L. Prasad et al., EVALUATION OF MUTAGENESIS FOR EPITOPE MAPPING - STRUCTURE OF AN ANTIBODY-PROTEIN ANTIGEN COMPLEX, The Journal of biological chemistry, 268(15), 1993, pp. 705-708
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.