the latter only with DX9 (). Despite cell-surface expression, neither mutant bound A24nef, consistent with an essential role for proline 163 and leucine 166 in ligand binding ().
Proline 199, in a flexible HLA-binding loop of D2, is predicted to make hydrophobic contact with alanine 150 of A*2402 and with cysteine 8 of the nef peptide. Proline 199 is present in all 3DL1 (except the rare 3DL1*040) but is replaced by leucine in 3DS1. A24nef binding of *015 was abrogated when proline 199 was replaced by leucine, whereas DX9 and Z27 antibodies bound more strongly and binding of the177407 antibody was decreased by ~20%. Although having no major effect on the expression and conformation of 3DL1, this substitution profoundly affected the functional interaction with HLA class I. Tryptophan 138, serine 163, arginine 166 and leucine 199 are all substitutions that distinguish 3DS1 from 3DL1; individually, three of them (positions 163, 166 and 199) abrogate A24nef binding and the fourth (position 138) effects a reduction. Although we have only examined a single combination of Bw4+ HLA class I and peptides, our results illustrate the potential for the 3DS1-specific residues to reduce avidity for HLA class I. ().
In summary, the properties of this series of site-directed mutants are consistent with the models of 3DL1*015 and its complex with A*2402. Although the results of such analysis cannot prove the validity of the models, they show their value as tools for the design of future experiments until such time as crystallographic structures for KIR3DL1 and 3DS1 are achieved.