The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Abstract

Background: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action.

Results: The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action.

Conclusions: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.

Keywords: Bacillus thuringiensis; Cry6; Hemolysin; Insecticidal toxin.

Fig. 1

Crystal structure of Cry6a toxin. Ribbon representation of trypsin-truncated Cry6Aa form showing two domain architecture: the “tail” domain consists of one helical bundle with five long α-helices, labeled αA, αC, αD, αG, and αH, and shorter helices, labeled αB, αE, αF, and αI; while several long and short loops form the “head” domain. N- and C-termini and the putative transmembrane region are labeled. The Cys88-Cys451 disulfide bond is shown and, in the insert box, the final 2Fo-Fc electron density map calculated at 1.5σ in the region of this bond is shown in blue mesh. Side and main chains of the amino acid residues are presented as sticks and colored by the atoms

Fig. 2

Comparison of Cry6Aa structures. a Superimposed ribbon representations of the crystal structures of the truncated (cyan) and the full-length (magenta) Cry6Aa forms. b The full-length Cry6Aa model is shown with the following features illustrated: wing-like intra-helical loops (red); putative transmembrane region (orange) with L259 shown in stick representation; WATIGAxI repeat sequence (green); TTNMTSNQY repeat sequence (cyan); WYNNSDWYNNSDW repeat (magenta); and the modeled Asn388–Lys450 in dark gray and cyan

Fig. 3

Charge state distributions (CSDs) of Cry6Aa. a Under non-reducing conditions the CSD centers around m/z 1690.84. b Following treatment with 1 mM dithiothreitol (DTT), the CSD centers around m/z 1021.34

Fig. 4

Comparison of Cry6Aa structure with structures of other related toxins. Superposition of Cry6Aa (cyan) with a HBL-B (yellow), b NheA (gray), and c HlyE (orange). In panel d, the surface representation of hydrophobic areas of the truncated Cry6Aa in ribbon representation are colored green and the remaining residues are colored gray. The putative transmembrane region is labeled (TM). C-terminal 463–472 residues are removed for simplicity

Fig. 5

Nematode bioassay: C. elegans were fed on E. coli transformed with the pET28b plasmid as a negative control (Vector); transformed with this vector containing the wild-type cry6Aa gene (Cry6Aa); or transformed with the vector with the L259D mutant of this gene (L259D). a Images of some nematodes chosen at random are shown and b results are presented as mean worm area (12 worms per bar, each bar average from three independent experiments). p values comparing each condition to vector control are shown. Error bars represent the standard errors of the means

Fig. 6

In vivo pore formation. a Bright field images (upper panels) and fluorescence images (lower panels) of the anterior regions of C. elegans fed on E. coli transformed with pQE9 vector or plasmids expressing Cry6Aa or Cry5Ba. b Percentage of treated worms showing propidium iodide (PI) uptake. The p values for the comparison between each toxin and the no-toxin control are shown

Fig. 7

Structural families of B. thuringiensis delta-endotoxins. Representatives of different structural classes of delta-endotoxins of B. thuringiensis are shown. Cry1Aa [PDB: 1CIY] [52] is a three-domain toxin; Cry6Aa is an alpha helical toxin (this work); Cry34 is an aegerolysin-like protein [PDB: 4JOX] [12] that acts as a binary toxin with Cry35, a Toxin_10 family protein [PDB: 4JP0] [12]; Cry51 is a member of the Etx/Mtx2 family [PDB: 4PKM] [11]; and Cyt1Aa, a member of the Bac_thur_toxin family [PDB: 3RON] [53]. All structures are to scale and colored according to secondary structure (alpha helix, red; beta strands, yellow)