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 ∼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 IC₅₀ 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.