Mutational analysis of an antigenic peptide shows recognition in a loop conformation.
We have analyzed the recognition between an antigenic undecapeptide and a monoclonal antibody through a mutational approach. Antibody mAb164 is directed against the native form of the TrpB2 subunit of Escherichia coli tryptophan synthase. It recognizes a synthetic peptide, P11, constituted of residues 273-HGRVGIYFGMK-283 of TrpB with high affinity. P11 was fused with a carrier protein, MalE, to facilitate its manipulation. The affinities between mAb164 and the MalE-P11 hybrids were measured by competition enzyme-linked immunosorbent assay (ELISA). The changes of the P11 residues into progressively shorter residues, the comparison of changes into Pro and Ala, and the study of double mutants showed the following. Four hydrophobic residues of P11, Val276, Ile278, Tyr279, and Phe280, were predominant in the interaction. For some residues, e.g., Tyr279, most groups of the side chain contributed to the interaction. For others, only some groups played a significant role, e.g., the Cdelta group of Ile278 or the Cbeta group of Phe280. The lack of side chain in position Gly281 and a tertiary interaction between the side chains of Ile278 and Lys283 were important. P11 was recognized in a loop conformation, close to that of residues 273-283 of TrpB in the crystal structure of the complete tryptophan synthase, TrpA2TrpB2. Comparison of our mutational data with NMR data on the conformation of the isolated peptide P11 and with kinetic data on its interaction with mAb164 indicate that mAb164 selects a conformer of P11 that represents only a small minority of the molecules. Our results provide useful information on the mechanisms by which linear epitopes and unconstrained peptides are recognized by receptors.
Rondard
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Unité de Biochimie Cellulaire, CNRS URA 1129, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Goldberg
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Bedouelle
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D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D014367:Tryptophan Synthase