The effect of NKT TCR alanine substitutions on CD1d–α-GalCer binding
| Residue . | mouse CD1d Kdeq . | human CD1d Kdeq . | human CD1d–α-GalCer contact . | Effect on affinity . |
|---|---|---|---|---|
| μM | μM | |||
| NKT WT | 0.94 ± 0.13 | 0.46 ± 0.05 | – | – |
| CDR1α | ||||
| Pro28α | 4.75 ± 0.13 | 5.44 ± 0.23 | α-GalCer Galactose; 6′-OH, 5′-O | *** |
| Ser30α | 1.16 ± 0.05 | 0.70 ± 0.07 | α-GalCer Galactose; 3′-OH, 4′-OH | * |
| CDR3α | ||||
| Asp94αa | >58 | >58 | α1-helix; Arg79 | *** |
| Arg95αa | >58 | >58 | α1-helix; Ser76, Arg79, Asp80 | *** |
| α-GalCer Galactose; 2′-OH, C-2 | ||||
| Sphingosine; 3′-OH, 4′-OH, C-3, C-4 | ||||
| Gly96αa | 1.19 ± 0.12 | 1.07 ± 0.13 | α2-helix; Gln150, Asp151 | * |
| α-GalCer Galactose; 2′-OH, 3′-OH, C-2 | ||||
| Ser97αa | >58 | >58 | α2-helix; Val147, Gln150 | *** |
| Thr98α | 0.90 ± 0.05 | 0.81 ± 0.07 | α2-helix; Gln150 | * |
| Leu99αa | >58 | >58 | α1-helix; Asp80, Phe84; | *** |
| α2-helix; Val147 | ||||
| Arg103αa | 2.56 ± 0.24 | 0.39 ± 0.06 | α1-helix; Arg79c | * |
| CDR2β | ||||
| Tyr48βb | >30 | >30 | α1-helix; Glu83, Lys86 | *** |
| Tyr50β | >25 | >25 | α1-helix; Glu83, Met87 | *** |
| Asn53β | 1.19 ± 0.12 | 0.73 ± 0.09 | α1-helix; Arg89c | * |
| Glu56βb | 0.72 ± 0.11 | 0.40 ± 0.03 | α1-helix; Lys86 | * |
| CDR3β | ||||
| Tyr103β | 0.82 ± 0.06 | 0.71 ± 0.13 | α2-helix; Gln150 | * |
| Residue . | mouse CD1d Kdeq . | human CD1d Kdeq . | human CD1d–α-GalCer contact . | Effect on affinity . |
|---|---|---|---|---|
| μM | μM | |||
| NKT WT | 0.94 ± 0.13 | 0.46 ± 0.05 | – | – |
| CDR1α | ||||
| Pro28α | 4.75 ± 0.13 | 5.44 ± 0.23 | α-GalCer Galactose; 6′-OH, 5′-O | *** |
| Ser30α | 1.16 ± 0.05 | 0.70 ± 0.07 | α-GalCer Galactose; 3′-OH, 4′-OH | * |
| CDR3α | ||||
| Asp94αa | >58 | >58 | α1-helix; Arg79 | *** |
| Arg95αa | >58 | >58 | α1-helix; Ser76, Arg79, Asp80 | *** |
| α-GalCer Galactose; 2′-OH, C-2 | ||||
| Sphingosine; 3′-OH, 4′-OH, C-3, C-4 | ||||
| Gly96αa | 1.19 ± 0.12 | 1.07 ± 0.13 | α2-helix; Gln150, Asp151 | * |
| α-GalCer Galactose; 2′-OH, 3′-OH, C-2 | ||||
| Ser97αa | >58 | >58 | α2-helix; Val147, Gln150 | *** |
| Thr98α | 0.90 ± 0.05 | 0.81 ± 0.07 | α2-helix; Gln150 | * |
| Leu99αa | >58 | >58 | α1-helix; Asp80, Phe84; | *** |
| α2-helix; Val147 | ||||
| Arg103αa | 2.56 ± 0.24 | 0.39 ± 0.06 | α1-helix; Arg79c | * |
| CDR2β | ||||
| Tyr48βb | >30 | >30 | α1-helix; Glu83, Lys86 | *** |
| Tyr50β | >25 | >25 | α1-helix; Glu83, Met87 | *** |
| Asn53β | 1.19 ± 0.12 | 0.73 ± 0.09 | α1-helix; Arg89c | * |
| Glu56βb | 0.72 ± 0.11 | 0.40 ± 0.03 | α1-helix; Lys86 | * |
| CDR3β | ||||
| Tyr103β | 0.82 ± 0.06 | 0.71 ± 0.13 | α2-helix; Gln150 | * |
* represents an effect in which NKT TCR alanine substitution results in a <4-fold reduction in binding to CD1d–α-GalCer compared to the WT value. *** represents an effect in which NKT TCR alanine substitution with a >10-fold reduction in binding to CD1d–α-GalCer when compared to the WT value.
Conserved between the human Jα18 gene segment and the mouse Jα18 gene segment homologue.
CDR2β framework residues.
Denotes contacts that are not maintained in the second NKT TCR–CD1d–α-GalCer complex within the asymmetric unit.