David Rose

drose@oci.utoronto.ca

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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