Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa

Susan P. Yates, Patricia L. Taylor, René Jørgensen, Dana Ferraris, Jie Zhang, Gregers R. Andersen, A. Rod Merrill

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)667-675
Number of pages9
JournalBiochemical Journal
Volume385
Issue number3
DOIs
StatePublished - Feb 1 2005
Externally publishedYes

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Keywords

  • Bacterial toxin
  • Competitive inhibitor
  • Exotoxin A
  • Mono-ADP- ribosyltransferase
  • Pseudomonas aeruginosa
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Yates, S. P., Taylor, P. L., Jørgensen, R., Ferraris, D., Zhang, J., Andersen, G. R., & Merrill, A. R. (2005). Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa. Biochemical Journal, 385(3), 667-675. https://doi.org/10.1042/BJ20041480