HETEROLIGATION OF A MOUSE MONOCLONAL IGE ANTIBODY (LA2) WITH SMALL MOLECULES, ANALYZED BY COMPUTER-AIDED AUTOMATED DOCKING

A mouse monoclonal anti-TNP IgE antibody (IgE-La2) was screened by a c ompetitive-binding ELISA with a random pool of over 2000 small molecul es, mostly drugs, drug derivatives and metabolites. Thirteen of these (naproxene, beta-carboxy-alpha-naphthol, oxolinic acid, hymecromone, 8 -aminoquinoline, beta-naphthylamine, alpha-nitrilo-cinnamic acid, 1,5- diaminonaphthaline, prolonium iodide, diaspirin, 3,4,5-trimethoxy-cinn amic acid, cycrimine, hemimellitic acid) were found to bind as strongl y, or stronger, to the antibody as the immunizing hapten. We have used a Monte Carlo search technique for simulated docking of the DNP and n on-DNP ligands to a model of the F-v region of IgE(La2). The validity of structural predictions made by the AutoDock program were tested on IgG(ANO2), the three-dimensional structure of which had been obtained previously by X-ray crystallography and 2D-NMR. The rms differences be tween the experimentally determined and auto-docked complexes in the e nergetically most favored binding modes were 0.31-0.44 Angstrom. Evalu ation of structures of IgE(La2)-ligand complexes [including 2,4-dinitr ophenol (DNP), 16 DNP amino acids, and the 13 non-DNP ligands listed a bove] obtained by computer-aided automated docking, suggested the exis tence of two subsites within an approximately 12 x 18 Angstrom(2) groo ve extending between the H and L CDRs. Some of the ligands (DNP-Glu, 8 -aminoquinoline, prolonium-I, beta-naphthylamine) were found to bind e xclusively to subsite 1, others (DNP-Ala, alpha-nitrilo-cinnamic acid, hemimellitic acid, beta-carboxy-alpha-naphthol) to subsite 2. The maj ority of DNP amino acids and other ligands (oxolinic acid, 3,4,5-trime thoxy-cinnamic acid, diaspirin, [R]-cycrimine) were found to occupy an overlapping area including subsites 1 and 2, while some of the compou nds (DNP-Asn, DNP-Pro, hymecromone, 1,5-naphthylenediamine) were predi cted to interact with either of these subsites with comparable probabi lities. When all of the docked La2-ligand complexes were taken into ac count, five tyrosine residues (H33, L32, L91, L92, L96) were found to provide the majority (53.4%) of all observed contact points. Thus, a m ultitude of interactions with aromatic residues, and a combinatorial t ype of interaction within the binding region, seem to be the major fac tors to explain the mechanism of heteroligation by IgE(La2).