Two-site autoinhibition of the ADP-ribosylating mosquitocidal toxin (MTX) from Bacillus sphaericus by its 70-kDa ricin-like binding domain.
The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is an approximately 97-kDa arginine-specific ADP-ribosyltransferase that is activated by proteolytic cleavage, thereby releasing the active 27-kDa enzyme (MTX(30-264)) and a 70-kDa C-terminal fragment (MTX(265-870)). In solution, the cleaved 70-kDa fragment is still a potent inhibitor of the ADP-ribosyltransferase activity of MTX. Here we studied the interaction of the 70-kDa fragment with the enzyme domain of MTX. Several C-terminal deletions of the 70-kDa fragment inhibited the enzymatic activity of MTX(30-264). However, the IC(50) values were about 2 orders of magnitude higher for the deletions than for the 70-kDa fragment. A peptide covering amino acid residues 265-285 of the holotoxin exhibited the same inhibitory potency as the C-terminal deletions of the 70-kDa fragment. MTX(265-285) contains several acidic residues, of which D273 and D275 were found to be essential for the inhibitory effect. Exchange of these residues in the 70-kDa fragment (MTX(265-870)) reduced its inhibitory potency. Kinetic analysis showed that the peptide MTX(265-285) had no effect on the V(max) of MTX(30-264) but increased the K(m) for NAD. By contrast, the 70-kDa fragment deleted of residues Ile265 through Asn285 inhibited the enzyme activity of MTX(30-264) mainly by decreasing the V(max) of the enzyme. A second binding site for interaction of MTX(265-870) with MTX(30-264) was localized to the C-terminus within the region of residues 750-870. The data support a two-site binding model for inhibition of the ADP-ribosyltransferase activity of MTX(30-264) by the 70-kDa fragment MTX(265-870) with an interaction of amino acid residues 265-285 at the active site and an allosteric inhibition by the C-terminal part of the 70-kDa fragment.
Carpusca
Irina
I
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany.
Schirmer
Jörg
J
Aktories
Klaus
K
D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001407:Bacillus / Q000737:chemistry*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D012276:Ricin / Q000737:chemistry*
D001427:Bacterial Toxins; D010446:Peptide Fragments; C068603:mosquitocidal toxin SSII-1; D012276:Ricin; D036002:ADP Ribose Transferases