ARF binds the C-terminal region of the Escherichia coli heat-labile toxin (LTA1) and competes for the binding of LTA2.
Cholera toxin (CT) and the heat-labile enterotoxin (LT) from Escherichia coli are highly related in terms of structure and biochemical activities and are the causative agents of cholera and traveler's diarrhea, respectively. The pathophysiological action of these toxins requires their activity as ADP-ribosyltransferases, transferring the ADP-ribose moiety from NAD onto the stimulatory, regulatory component of adenylyl cyclase, Gs. This reaction is highly dependent on the protein cofactor, termed ADP-ribosylation factor (ARF), that is itself a 20 kDa regulatory GTPase. In this study, we define sites of interaction between LTA and human ARF3. The residues identified as important to ARF binding include several of those previously shown to bind to the A2 subunit of the toxin and those important to the organization of two flexible loops, previously implicated as regulators of substrate entry. A model for how ARF acts to enhance the catalytic activity is proposed. A critical portion of the overlap between ARF and LTA(2) in binding LTA(1) includes a short region of sequence homology between LTA(2) and the switch II region of ARF. LTA(2) also interacted with ARF effectors in two-hybrid assays, and thus, we discuss the possibility that the LTA(2) subunit may function in cells as a partial ARF mimetic to compete for the binding of ARF to LTA(1) or regulate aspects of the toxin's transport from the cell surface to the ER.
Zhu
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X
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322-3050, USA.
Kim
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E
Boman
A L
AL
Hodel
A
A
Cieplak
W
W
Kahn
R A
RA
D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli; D011065:Poly(ADP-ribose) Polymerases; C116687:ARF3 protein, human; D020727:ADP-Ribosylation Factors