Evaluation of mutagenesis for epitope mapping. Structure of an antibody-protein antigen complex.
The location and description of epitopes on proteins describe the basis of immunological specificity. The 2.8-A structure of the phosphocarrier protein, HPr from Escherichia coli, complexed to the Fab fragment of the monoclonal antibody, Jel42, has been determined. This allows the first comparison of epitope predictions from extensive site-directed mutagenesis experiments, coupled with biological activity studies (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, 4877-4881), with those from x-ray analysis. There are 14 amino acid residues 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 appear to have critical roles in determining protein conformation; the other 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 chains 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 that mutations which caused greater than 10-fold changes in antibody binding affinity were correctly assigned to the epitope by the mutagenesis experiments. Guidelines are also presented in order to minimize incorrect assignments.
Prasad
L
L
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Sharma
S
S
Vandonselaar
M
M
Quail
J W
JW
Lee
J S
JS
Waygood
E B
EB
Wilson
K S
KS
Dauter
Z
Z
Delbaere
L T
LT
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D000906:Antibodies; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins; D000939:Epitopes; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr