(A) CD spectra of CD1c and CD1copt at 20°C and 95°C. (B) The CD1c and CD1copt constructs both bind the F10/21A3 single-chain antibody as assessed by size exclusion chromatography. SDS-PAGE analysis of the shifted peak fractions confirms the presence of complexes between both CD1c and CD1copt with F10/21A3. (C) Both CD1c and CD1copt bind to the CD1c specific monoclonal antibody L161 (lanes 2 and 4, respectively) as evidenced by native gel shift compared to L161, CD1c or CD1copt alone (lanes 1, 3 and 5, respectively). (D) Loading efficiency of a variety of lipids is conserved between CD1c and CD1copt: CD1c or a fully glycosylated version of CD1copt are shown on a native isoelectric focusing gels as unloaded or loaded with the charged lipid GT1b (left box). Addition of saposins and a series of lipids resulted in similar shifting of the CD1c-GT1b and CD1copt-GT1b complexes upon displacement of GT1b (right box). Shown are: lactosylceramide (LC), PBS57, sulfatide, GM3 or GSL-1 and the indicated saposin. (E) CD1c-restricted T cell recognition of wild- type and optimized CD1c molecules. Wild-type and CD1copt proteins were coated on a micro-titer plate and pulsed with the indicated concentrations of synthetic MPM lipid (filled symbols), then washed and used to stimulate IFN-γrelease by the CD8–1 T cell line. Open symbols show CD8–1 T cell responses to wild-type and CD1copt molecules that were pulsed with vehicle alone. Shown are the means and standard deviations of IFN-γconcentrations from 3 replicates, as quantified by a standardized ELISA.

(A) Top view of the ligand binding domain as a ribbon (top panel) and surface diagram (bottom panel). The CD1c heavy chain is shown in light magenta, MPM in yellow and C₁₂ in light green. MPM is bound in the A′ pocket while a C₁₂ is bound in the open F′ groove. Two ordered water molecules that solvate the MPM head group are shown as purple spheres. Omit map (green) and 2Fo-Fc (white) densities at a σ=1 and 1.4, respectively are shown for both ligands. (B) Side view of CD1c shown as a ribbon diagram with β₂m colored light purple. The alkyl chain of MPM is buried underneath the α1 helix, with its phosphomannose head group reaching out of the ligand-binding groove, accessible for recognition by a TCR. The chemical structures of MPM and C₁₂ are shown in the bottom panel.

Top and side views of the binding cavities of CD1c (light magenta) with MPM and C₁₂, CD1a (yellow) with mycobactin (1XZ0), CD1b (cyan) with GM2 and spacer lipids (1GZQ) and CD1d (light purple) with α-galactosylceramide (1ZT4), shown as surface cavity representations. Red, blue and yellow surface colors represent the contribution of oxygen, nitrogen and sulfur atoms to the surface cavity, whereas ligands are colored with the following atom designations: carbon=yellow, oxygen=red, nitrogen=blue, and phosphorus=orange. Residues forming conserved features in CD1c are shown as sticks and colored as described above. Cavity volumes for each CD1 isoform were calculated with Pocket Finder(Hendlich et al., 1997) and are indicated in ų.

(A) Ribbon (left panel) and surface diagram (right panel) of the D′ and E′ portals in CD1c (magenta) and their comparison to CD1b (cyan). Residues forming the D′ and E′ portals in CD1c and corresponding residues in CD1b are shown in yellow and orange, respectively. (B) Overlay of ribbon diagrams of CD1c and CD1b reveal an increased distance between the α1 helix and the S1 strand of the α2 β-sheet in CD1c. The distances are shown as yellow dashes and the increase is indicated at three positions corresponding to the location of the D′ and E′ portals in CD1c.

Top view of a surface representation (left panel) and ribbon diagram (right panel) of CD1c (magenta, top panel) and CD1b (cyan, bottom panel). Residues lining the F′ groove in CD1c are shown in light pink and the corresponding residues in CD1b are shown in teal. The unresolved residues in the flexible loop connecting the CD1c α1 helix to the β-sheet are shown as a dashed line.

CD1c is shown as a ribbon diagram with residues involved in hydrogen bonding with MPM shown as sticks and labeled. Atoms are colored as such: yellow, carbon; blue, nitrogen; red, oxygen; orange, phosphorus. Two bound water molecules are shown as purple spheres. Highly probable hydrogen bonds are displayed as large dashes, potential ones as small dashes.

A fixed concentration of CD1c was incubated with increasing concentrations of lipo-12 and the tryptophan fluorescence signal of the samples was measured. The quenching of tryptophan fluorescence with increasing amounts of lipo-12 was expressed as a ratio (E-E_o)/E_o, the change in fluorescence divided by the control fluorescence in the absence of added lipo-12. The data were fit to a one-site binding model to determine the apparent K_D of this interaction.