title: Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors.
abstract: Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.
authors.affiliation: Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Stevens
authors.forename: J
authors.initials: J
authors.lastname: Moss
authors.forename: M
authors.initials: M
authors.lastname: Vaughan
authors.forename: M
authors.initials: M
authors.lastname: Pizza
authors.forename: R
authors.initials: R
authors.lastname: Rappuoli
mesh_terms: D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004768:Enterotoxins / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics; D014633:Valine / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D000246:Adenosine Diphosphate Ribose; D014443:Tyrosine; C027538:heat-labile enterotoxin, E coli; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D014633:Valine; D008239:Lysine