Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: Implications for the activation process and for ADP ribosylation

Mi Li, Fred Dyda, Itai Benhar, Ira Pastan, David R. Davies*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

88 Scopus citations

Abstract

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, β-methylene-thiazole-4- carboxamide adenine dinucleotide (β-TAD), and refined the complex structure at 2.3 Å resolution. There are two independent molecules of PEIII in the crystal, and the conformations of β-TAD show some differences in the two binding sites. The β-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However, molecule 1 binds to an intact β-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.

Original languageEnglish
Pages (from-to)6902-6906
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number14
DOIs
StatePublished - 9 Jul 1996
Externally publishedYes

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