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

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

Structural and Biophysical Characterization of <i>Bacillus thuringiensis</i> Insecticidal Proteins Cry34Ab1 and Cry35Ab1

<div><p><i>Bacillus thuringiensis</i> strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from <i>B. thuringiensis</i> strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) <i>Diabrotica virgifera virgifera</i> Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.</p></div

Crystal structures of Cry34Ab1 and Cry35Ab1.

<p>(A) The structure of Cry34Ab1 is a β-sandwich of 10 strands. (B) Cry35Ab1 contains two domains. The N-terminal trefoil domain contains α-helices and three β-sheets. The C-terminal domain is terminated with a three helix fold which is not required for activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-Gao1" target="_blank">[17]</a>. This figure, and all subsequent structure representations, were made with PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-DeLano1" target="_blank">[66]</a>.</p

<b>Table 3.</b> Measured and theoretical values for CCS for Cry34Ab1 and trCry35Ab1.

<p>Theoretical values were calculated using the projection approximation (PA), exact hard sphere scattering (EHSS) and trajectory method (TM) with helium as the collision gas. Experimentally measured CCS used nitrogen as the collision gas.</p><p><b>Table 3.</b> Measured and theoretical values for CCS for Cry34Ab1 and trCry35Ab1.</p

Scattering curve of the SAXS data and experimental fit.

<p>(A) Scattering curves for Cry34Ab1 (blue line) from the SAXS experiment and the fit made by the GNOM program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-Semenyuk1" target="_blank">[20]</a> (red line) to scattering curve. (B) Scattering curves for trCry35Ab1. The X axis is s in arbitrary units where s = 4πsinθ/λ and the Y axis is the log of intensity in arbitrary units.</p

Comparison of proteins structurally related to Cry35Ab1.

<p>Cry35Ab1 is structurally related to a wide variety pore-forming proteins as assessed by combinatorial extension. All structures contain a conserved beta-sheet core and varying loop regions.</p

<b>Table 1.</b> Cry34Ab1 (4JOX) data processing, model and refinement statistics.

a<p> R_merge  =  100Σ(h)Σ(i)|I(i)-<i>|/Σ(h)Σ(i)I(i) where I(i) is the <b>i</b>th intensity measurement of reflection h, and <i> is the average intensity from multiple observations.</i></i></p><i><i>b<p> R_cryst  =  Σ||<b>F</b>_obs|-|<b>F</b>_calc||/Σ|<b>F</b>_obs|. Where <b>F</b>_obs and <b>F</b>_calc are the structure factor amplitudes from the data and the model, respectively. R_free is R_cryst with 10% of the structure factors.</p>c<p> Number of residues in favored/additionally favored outlier region. Calculated using PROCHECK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-Laskowski1" target="_blank">[14]</a>.</p><p><b>Table 1.</b> Cry34Ab1 (4JOX) data processing, model and refinement statistics.</p></i></i

<b>Table 5.</b> Combinatorial extension analysis of PDB submitted structures against Cry35Ab1 coordinates.

<p>Results of the top 10 unique hits ranked according to Z-score.</p><p><b>Table 5.</b> Combinatorial extension analysis of PDB submitted structures against Cry35Ab1 coordinates.</p

<b>Table 2.</b> Cry35Ab1 (4JP0) data processing, model and refinement statistics.

a<p> R_merge  =  100Σ(h)Σ(i)|I(i)-<i>|/Σ(h)Σ(i)I(i) where I(i) is the <b>i</b>th intensity measurement of reflection h, and <i> is the average intensity from multiple observations.</i></i></p><i><i>b<p> R_cryst  =  Σ||<b>F</b>_obs|-|<b>F</b>_calc||/Σ|<b>F</b>_obs|. Where <b>F</b>_obs and <b>F</b>_calc are the structure factor amplitudes from the data and the model, respectively. R_free is R_cryst with 10% of the structure factors.</p>c<p> Number of residues in favored/additionally favored outlier region. Calculated using PROCHECK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112555#pone.0112555-Laskowski1" target="_blank">[14]</a>.</p><p><b>Table 2.</b> Cry35Ab1 (4JP0) data processing, model and refinement statistics.</p></i></i