Molecular and structural basis of the specificity of a neutralizing acetylcholine receptor-mimicking antibody, using combined mutational and molecular modeling analyses.
The antagonist activity of short-chain toxins from snake venoms toward the nicotinic acetylcholine receptor (nAChR) is neutralized upon binding to a toxin-specific monoclonal antibody called Malpha2-3 (1). To establish the molecular basis of this specificity, we predicted from both mutational analyses and docking procedures the structure of the Malpha2-3-toxin complex. From knowledge of the functional paratope and epitope, and using a double-mutation cycle procedure, we gathered evidence that Asp(31) in complementarity determining region 1H is close to, and perhaps interacts with, Arg(33) in the antigen. The use of this pair of proximate residues during the selection procedure yielded three models based on docking calculations. The selected models predicted the proximity of Tyr(49) and/or Tyr(50) in the antibody to Lys(47) in the toxin. This was experimentally confirmed using another round of double-mutation cycles. The two models finally selected were submitted to energy minimization in a CHARMM22 force field, and were characterized by a root mean square deviation of 7.0 +/- 2.9 A. Both models display most features of antibody-antigen structures. Since Malpha2-3 also partially mimics some binding properties of nAChR, these structural features not only explain its fine specificity of recognition, but may also further clarify how toxins bind to nAChR.
Germain
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N
Department d'Ingenierie et d'Etudes des Proteins, Commissariat à l'Energie Atomique, Saclay, Gif-sur-Yvette Cedex 91191, France.
Mérienne
K
K
Zinn-Justin
S
S
Boulain
J C
JC
Ducancel
F
F
Ménez
A
A
D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D001224:Aspartic Acid; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018733:Nicotinic Antagonists / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012910:Snake Venoms / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D012911:Snakes; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D012910:Snake Venoms; D001224:Aspartic Acid