the crystal structure of human cd1d with and without -galcer vicki stronge vincenzo cerundolo terry...
TRANSCRIPT
![Page 1: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/1.jpg)
The crystal structure of human CD1d with and without
-GalCer
Vicki Stronge
Vincenzo Cerundolo Terry Butters
Raymond Dwek
Institute of Molecular MedicineGlycobiology Institute
![Page 2: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/2.jpg)
CD1d
Large, ligand-binding pocket lined with hydrophobic residues
Non-classical antigen-presenting molecule
Five isoforms in humans - CD1a, b, c, d and e
![Page 3: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/3.jpg)
-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
![Page 4: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/4.jpg)
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
![Page 5: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/5.jpg)
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
![Page 6: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/6.jpg)
Structure of human CD1d with -GalCer
Top ViewSide View
![Page 7: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/7.jpg)
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’)
![Page 8: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/8.jpg)
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
![Page 9: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/9.jpg)
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
![Page 10: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/10.jpg)
Overlay of Human and Mouse CD1d
• Overlay of C traces superimposes well
• -GalCer fits well into the mouse CD1d groove
![Page 11: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/11.jpg)
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”)
![Page 12: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/12.jpg)
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
![Page 13: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/13.jpg)
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
![Page 14: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/14.jpg)
Overlay of CD1 and MHC class I structures
CD1d--GalCer
Empty CD1d
MHC class I
Mouse CD1d
![Page 15: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/15.jpg)
Lipid binding vs Non-lipid Binding hCD1d molecule
Lipid binding molecule
Non-lipid binding molecule
Side ViewTop View
![Page 16: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/16.jpg)
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
![Page 17: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/17.jpg)
Acknowledgements
Michael KochYvonne Jones
Vincenzo Cerundolo Terry Butters Fran Platt
![Page 18: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/18.jpg)
Equivalent Residues of Human and Mouse CD1d
• mutations cause alteration in iNKT hybridomarecognition
![Page 19: The crystal structure of human CD1d with and without -GalCer Vicki Stronge Vincenzo Cerundolo Terry Butters Raymond Dwek Institute of Molecular Medicine](https://reader033.vdocuments.mx/reader033/viewer/2022052701/56649f3e5503460f94c5eae5/html5/thumbnails/19.jpg)
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