Structural model of the anti-snake-toxin antibody, Mα2,3

Abstract

We present results of structural modeling of the variable fragment of M alpha 2,3, an antibody capable of neutralizing all short snake toxins. Three different methods were used to model the hypervariable loops: the conformational search algorithm CONGEN (Bruccoleri and Karplus, Biopolymers 26:137-168, 1987), high-temperature molecular dynamics (Bruccoleri and Karplus, Biopolymers 29:1847-1862, 1990), and a combined knowledge-based and energy-based algorithm (Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272, 1989). Ninety plausible conformations were generated and were clustered into 13 classes. The clustering results indicate that there was little overlap of the conformational space explored by the different methods. Canonical loop structures were found by all methods for two of the loops, in agreement with previously established empirical modeling criteria. Nine of the 13 classes of structure were rejected on the ground of their lacking common features of antibody combining-site structure. The remaining four models were refined using restrained molecular dynamics. It was found that interconversion between the four resulting structures is possible with no significant energy barriers, suggesting that they are in thermodynamic equilibrium at 300 K. Features of the combining-site structure likely to be particularly important for antigen binding are discussed.

The Functional Architecture of an Acetylcholine Receptor-mimicking Antibody

“…In the absence of crystallographic data, the structure of M␣2-3 has been previously predicted by modeling analysis (9). Thus, 31 residues of a putative combining site were proposed to be exposed to solvent.…”

“…First, a predictive model of the antibody has to be established. Using the amino acid sequence of M␣2-3 deduced from its cloned cDNA, four predictive models of its combining antigen site including CDRs were previously selected (9). All models suggested that the putative combining site of M␣2-3 possesses 31 solvent exposed residues that presumably include most functional elements of the antibody combining site.…”

“…Site-directed Mutagenesis-Numbering of the amino acids forming the scFv of M␣2-3 is that of Tenette et al (9). The following nucleotide probes were used for mutagenesis: VLN31A (5Ј-CAGAGTGTGAGTGC- VHY33F (5Ј-TTCACTGACTAC-TTTATAAACTGG-3Ј), VHY33S (5Ј-TTCACTGACTACTCTATAAACT-GG-3Ј), VHY52F (5Ј-ATTGGATGGATTTTTCCTGCAAGCGGT-3Ј), VHY52S (5Ј-ATTGGATGGATTTCTCCTGCAAGCGGT-3Ј), VHA54L (5Ј-TGGATTTATCCTCTGAGCGGTAATACT-3Ј), VHA54G (5Ј-TGGAT-TTATCCTGGTAGCGGTAATACT-3Ј), VHS55A (5Ј-ATTTATCCTGCA-GCCGGTAATACTAAG-3Ј), VHN57A (5Ј-CCTGCAAGCGGTGCTACT-AAGTACAAT-3Ј), VHM100D (5Ј-TGTGCAAGAGCTGACGGGGCTAC-GGCT-3Ј), VHG101S (5Ј-GCAAGAGCTATGAGCGCTACGGCTACA--3Ј), VHG101A (5Ј-GCAAGAGCTATGGCGGCTACGGCTACA-3Ј), VHA102V (5Ј-AGAGCTATGGGGGTTACGGCTACACTT-3Ј), VHA-102G (5Ј-AGAGCTATGGGGGGTACGGCTACACTT-3Ј), VHT103S (5Ј-GCTATGGGGGCTTCGGCTACACTTTTG-3Ј), VHT103A (5Ј-GCTATG-GGGGCTGCGGCTACACTTTTG-3Ј), and VHA104S (5Ј-ATGGGGGCT-ACGTCTACACTTTTGGAC-3Ј).…”

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

“…Two models of the M␣2-3 variable segment were selected from the four models published by Tenette et al (6) on the basis of the canonicity of their hypervariable loop structures. Docking of the structure of toxin ␣ onto the structures of the M␣2-3 variable segment was carried out using the program DOCK (18).…”

“…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).…”