Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa.
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 K(i) 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 A (1 A=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.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1.
Susan P
SP
Yates
Patricia L
PL
Taylor
René
R
Jørgensen
Dana
D
Ferraris
Jie
J
Zhang
Gregers R
GR
Andersen
A Rod
AR
Merrill
D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005098:Exotoxins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000201:enzymology; D012995:Solubility; D013329:Structure-Activity Relationship; D037521:Virulence Factors / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000737:chemistry*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D005098:Exotoxins; D010446:Peptide Fragments; D037521:Virulence Factors; D014867:Water; D000097668:Pseudomonas aeruginosa Exotoxin A