Crystallization of the Bacillus thuringiensis toxin Cry1Ac and its complex with the receptor ligand N-acetyl-D-galactosamine.
Cry1Ac from Bacillus thuringiensis ssp. kurstaki HD-73 is a pore-forming protein specifically toxic to lepidopteran insect larvae. It binds to the cell-surface receptor aminopeptidase N in Manduca sexta midgut via the sugar N-acetyl-D-galactosamine (GalNAc). By using 1,3-diaminopropane (DAP) as the buffer throughout protoxin activation and chromatography on Q-Sepharose at pH 10.3, trypsin-activated Cry1Ac has been purified in a monomeric state, which was crucial to obtaining single crystals of Cry1Ac and of the Cry1Ac-GalNAc complex. Crystals of Cry1Ac alone are triclinic, with unit-cell parameters a = 51.78, b = 113.23, c = 123.41 A, alpha = 113.11, beta = 91.49, gamma = 100.46 degrees; those of the Cry1Ac-GalNAc complex show P2(1) symmetry, with unit-cell parameters a = 121.36, b = 51.19, c = 210.56 A, beta = 105.75 degrees. Data sets collected to 2.36 and 2.95 A resolution, respectively, show that both crystal forms contain four molecules of the 66 kDa toxin in the asymmetric unit and have related packing arrangements. The deaggregating effect of DAP may be explained by its capacity for bivalent hydrogen bonding and hydrophobic interactions at protein interfaces.
Derbyshire
D J
DJ
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England.
Ellar
D J
DJ
Li
J
J
D000116:Acetylgalactosamine / Q000737:chemistry*; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D001427:Bacterial Toxins; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D004731:Endotoxins / Q000737:chemistry* / Q000302:isolation & purification; D006460:Hemolysin Proteins; D008871:Microvilli / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000116:Acetylgalactosamine