EVALUATION OF MUTAGENESIS FOR EPITOPE MAPPING - STRUCTURE OF AN ANTIBODY-PROTEIN ANTIGEN COMPLEX

The location and description of epitopes on proteins describe the basi s of immunological specificity. The 2.8-angstrom structure of the phos phocarrier protein, HPr from Escherichia coli, complexed to the Fab fr agment of the monoclonal antibody, Jel42, has been determined. This al lows the first comparison of epitope predictions from extensive site-d irected mutagenesis experiments, coupled with biological activity stud ies (Sharma, S., Georges, F., Klevit, R. E., Delbaere, L. T. J., Lee, J. S., and Waygood, E. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4 877-4881), with those from x-ray analysis. There are 14 amino acid res idues of E. coli HPr that interact with the Jel42 antigen-binding site . Nine of these were correctly assigned by the mutagenesis studies. Of the 5 remaining residues, Met-1 could not be altered; two others appe ar to have critical roles in determining protein conformation; the oth er 2 residues have a minimal effect on antibody binding since they are located on the periphery of the epitope with one face of their side c hains in van der Waals contact with the antibody and the other face in contact with solvent. Four residues were incorrectly assigned to the epitope. These residues were located adjacent to epitope residues that were likely perturbed by these mutations. This study demonstrates tha t mutations which caused greater than 10-fold changes in antibody bind ing affinity were correctly assigned to the epitope by the mutagenesis experiments. Guidelines are also presented in order to minimize incor rect assignments.