The Functional Architecture of an Acetylcholine Receptor-mimicking Antibody

Abstract

M␣2-3 is a monoclonal antibody that partially mimics the nicotinic acetylcholine receptor (AChR). Its threedimensional 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 M␣2-3 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 Å 2 , 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.

Molecular and Structural Basis of the Specificity of a Neutralizing Acetylcholine Receptor-mimicking Antibody, Using Combined Mutational and Molecular Modeling Analyses

“…A total of 44 scFv(M␣2-3) mutants have been prepared to identify the paratope by which M␣2-3 binds to a snake toxin (7). We have since further refined this surface using 13 new antibody mutations (data not shown).…”

“…The greatest moving amplitude of the C␣ of Arg 33 in loop II is only 4.7 Å, whereas it is 8.9 Å and 10.7 Å for Gln 7 and Lys 47 , respectively. Trp 29 occupies a well fixed position on the paratope, with a maximum distance of 3.5 Å within all the models, its indole side chain being a sort of pivot around which the toxin seems to move in all complexes (Fig.…”

“…First, they bind to it with similar affinities, with respective K d of 10 nM and 35 nM for Ea and toxin ␣ (7). Second, the two toxins have in common 9 (Gln 7 47 , and Ile 50 ) of the 10 residues that constitute the epitope of Ea, the differential residue being Phe 32 in Ea, which is replaced by His 32 in toxin ␣. Third, all common side chains adopt virtually identical conformations and hence nicely superimpose in space (9).…”

“…A structural model of the variable fragment of M␣2-3 was known from a homology modeling-based protocol (6). Residues forming the paratope of the antibody were also known from mutational analyses of its complementarity determining region (CDR) residues (7). Additionally, the three-dimensional structures of various antigens were known, like those of erabutoxin a (Ea) from Laticauda semifasciata (8) and toxin ␣ from Naja nigricollis (9).…”

An Antibody as Surrogate Receptor Reveals Determinants of Activity of an Innate Immune Peptide Antibiotic

“…A growing body of literature exists wherein antibodies raised against biological ligands have been found to possess physiological properties of their natural receptors (41)(42)(43)(44). In the present case, the potent activity of IN-411, testifies to the "biological target-like" properties of the mAb surrogate.…”