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Applied and Environmental Microbiology, October 1999, p. 4513-4520, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification of Residues in Domain III of Bacillus thuringiensis Cry1Ac Toxin That Affect Binding and Toxicity

Mi Kyong Lee,1 Taek H. You,1 Fred L. Gould,2 and Donald H. Dean1,*

Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210,1 and Department of Entomology, North Carolina State University, Raleigh, North Carolina 276952

Received 1 April 1999/Accepted 5 August 1999

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 503SS504, 506NNI508, 509QNR511, 522ST523, and 524ST525. Single alanine substitutions were made at the residues 509Q, 510N, 511R, and 513Y. All mutant proteins produced stable toxic fragments as judged by trypsin digestion, midgut enzyme digestion, and circular dichroism spectrum analysis. The mutations, 503SS504-AA, 506NNI508-AAA, 522ST523-AA, 524ST525-AA, and 510N-A affected neither the protein's toxicity nor its binding to brush border membrane vesicles (BBMV) prepared from these insects. Toward L. dispar and M. sexta, the 509QNR511-AAA, 509Q-A, 511R-A, and 513Y-A mutant toxins showed 4- to 10-fold reductions in binding affinities to BBMV, with 2- to 3-fold reductions in toxicity. Toward H. virescens, the 509QNR511-AAA, 509Q-A, 511R-A, and 513Y-mutant toxins showed 8- to 22-fold reductions in binding affinities, but only 509QNR511-AAA and 511R-A mutant toxins reduced toxicity by approximately three to four times. In the present study, greater loss in binding affinity relative to toxicity has been observed. These data suggest that the residues 509Q, 511R, and 513Y in domain 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 more than fivefold.

* Corresponding author. Mailing address: Department of Biochemistry, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210. Phone: (614) 292-8829. Fax: (614) 292-3206. E-mail: dean.10{at}osu.edu.

Applied and Environmental Microbiology, October 1999, p. 4513-4520, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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