David Rose [email protected]
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Transcript of David Rose [email protected]
David Rose
Three-Dimensional Structures of Immunoglobulins
- Antibody Domain and Fragment Nomenclature
- Structural basis of Diversity - Immunoglobulin Fold
- Shapes / Categories of Binding Sites
- Biochemical Basis of Binding / Recognition
- Conformational Changes on Binding - Antibody Flexibility
- Other Immunoglobulin- like Molecules
R.R. Porter, Nobel Lecture, 1972
(Fab’)2
Fv fragment
Antibody Domain and Fragment Nomenclature
- Variable, Constant RegionsDefined by similarity of amino-acid sequence between antibodiesEach forms a structural unit (Ig fold)
- Heavy Chains1 variable domain (N-terminus): Vh3 (IgG) or more constant domains: Ch1 - Ch3
constant domains define antibody class
- Light Chains1 variable domain (N-terminus): Vl1 constant domain: Cl
- Antigen Binding Fragment (Fab)Vl/Vh - Cl/Ch1 heterodimer
- Constant (crystallizable) Fragment (Fc)Ch2-Ch3 homodimer
- Variable Fragment (Fv)Vl/Vh heterodimer
- Epitope: Part of the antigen recognized by the antibody- Paratope: Antibody recognition region
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- Variability Plotsv = num different residue types / Freq (most common)
- Relationship to V(D)J gene segments
Kappa light chain V regions
IgG heavy chain V regions
V V V V
V
V
J
J
J J J J CL
CL
CL
V J CD
Light Chain
Heavy Chain
Light Chain Variable Region
Immunoglobulin Fold:Each region (V,C) forms a -sheet sandwich110-120 residues
Light Chain Constant Region
- Canonical CDR structuresChothia and Lesk
L1-3, H1, H2 well defined by loop structures
- Framework / Complementarity -Determining Regions (CDRs)(Hypervariable regions)
assemble to make up the antibody binding region.
- Fab/Fv binding site (combining site)
6 CDRs: 3 Light Chain + 3 Heavy ChainL1-3, H1-3
Canonical forms of CDR loops.
Al-lazikani, Lesk & Chothia, J Mol Biol (1997) 273:927
Canonical forms, chain L1 CDRs
H3 CDR’s: 12-residue length
Al-lazikani, Lesk & Chothia J Mol Biol (2000) 295:979
Shapes / Categories of Binding sites
- Flat: mostly surface residues from both antibody and antigen. Frequently discontinuous regions of antigen
- Groove / Crevice:Binds to stretches of antigen, usually continuous
- Pocket:Usually small molecule antigens, tight loops, or ends of polymers that penetrate a small pocket.
Pocket Groove Flat
Combining Site Shapes
Types of Antigens:
1. Small Molecules (haptens)
Pocket-shapedshape complementarityelectrostatics / hydrogen bonds (enthalpy-driven)High association constants (108 - 109 M-1)
2. Proteins
a. discontinuousflat shapedhydrophobic (elimination of water)Van der Waalshydrogen bondssome entropic contribution
Buried surface ~700-800 Å2
Moderate - high association constants (106 - 108 M-1)
b. continuousgroove / creviceextended loop on antigen
(usually -turn but can be -helix)higher entropic cost
Hydrophobic, entropic, van der Waalsshape complementarity
Induced fit of antibody and/or antigen
Fab F11.2.32 HIV-1 protease peptide complex
Lescar et al, J Mol Biol (1997) 267: 1207
Fab 17/9 complex with peptide from Influenza virus hemagglutinin
Rini, Schulze-Gahmen & Wilson (1992) Science 255:959
MRK-16 Fab structure: Vasudevan, Tsuruo and Rose, J. Biol Chem (1998) 273:25413
Jean M. H. van den Elsen, Douglas A. Kuntz, Flip J. Hoedemaeker, and David R. Rose Antibody C219 recognizes an -helical epitope on P-glycoprotein PNAS 96: 13679-13684
3. Carbohydrate / polysaccharide
Groove - shaped (chain binder)or pocket-shaped (end binder)
Hydrophobic (especially aromatic)some hydrogen bondswater can be used as coordinating ligand
High entropic costLower buried surface (500-600 Å2)Lower association constants (104 - 105 M-1)
Fab Se155.4 complex with Salmonella cell-surface antigen
Cygler, Rose & Bundle (1991) Science 253:442
Binding of cholera O1 antigen to Fab S-20-4
Villeneuve et al, PNAS (2000) 97:8433
Conformational changes / flexibility
Induced fitantibody binding site plasticityepitope
Antibody flexibilitydomain:domain interaction changesantigen bindingtether links in intact IgG structure
Antibody conformational change on complexation: Fab 17-IA with HRV-14Smith et al (1996) Nature 383:350
Epitope Conformational change on complexation : HIV Protease
Epitope conformational change on complexation: Flu hemagglutinin
Conformational Change on Antigen Binding
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Harris LJ, Skaletsky E, McPherson A. (1998) J Mol Biol 275:861
Crystal Structure of an Intact IgG
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Same antibody binds to two different antigens
Antibody D3H44 uses different regions of the combining site to bind to 6A6 and TF. “open book” view coloured by electrostatics.
Eigenbrot et al, (2003) J. Mol. Biol. 331:433-446
D3H44 residues contacting 6A6 (green) and TF (pink)
Grafting loops from D1.3 onto the F8 framework
Donini et al (2003), J. Mol. Biol. 330:323-332
Affinities of reduced scFv’s from periplasm (l) and cytoplasm (r) to HEL
Residues the same in F8 (grey) or D1.3 (blue)
Grafted loops in P1(red),P2(orange), P3(yellow),P4(green), P5(cyan)
Evolution of affinity to same antigen epitopeAntibodies to hen egg lysozyme differing due to somatic mutation.
Li et al (2003), Nature Str. Biol. 10:482-488
Shape complementarity (left) and contact residues (rt) for H26 (top) and H8
Progression from weakest (H26 red) to strongest (H8 blue) comparison to unliganded H63 (yellow). H8 complex least distorted from unliganded
Same antibody, different specificities
James et al (2003), Science 299:1362-1367
Other Immunoglobulin Family Proteins
IgG Fc Complexes
Protein A domain B1 Protein G domain C2
Rheumatoid Factor Neonatal Fc Receptor
DeLano et al, Science (2000) 287:1279
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T-cell Receptor
T-cell Receptor Antibody Fab
D.N.Garboczi, P.Ghosh, U.Utz, Q.R.Fan, W.E.Biddison, D.C.Wiley. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2 Nature 384, 134 (1996)
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