The
G-protein-coupled receptor BT-R1 in the moth Manduca sexta represents a class of single-membrane-spanning
α-helical proteins within the cadherin family that regulate
intercellular adhesion and contribute to important signaling activities
that control cellular homeostasis. The Cry1A toxins, Cry1Aa, Cry1Ab,
and Cry1Ac, produced by Bacillus thuringiensis bind BT-R1 very tightly (K
d = 1.1 nM) and trigger a Mg2+-dependent signaling pathway
that involves the stimulation of G-protein α-subunit, which
subsequently launches a coordinated signaling cascade, resulting in
insect death. The three Cry1A toxins compete for the same binding
site on BT-R1, and the pattern of inhibition of insecticidal
activity against M. sexta is strikingly
similar for all three toxins. The binding domain is localized in the
12th cadherin repeat (EC12: Asp1349 to Arg1460, 1349DR1460) in BT-R1 and to various truncation fragments
derived therefrom. Fine mapping of EC12 revealed that the smallest
fragment capable of binding is a highly conserved 94-amino acid polypeptide
bounded by Ile1363 and Ser1456 (1363IS1456),
designated as the toxin-binding site (TBS). Logistical regression
analysis revealed that binding of an EC12 truncation fragment containing
the TBS is antagonistic to each of the Cry1A toxins and completely
inhibits the insecticidal activity of all three. Elucidation of the
EC12 motif of the TBS by X-ray crystallography at a 1.9 Å resolution
combined with results of competitive binding analyses, live cell experiments,
and whole insect bioassays substantiate the exclusive involvement
of BT-R1 in initiating insect cell death and demonstrate
that the natural receptor BT-R1 contains a single TBS.