SPR analysis of the binding of CD1d-αGalCer to the Vα24− and Vα24+ NKT TCRs.

<p>(A) SPR binding curves of CD1d-αGalCer. Shown are the curves and fits used for kinetic analysis to surface-immobilized Vα24− (clone J24N.22) (top) and Vα24+ (clone J24L.17) (bottom) NKT TCRs. For the Vα24− TCR: k_on = 1.62×10⁵±0.11×10⁵ (Ms)⁻¹, k_off = 0.342±0.007 s⁻¹, and K_D = 2.1 µM; for Vα24+ TCR: k_on = 1.94×10⁵±0.16×10⁵ (Ms)⁻¹, k_off = 0.414±0.010 s⁻¹, and K_D = 2.1 µM. Grey traces represent experimental data and black lines fittings to a Langmuir 1∶1 kinetic model. Curves represent the following concentrations of analyte: 0, .037, .111, .333, 1, and 3 µM. In addition, equilibrium analysis was performed on these curves and those for 9 µM and 27 µM; the fits and calculated K_Ds for this analysis are shown as inserts. (B) Alanine scanning mutants of key residues in the Vα24− TCR interface were screened by SPR, and their k_on and k_off values are shown plotted on a k_on versus k_off plot with K_D isotherms shown along with the values for the wild-type Vα24− TCR and the Vα24+ iNKT TCR (J24.L17).</p

Complex structure of the Vα24− TCR with CD1d-αGalCer.

<p>(A) Left panel, ribbon representation of the human Vα24− TCR J24.N22 (slate, α chain; orange, β chain) in complex with human CD1d-β₂m (ribbon, white) and αGalCer (sticks, yellow). Right panel, the Vα24− TCR-CD1d-αGalCer complex (orange) is shown superimposed with a Vα24+ NKT TCR-CD1d-αGalCer complex (PDB ID: 3HUJ; TCR in green) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001412#pbio.1001412-Pellicci1" target="_blank">[19]</a>. Complexes were aligned via the main-chain CA carbons of the CD1d heavy chain. (B) Positioning of the four different human NKT TCR loops on the CD1d-ligand surface: purple, Vα24− TCR; orange, Vα24+ NKT TCR-CD1d-βGalCer (PDB ID: 3SDX) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001412#pbio.1001412-Pellicci2" target="_blank">[23]</a>; berry, Vα24+ NKT TCR-CD1d-αGalCer complex; and green, the iNKT TCR-CD1d-LPC complex (PDB ID: 3TZV) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001412#pbio.1001412-LopezSagaseta1" target="_blank">[25]</a>. Shown is CD1d (white)-αGalCer (yellow) from the Vα24− TCR-CD1-αGalCer complex. (C) Upper panel, footprint of the Vα24− TCR on the surface of CD1d-αGalCer. Residues that are contacted by the TCR α chain, β chain, or both are colored in blue, orange, and green, respectively. Lower panel, footprint of the Vα24+ NKT TCR on the surface of CD1d-αGalCer; colors of CD1d are as for the Vα24− TCR. (D) Electron density of the αGalCer ligand in the Vα24− TCR-CD1d-αGalCer complex. Electron density, shown as a blue mesh, corresponds to a composite omit map (2Fo–Fc) contoured at 1σ around the αGalCer ligand (yellow). CD1d is shown in grey ribbons, and the α2 helix has been omitted to facilitate the visualization of the ligand. The TCR α and β chains in light blue and yellow-orange, respectively.</p

The role of the TCR β chain in Vα24− TCR engagement of CD1d-αGalCer.

<p>(A) Contacts made by the CDR2β loops of the Vα24− and Vα24+ NKT TCRs with CD1d. CD1d is shown as grey ribbons and the Vα24− and Vα24+ NKT TCR CDR2β loops in yellow-orange and pale-green color, respectively. Hydrogen bonds (≤3.3 Å) and salt bridges are shown as yellow dotted lines for the Vα24− and Vα24+ NKT TCRs. (B) Electron density (Fo-Fc omit map, contoured at 3σ) for the CDR3β loop of the Vα24− TCR is shown as blue mesh together with the CDR3β in stick representation in yellow-orange; CD1d is shown in grey ribbons and αGalCer in yellow sticks. Potential H-bond is displayed as dotted, yellow line.</p

Atomic contact comparison of iNKT-TCRs, CD1d, and lipid ligands.

<p>Atomic contact comparison of iNKT-TCRs, CD1d, and lipid ligands.</p

Unique and conserved contacts of the Vα24− TCR CDRα loops with CD1d-αGalcer.

<p>Contacts made by the CDR1α, CDR2α, and CDR3α loops to CD1d− αGalCer are represented in (A), (B), and (C) respectively. CD1d is shown as grey ribbons, TCR CDRα loops in light blue, and αGalCer is represented as yellow sticks. A model of αGlcCer generated via superposition of the CD1d/αGlcCer structure (PDB ID: 3ARG) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001412#pbio.1001412-Wun2" target="_blank">[35]</a> is shown in cyan in (A) in comparison with αGalCer. Positions of the 4′OH and 6′OH of αGlcCer are indicated. Water molecules in (B) are displayed as pale-green spheres. Hydrogen bonds (≤3.3 Å) are shown as yellow dashed lines. (C) Conserved hydrogen bonds between the Vα24− and Vα24+ NKT TCR CDR3α loops are shown as dashed lines colored cyan.</p

Data collection and refinement statistics (molecular replacement).

<p>Values in parentheses are for highest resolution shell.</p

Alignment of the Vα24− and Vα24+ NKT TCR CDR loop sequences.

<p>Alignment of the Vα24− and Vα24+ NKT TCR CDR loop sequences.</p