Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Abstract

Bacillus thuringiensis 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 B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera 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.

Ion Mobility Collision Cross Section Compendium

“…72 While most of the CCS values have been for tryptic peptides, there is recent and significant efforts being made in the quantitative IM analysis of structurally-interesting peptide and protein classes, including helical peptides, 174–176 metalloproteins, 177–180 intrinsically-disordered proteins, 181–184 metamorphic proteins, 185,186 amyloids, 187–194 and membrane-bound proteins and assemblies. 117,195–198 The last three years has seen a balance of cross section reporting across most of the chemical classes, including lipids and carbohydrates. The exception is nucleotide CCS values, which, aside for the 2009 study from McLean and coworkers, 68 have been published in small numbers spread across several studies and years, and currently comprise about 1% all CCS values reported.…”

Structural classification of insecticidal proteins – Towards an in silico characterisation of novel toxins

“…All of the other Toxin_10 proteins act with their specific partner proteins to form binary toxins as follows: BinA and BinB (both Toxin_10 proteins) (Broadwell et al, 1990;Oei et al, 1990); Cry34 (aegerolysin-like) and Cry35 (Toxin_10) (Kelker et al, 2014;Masson et al, 2004); Cry48 (3-domain) and Cry49 (Jones et al, 2007). The role of each protein in these binary pairs is clearly significant to understanding the specificity of the toxins and potential binding of both components presents further challenges to prediction.…”

“…Cry21 The structures of the insecticidal toxins BinB (PDB 3WA1 (Srisucharitpanit et al, 2014)), Cry35 (PDB 4JP0 (Kelker et al, 2014)), Cry23 (PDB 4RHZ), Cry45 (PDB 2D42 (Akiba et al, 2006)), Cry46 (PDB 2ZTB (Akiba et al, 2009)) and Cry51 (PDB 4PKM (Xu et al, 2015)) are…”

“…Other groups include the Cyt toxins, the ADP-ribosyl transferase toxins Mtx1 and Vip1/2 (along with the Vip1-like Vip4 protein). Cry34 is an aegerolysin like protein and with Cry35 is part of a two-component toxin (Kelker et al, 2014). Cry37, which itself is part of a two-component toxin with Cry23, which shows structural homology with Cry34 (Rydel et al, 2001).…”

“…These include Cry37 (de Maagd et al, 2003) and the aegerolysin-like Cry34 (Kelker et al, 2014) which both form part of binary toxins (with Cry23 and Cry35 respectively, discussed below). The presence of extended beta sheets also characterises the sphaericolysin/anthrolysin family of toxins that is highly conserved across isolates from (Berry, 2012;Bourdeau et al, 2009) and appear to act as cholesterol dependent cytolysins (From et al, 2008;Nishiwaki et al, 2007).…”