the crystal structure of human cd1d with and without -galcer vicki stronge vincenzo cerundolo terry...

The crystal structure of human CD1d with and without

-GalCer

Vicki Stronge

Vincenzo Cerundolo Terry Butters

Raymond Dwek

Institute of Molecular MedicineGlycobiology Institute

CD1d

Large, ligand-binding pocket lined with hydrophobic residues

Non-classical antigen-presenting molecule

Five isoforms in humans - CD1a, b, c, d and e

-Galactosylceramide

• -GalCer binds human and mouse CD1dwith high affinity

• It is the most potent activator of invariant NKT cells (iNKT) to date

• Stimulates the secretion of large amounts

of IFN- and IL-4 (TH1 and TH2).

• Capable of enhancing or suppressing immune responses

Structures of CD1 molecules

Molecule Ligand Reference

Mouse CD1d not defined Zeng et. al., Science, 1997

CD1b PI Gadola and Zaccai et. al., Nat. Immunol., 2002

CD1b GM2 Gadola and Zaccai et. al., Nat. Immunol., 2003

CD1a Sulphatide Zajonc et. al., Nat. Immunol., 2003

CD1b GMM Batuwangala and Shepherd et. al., J. Immunol., 2004

CD1a DDM lipopeptide Zajonc et. al., Immunity, 2005

Refolding of CD1d monomers

• Oxidative refolding chromatography using chaperone-containing resin

• Enables CD1d to refold around a single lipid

• Used for the generation of CD1d tetramers for iNKT cell staining

• Needed to remove the biotinylation site in order to crystallize the protein

Structure of human CD1d with -GalCer

Top ViewSide View

Antigen-binding Pockets of hCD1dSide View Top View

• Pole is formed by Phe70 and Cys12 (and Trp40)

• T’ tunnel is blocked by Val98, Phe114 and Val116

• F’ pocket is blocked by Phe84 (Leu84)

• Alkyl chains extend to end of pockets and fill available space (A’ and C’)

Hydrogen Bonds of hCD1d to -GalCer

• H bonds hold polar head of -GalCer in correct position and orientation for T cell recognition

• Longer chain lipids may not fit into the groove as the branch point would not be held in place

Modelling of TCR on CD1d with -GalCer

• green box covers areas where mutations of the mouse residues result in loss or change in TCR function

• S76G• R79E, D80A• D153Y

Overlay of Human and Mouse CD1d

• Overlay of C traces superimposes well

• -GalCer fits well into the mouse CD1d groove

The asymmetric unit of the CD1d crystal has two molecules

• one molecule had a-GalCer present

(“lipid binding molecule”)

• other molecule had

very little density – no lipid bound

(“Non-lipid binding

molecule”)

Electron Density of -GalCer in Groove of hCD1d

Lipid binding molecule Non-lipid binding molecule

O

OH

OHNH

O

O

OH

OH

OH

OH

-GalCer

hCD1d-2m monomers are unstable in the absence of lipid

0.0

1.0

2.0

3.0

4.0

UV

Abs

orba

nce

(mA

U)

50 100 150 200

Volume (ml)

hCD1d-2M complex

+ -GalCer

- -GalCer

Overlay of CD1 and MHC class I structures

CD1d--GalCer

Empty CD1d

MHC class I

Mouse CD1d

Lipid binding vs Non-lipid Binding hCD1d molecule

Lipid binding molecule

Non-lipid binding molecule

Side ViewTop View

Future Experiments

1. Co-crystallization of CD1d--GalCer with the iNKT TCR

2. Analysis of the loading requirements of -GalCer on CD1d using a complex-specific antibody

3. Confirm the identity of the GSLs bound to secreted forms of CD1b and CD1d

Acknowledgements

Michael KochYvonne Jones

Vincenzo Cerundolo Terry Butters Fran Platt

Equivalent Residues of Human and Mouse CD1d

• mutations cause alteration in iNKT hybridomarecognition

Overlay of Antigens of hCD1 Structures

Green: GMM in hCD1bPurple: Sulfatide in hCD1aOrange: -GalCer in hCD1d

• -GalCer fills A’ and C’ pocket

• GMM wraps other way around pole to enter T’ tunnel

• sulfatide is barely above TCR recogntion surface