Participation of lysine 516 and phenylalanine 530 of diphtheria toxin in receptor recognition.
To identify amino acid residues within diphtheria toxin that participate in receptor recognition, we made alanine replacements for each of 12 solvent-accessible residues within a loop (residues 516-530) of the toxin's R domain, which prominently extends from the main surface of the domain. Amino acid replacements were generated in an enzymatically attenuated form of the toxin (in compliance with regulations for cloning in Escherichia coli), and the mutant toxins were purified and assayed for toxicity on Vero cells. The largest effects were seen with K516A and F530A, which caused approximately 22- and approximately 10-fold increases, respectively, in the toxin concentration required for half-maximal inhibition of protein synthesis (IC50). Smaller effects were seen at certain other sites and no effect at still others. K516A caused approximately 500-fold reduction in ability to compete with radiolabeled wild-type toxin for receptors, and F530A gave approximately 100-fold reduction. The small differences in IC50 with the mutants, compared with differences in receptor binding, are attributable to nonlinearity in the cytotoxicity assay with the enzymatically attenuated toxin. K516A and F530A also inhibited the receptor-blocking activity of the isolated R domain. Neither mutation caused a change in the circular dichroism spectrum of the R domain. These results indicate important roles for Lys-516 and Phe-530 in receptor recognition. In addition, on the basis of cytotoxicity data, four other residues (Tyr-514, Val-523, Asn-524, and Lys-526) are proposed play roles in receptor binding. These findings support the notion that the toxin's receptor binds to the solvent-exposed face of the R domain opposite the catalytic domain.
Shen
W H
WH
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Choe
S
S
Eisenberg
D
D
Collier
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D004167:Diphtheria Toxin / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D014709:Vero Cells
D004167:Diphtheria Toxin; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D010649:Phenylalanine; D008239:Lysine