Transcript Antibody domain exchange is an immunological solution to

Antibody domain
exchange is an
immunological solution
to carbohydrate cluster
recognition.
Daniel Calarese … Ian A. Wilson
(16 authors from
Scripps, Oxford, FSU, U Ag Vienna)
Science June 27, 2003 (300:2065)
Burton, Wilson, and Calarese
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What is “domain exchange”?
Paratope
• 2 Ordinary Fabs
Variable Domains
H
L
Constant Domains
• VH domains exchanged
Forming a Fab dimer with
two ordinary VH –VL paratopes
+ two new VH –VH paratopes
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“Toober” models
Two
Conventional
Fab’s
2G12
Fab’s
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What is 2G12?
• Human monoclonal IgG1 from patient B cells.
• Neutralizes unusually broad range of known HIV
strains (25-50%).
• Binds to dense cluster of carbohydrate epitopes
on the “silent” face of gp120.
• Unusually high affinity among anti-carbohydrate
antibodies.
• Antibodies to the 2G12 epitope are unusual in
sera from HIV+ patients (by competition).
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The “Silent” face of gp120
• Is an unusual epitope among HIV+ patient antibodies.
• Potential protein epitopes beneath the carbohydrate are
shielded.
• Carbohydrate is made by host (not virus enzymes)
hence may be “self” (though the dense cluster of oligomannose
residues has not been described on mammalian glycoproteins).
• Has multiple glycoforms diluting any one epitope.
• Antibodies to carbohydrate epitopes usually have low
affinities (Kd micromolar; but 2G12 Kd is nanomolar).
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Why is the structure of 2G12
important?
• Paradigm shift: functional framework changes
via somatic hypermutation.
• Paradigm shift: VH-VH paratope.
• Exception to the rule that Fab:antigen
interactions are monovalent.
• Domain-swapped dimer is unprecedented
among >250 published Fab structures.
• Explains high affinity and ability to bind closelyspaced epitopes.
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Protein Crystallography
• Accounts for 85% of published structures.
• Has an overall success rate of ~3%.
• <1% of the human proteome has
crystallographically known structure.
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Methods
•
•
•
•
Express full IgG1 in CHO cells.
Papain  Fab (completeness confirmed by SDS-PAGE).
Purif. Prot. A/G columns, 30 mg/ml.
1 μl (ligand 5 to 1 molar ratio) + 1 μl cryst. sol’n
(ammonium/sulfate/phosphate, PEG, imidazole malate,
pH 6/7), sitting drop crystallization.
• Diffract Stanford Synchrotron cryopres. in Liq. N2
• Phase solved by molecular replacement with best-fitting
existing Fab structure (1fvd).
• Model of known amino acid sequence built into electron
density map and refined (with restraints) for best fit.
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Crystallographic Results
Unliganded
2G12 Fab*
2.2 Å
+Man2
+Man9GlcNAc2
1.75 Å
3.0 Å
Number of
Reflections
R
60,000
126,000
30,000
0.22
0.23
0.25
Rfree (5%)
0.27
0.25
0.33
PDB ID
1OM3
1OP3
1OP5
Resolution
* ~6,600 atoms
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Framework Somatic Mutations
Facilitate Domain Exchange
1. Weaken VH-VL
2. Alter H Chain Elbow
Conserved ball and socket is not mutated but is dislocated.
Mutated residues add stabilization.
3. Strengthen VH-VH’
See details in Protein Explorer
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Is this bizarre Fab dimer for real?
It is not observed in >250 published Fab crystal structures.
Fab: about 80% dimer + 20% monomer in solution by
• Sedimentation equilibrium analytical ultracentrifugation.
• Gel filtration.
Intact IgG1 has compact conformation by
• Velocity sedimentation coefficient.
• Negative staining electron microscopy.
– With and without gp140 ligand
Apparently the dimer is for real.
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Fig. 2c: Electron microscopy of intact 2G12 IgG1 shows a
compact form (neither Y nor T) with or without bound
antigen.
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Mutants of 2G12
reducing gp120 binding affinity ~100-1,000 fold
• Primary combining site (VH-VL) 5/11
• Secondary binding site (VH-VH) 4/5
• Domain exchange-facilitating residues
– VH-VH 4/4
– Elbow 3/3
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Function of HIV oligomannose
• DC-SIGN is a human lectin.
(Dendritic Cell-Specific ICAM-3 Grabbing Nonintegrin)
• Facilitates infection of CD4+ cells by binding HIV
carbohydrate.
• Speculation: HIV-1 evolved oligomannose to
utilize DC-SIGN.
• 2G12 exploits this “Achilles heel” of HIV-1.
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Conclusions
• 2G12 forms a novel domain-swapped dimer (in 3 crystals and in
solution) in both Fab and intact IgG1.
• A novel form of paratope occurs at the VH-VH’ interface.
• The novel conformation is enabled by somatic mutations to
framework residues.
• The resulting 4-paratope array accommodates closely spaced
carbohydrate epitopes (35 Å, vs. 50-140 Å for Y or T) achieving
nanomolar avidity. An epitope on gp120 is predicted.
• Vaccines mimicking oligomannose cluster may elicit protective Abs.
• 2IG2: scaffold for designing Abs to other epitope clusters?
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Alternative Antibody Lifestyles
Naturally Occurring
• Camel H chain dimer (no L; “VHH”)
• (Muyldermans et al., TiBS 26:230, 2001)
• VH domain-swapped dimer
• (Calarese et al., Science 300:2065, 2003)
Engineered
• Fv (VH-VL with no C domains)
• Fv domain-swapped “triabody” (trimer ring)
• (Pei et al. PNAS 95:9637, 1997)
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Some High-Impact Crystal Structures
• Myoglobin (1960): first example of protein structure.
• Lysozyme (1965): first enzyme.
• Fab (1973): first immunoglobulin fold (shared in unrelated proteins,
first SOD).
• tRNA (1975): first RNA structure.
• MHC (1987): explained restriction of T cell recognition.
• Ribosome (2000): peptidyl transferase is a ribozyme.
• 2G12 (2003): unprecedented dimeric paratope with framework
somatic mutations.
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First crystal structures
without big surprises (less impact)
• DNA double helix (1973-80): predicted
correctly in 1953.
• T cell antigen receptor (, 1996).
• TCR  (2001)
• CD1 with phospholipid ligand (2002)
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Fig. 3D
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Fig. 3B
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Fig. 6: Model of 2G12 + gp120
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