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David Rose drose@oci.utoronto

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

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David Rose drose@oci.utoronto

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  1. 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

  2. R.R. Porter, Nobel Lecture, 1972

  3. (Fab’)2 Fv fragment

  4. Antibody Domain and Fragment Nomenclature - Variable, Constant Regions Defined by similarity of amino-acid sequence between antibodies Each forms a structural unit (Ig fold) - Heavy Chains 1 variable domain (N-terminus): Vh 3 (IgG) or more constant domains: Ch1 - Ch3 constant domains define antibody class - Light Chains 1 variable domain (N-terminus): Vl 1 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

  5. - Variability Plots v = num different residue types / Freq (most common) - Relationship to V(D)J gene segments

  6. Kappa light chain V regions

  7. IgG heavy chain V regions

  8. V V V V CL J J J J J CL V V J CL Light Chain V D J C Heavy Chain

  9. Immunoglobulin Fold: Each region (V,C) forms a -sheet sandwich 110-120 residues Light Chain Variable Region

  10. Light Chain Constant Region

  11. - 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 Chain L1-3, H1-3 - Canonical CDR structures Chothia and Lesk L1-3, H1, H2 well defined by loop structures

  12. Canonical forms of CDR loops. Al-lazikani, Lesk & Chothia, J Mol Biol (1997) 273:927

  13. Canonical forms,  chain L1 CDRs

  14. H3 CDR’s: 12-residue length Al-lazikani, Lesk & Chothia J Mol Biol (2000) 295:979

  15. 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.

  16. Combining Site Shapes Pocket Groove Flat

  17. Types of Antigens: 1. Small Molecules (haptens) Pocket-shaped shape complementarity electrostatics / hydrogen bonds (enthalpy-driven) High association constants (108 - 109 M-1)

  18. 2. Proteins a. discontinuous flat shaped hydrophobic (elimination of water) Van der Waals hydrogen bonds some entropic contribution Buried surface ~700-800 Å2 Moderate - high association constants (106 - 108 M-1)

  19. b. continuous groove / crevice extended loop on antigen (usually -turn but can be -helix) higher entropic cost Hydrophobic, entropic, van der Waals shape complementarity Induced fit of antibody and/or antigen

  20. Fab F11.2.32 HIV-1 protease peptide complex Lescar et al, J Mol Biol (1997) 267: 1207

  21. Fab 17/9 complex with peptide from Influenza virus hemagglutinin Rini, Schulze-Gahmen & Wilson (1992) Science 255:959

  22. MRK-16 Fab structure: Vasudevan, Tsuruo and Rose, J. Biol Chem (1998) 273:25413

  23. 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

  24. 3. Carbohydrate / polysaccharide Groove - shaped (chain binder) or pocket-shaped (end binder) Hydrophobic (especially aromatic) some hydrogen bonds water can be used as coordinating ligand High entropic cost Lower buried surface (500-600 Å2) Lower association constants (104 - 105 M-1)

  25. Fab Se155.4 complex with Salmonella cell-surface antigen Cygler, Rose & Bundle (1991) Science 253:442

  26. Binding of cholera O1 antigen to Fab S-20-4 Villeneuve et al, PNAS (2000) 97:8433

  27. Conformational changes / flexibility Induced fit antibody binding site plasticity epitope Antibody flexibility domain:domain interaction changes antigen binding tether links in intact IgG structure

  28. Antibody conformational change on complexation: Fab 17-IA with HRV-14 Smith et al (1996) Nature 383:350

  29. Epitope Conformational change on complexation : HIV Protease

  30. Epitope conformational changeon complexation: Flu hemagglutinin

  31. Conformational Change on Antigen Binding

  32. Crystal Structure of an Intact IgG Harris LJ, Skaletsky E, McPherson A. (1998) J Mol Biol 275:861

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