PARTICIPATION OF LYSINE-516 AND PHENYLALANINE-530 OF DIPHTHERIA-TOXININ RECEPTOR RECOGNITION

To identify amino acid residues within diphtheria toxin that participa te in receptor recognition, we made alanine replacements for each of 1 2 solvent-accessible residues within a loop (residues 516-530) of the toxin's R domain, which prominently extends from the main surface of t he domain. Amino acid replacements were generated in an enzymatically attenuated form of the toxin (in compliance with regulations for cloni ng in Escherichia coli), and the mutant toxins were purified and assay ed for toxicity on Vero cells. The largest effects were seen with K516 A and F530A, which caused similar to 22- and similar to 10-fold increa ses, respectively, in the toxin concentration required for half-maxima l inhibition of protein synthesis (IC50). Smaller effects were seen at certain other sites and no effect at still others. K516A caused simil ar to 500-fold reduction in ability to compete with radiolabeled wildt ype toxin for receptors, and F530A gave similar to 100-fold reduction. The small differences in IC50 with the mutants, compared with differe nces in receptor binding, are attributable to nonlinearity in the cyto toxicity assay with the enzymatically attenuated toxin. K516A and F530 A also inhibited the receptor-blocking activity of the isolated R doma in. Neither mutation caused a change in the circular dichroism spectru m of the R domain. These results indicate important roles for Lys-516 and Phe-530 in receptor recognition. In addition, on the basis of cyto toxicity data, four other residues (Tyr-514, Val-523, Asn-524, and Lys -526) are proposed play roles in receptor binding. These findings supp ort the notion that the toxin's receptor binds to the solvent exposed face of the R domain opposite the catalytic domain.