Two-Site Autoinhibition of the ADP-Ribosylating Mosquitocidal Toxin (MTX) from Bacillus sphaericus by Its 70-kDa Ricin-like Binding Domain

Abstract

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.

Structure and Mode of Action of a Mosquitocidal Holotoxin

“…7 This observation, in conjunction with the showing the three RB repeats in orange, green and blue. Note that each RB repeat has three strands in one β-sheet and the fourth strand in another β-sheet.…”

“…8 Moreover, it was shown that the four RBL domains of MTX holo cause an autoinhibition that is non-competitive with respect to NAD + . 7 Here, we report the structure of MTX holo and discuss the autoinhibition as well as the association and the possible functions of the RBL domains.…”

“…3,4 MTX is produced as a single polypeptide chain of 870 residues consisting of a putative signal sequence (residues 1-29), a catalytic domain (residues 30-264), a linker (265-295) and a further 575 residues. [3][4][5][6][7][8] The catalytic domain is homologous to those of other toxins such as cholera toxin, pertussis toxin, Escherichia coli heat-labile enterotoxin, Pseudomonas exotoxin A and diphtheria toxin [9][10][11][12][13][14][15] as well as to ADPribosylating enzymes such as poly(ADP-ribosyl) polymerase (PARP) and ART2.2. 16,17 The catalytic domain of MTX ADP-ribosylates arginines of various proteins.…”

“…5,6 In vitro, MTX holo can be activated by a single proteolytic cut with chymotrypsin and a subsequent removal of the C-terminal fragment (residues 265-870). [5][6][7] Previously, it was shown that residues 265-285 of the C-terminal fragment inhibit ADP-ribosylation in competition with NAD + . 7 This kinetic result was confirmed by the structure of the catalytic domain plus extended linker (MTX cali ) showing that residues 270-283 occupy the characteristic NAD + binding site.…”

“…[5][6][7] Previously, it was shown that residues 265-285 of the C-terminal fragment inhibit ADP-ribosylation in competition with NAD + . 7 This kinetic result was confirmed by the structure of the catalytic domain plus extended linker (MTX cali ) showing that residues 270-283 occupy the characteristic NAD + binding site. 8 Moreover, it was shown that the four RBL domains of MTX holo cause an autoinhibition that is non-competitive with respect to NAD + .…”