Structural requirements of the unique disulphide bond and the proline-rich motif within the alpha4-alpha5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa delta-endotoxin.
Both the disulphide bond (Cys192-Cys199) and the proline-rich motif (Pro193ProAsnPro196) in the long loop connecting the alpha4-alpha5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry delta-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes aegypti larvae were determined, Escherichia coli cells expressing each mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A mutants. Increasing the flexibility of the alpha4-alpha5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations significantly decreased the larvicidal activity. Similar to the wild-type protoxin, all mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These findings are the first biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the alpha4-alpha5 loop of the Cry4Aa toxin.
Tapaneeyakorn
Satita
S
Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Pornwiroon
Walairat
W
Katzenmeier
Gerd
G
Angsuthanasombat
Chanan
C
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers; D004220:Disulfides / Q000737:chemistry*; D004731:Endotoxins / Q000737:chemistry* / Q000494:pharmacology; D006460:Hemolysin Proteins; D007814:Larva / Q000187:drug effects*; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004220:Disulfides; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D011392:Proline