Interaction of diphtheria toxin with adenylyl-(3',5')-uridine 3'-monophosphate. II. The NAD-binding site and determinants of dinucleotide affinity.

C M Collins, R J Collier

The Journal of biological chemistry 1984 Dec 25

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Diphtheria toxin (DT) binds NAD with a KD of about 10 microM and adenylyl-(3',5')-uridine 3'-monophosphate (ApUp) with KD values ranging from 9 pM to 1.8 nM, depending on temperature (Collins, C. M., Barbieri, J. T., and Collier, R. J. (1984) J. Biol. Chem. 259, 15154-15158). Here we report experiments to explore relationships between ApUp binding and NAD binding to DT and to identify structural features of ApUp that determine its high affinity for DT. NAD, adenine, and nicotinamide competitively inhibited ApUp binding to DT, and we confirmed that ApUp blocked the binding and hydrolysis of NAD. Binding of P-site ligands to the toxin blocked interactions with ApUp. CRM197, a mutant form of DT defective in NAD binding and hydrolysis, bound ApUp 5,000-fold less tightly than did DT. These results are consistent with models in which the ApUp- and NAD-binding sites on DT overlap or are identical. Various mono-, di-, and oligonucleotides were studied as competitors of ApUp binding or the NAD-glycohydrolase reaction. The results imply that the high affinity of ApUp for DT depends on the presence of the 3'-terminal phosphate and a 3'-5' internucleoside linkage. There was strong specificity for adenine as the 5' base, but only weak specificity for uracil as the 3' base. Oligoribonucleotides containing additional nucleotides at either or both ends of ApUp sequences bound to the toxin 1-3 orders of magnitude less avidly than ApUp. Oligodeoxyribonucleotides containing dApdT sequences bound with still lower affinities. In contrast to the case with whole toxin, ApUp bound to fragment A less avidly than did NAD, and elimination of the 3'-terminal phosphate of ApUp resulted in increased affinity for the protein. These differences may reflect the absence in free fragment A of interactions with the cationic P-site, located on the toxin's B moiety.

Citation

C M Collins, R J Collier. Interaction of diphtheria toxin with adenylyl-(3',5')-uridine 3'-monophosphate. II. The NAD-binding site and determinants of dinucleotide affinity. The Journal of biological chemistry. 1984 Dec 25;259(24):15159-62