Molecular analysis of the combining site of a monoclonal antibody against spermine

The structural basis of the binding of the polyamine spermine to the monocl onal antibody SPM8-2 was studied using computer modelling, ELISA methods an d chemical modifications of thr binding site residues. Paratope modelling s howed that the antibody combining site forms a highly negatively charged ca vity mainly shaped by aspartic acid and tyrosine residues which contact the terra-positively charged spermine molecule by electrostatic interactions a nd hydrogen bondings. The importance of the electrostatic environment for s permine binding to SPM8-2 is emphasised by the strong dependency on pH and ionic strength, Specific chemical modifications of carboxylate groups and t yrosine residues of the antibody adsorbed to microtiter plates resulted in decreased binding of the N-1-biotin-spermine conjugate used to monitor the activity of the antibody. These observations are consistent with a key role of aspartate and tyrosine residues in complex formation with spermine. The se studies, important to our understanding of antibody-hapten specificity, may also shed light on important motifs responsible for protein-polyamine i nteractions. (C) 1999 Elsevier Science Ltd. All rights reserved.