CRYSTAL STRUCTURE OF THE CATALYTIC DOMAIN OF PSEUDOMONAS AERUGINOSA EXOTOXIN A COMPLEXED WITH AN NAD ANALOGUE: IMPLICATIONS FOR THE ACTIVATION PROCESS AND FOR ADP-RIBOSYLATION. Mi Li^*, Fred Dyda^*, Itai Benhar^_, Ira Pastan^_, David R. Davies^*, Laboratory of Molecular Biology, NIDDK and ^_Laboratory of Molecular Biology, DCBDC, NCI, National Institutes of Health, Bethesda MD 20892

The isolated third domain of pseudomonas aeruginosa exotoxin A (PEIII) catalyses the transfer of ADP-ribose from NAD to elongation factor-2 in eukaryotic cells. A previous structure of PEIII crystallized in the presence of NAD showed only hydrolysis products of NAD bound to the enzyme. In order to define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analogue, ß-thiazole-4-carboxamide adenine dinucleotide (ß-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 ß-TAD show some differences in the two binding sites. The TAD attached to monomer 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. The other molecule, monomer 1, shows an intact TAD and has no crystal packing contacts in the vicinity of ß-TAD, so that the observed conformation probably most resembles that in the free PEIII in solution.

Li, Dyda, Benhar, Pastan & Davies: The Crystal Structure of pseudomonas aeruginosa exotoxin Domain III with nicotinamide and AMP: Conformational differences with the intact extoxin. PNAS, 92: 9308-9312, 1995.