The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant Ki of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 Å (1 Å=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.
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Research Article|
January 24 2005
Structure–function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa
Susan P. YATES;
Susan P. YATES
*Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1
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Patricia L. TAYLOR;
Patricia L. TAYLOR
*Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1
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René JØRGENSEN;
René JØRGENSEN
†Macromolecular Crystallography, Department of Molecular Biology, University of Aarhus, Gustav Wieds vej 10C, DK8000 Aarhus, Denmark
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Dana FERRARIS;
Dana FERRARIS
‡Guilford Pharmaceuticals, Baltimore, MD 21224, U.S.A.
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Jie ZHANG;
Jie ZHANG
‡Guilford Pharmaceuticals, Baltimore, MD 21224, U.S.A.
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Gregers R. ANDERSEN;
Gregers R. ANDERSEN
1
†Macromolecular Crystallography, Department of Molecular Biology, University of Aarhus, Gustav Wieds vej 10C, DK8000 Aarhus, Denmark
1Correspondence may be addressed to either of the authors (email rmerrill@uoguelph.ca and grand@bioxray.dk).
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A. Rod MERRILL
A. Rod MERRILL
1
*Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1
1Correspondence may be addressed to either of the authors (email rmerrill@uoguelph.ca and grand@bioxray.dk).
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Publisher: Portland Press Ltd
Received:
August 31 2004
Revision Received:
September 24 2004
Accepted:
September 30 2004
Accepted Manuscript online:
September 30 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (3): 667–675.
Article history
Received:
August 31 2004
Revision Received:
September 24 2004
Accepted:
September 30 2004
Accepted Manuscript online:
September 30 2004
Citation
Susan P. YATES, Patricia L. TAYLOR, René JØRGENSEN, Dana FERRARIS, Jie ZHANG, Gregers R. ANDERSEN, A. Rod MERRILL; Structure–function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa. Biochem J 1 February 2005; 385 (3): 667–675. doi: https://doi.org/10.1042/BJ20041480
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