The functional architecture of an acetylcholine receptor-mimicking antibody.
Malpha2-3 is a monoclonal antibody that partially mimics the nicotinic acetylcholine receptor (AChR). Its three-dimensional structure has been previously predicted by molecular modeling, suggesting that 29 complementarity determining region (CDR) residues and 2 framework residues are exposed to solvent. To identify the antibody residues that bind to the antigen, i.e. snake toxin that binds specifically to AChR, we (i) produced the scFv form of Malpha2-3 fused to alkaline phosphatase, in the periplasmic space of Escherichia coli; (ii) submitted approximately 75% of exposed residues of the fused scFv to individual or combined mutations, and (iii) identified the residues whose mutations affect scFv binding to the toxin, using a sensitive enzyme-linked immunosorbent assay. 11 critical residues were identified, including 8 heavy chain residues, 2 framework residues, and 1 light chain residue. They cover a surface of approximately 800 A2, with a subset of most critical residues (VHD31, VHY32, and VHG101) and several aromatic residues. This functional architecture not only constitutes a plausible complementary binding surface for the snake toxin but also offers a structural basis to ultimately understand the capacity of the antibody to partially mimic AChR.
Mérienne
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K
Département d'Ingénierie et d'Etudes des Protéines, Direction des Sciences du Vivant, Commissariat à l'Energie Atomique, Saclay, 91191 Gif-sur-Yvette Cedex, France.
Germain
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N
Zinn-Justin
S
S
Boulain
J C
JC
Ducancel
F
F
Ménez
A
A
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001666:Binding Sites, Antibody; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D014101:Torpedo
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase