Identification of residues in domain III of Bacillus thuringiensis Cry1Ac toxin that affect binding and toxicity

Alanine substitution mutations in the Cry1Ac domain III region, from amino acid residues 503 to 525, were constructed to study the functional role of domain III in the toxicity and receptor binding of the protein to Lymantria dispar, Manduca sexta, and Heliothis virescens. Five sets of alanine block mutants were generated at the residues (SS504)-S-503, (NNI508)-N-506, (509 )QNR(511), (ST523)-S-522, and (ST525)-S-524. Single alanine substitutions w ere made at the residues (509)Q, N-510, R-511, and Y-513. All mutant protei ns produced stable toxic fragments as judged by trypsin digestion, midgut e nzyme digestion, and circular dichroism spectrum analysis. The mutations, ( 50)3SS(504)-AA, (NNI508)-N-506-AAA, (ST523)-S-522-AA, (ST525)-S-524-AA, and N-510-A affected neither the protein's toxicity nor its binding to brush b order membrane vesicles (BBMV) prepared from these insects. Toward L. dispa r and M. sexta, the (509)QNR(511)-AAA, (509)Q-A, R-511-A, and Y-513-A mutan t toxins showed 4- to 10-fold reductions in binding affinities to BBMV, wit h 2- to 3-fold reductions in toxicity. Toward H. virescens, the (509)QNR(51 1)-AAA, R-511-A, R-511-A, and Y-513-mutant toxins showed 8- to 22-fold redu ctions in binding affinities, but only (509)QNR(511)-AAA and R-511-A mutant toxins reduced toxicity by approximately three to four times. In the prese nt study, greater loss in binding affinity relative to toxicity has been ob served. These data suggest that the residues (509)Q, R-511, and Y-513 in do main III might be only involved in initial binding to the receptor and that the initial binding step becomes rate limiting only when it is reduced mor e than fivefold.