Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

Significance Pesticidal proteins from bacteria, such as Bacillus thuringiensis and Lysinibacillus sphaericus, are widely used as biocontrol agents against various mosquito vectors of human disease. Tpp49Aa1/Cry48Aa1 from L. sphaericus are required as a pair to exert toxicity and are able to overcome resistance to currently marketed bioinsecticides. We used an emerging technique to obtain a high-resolution structure of Tpp49Aa1 from natively produced crystals. Cellular models confirmed that both proteins are required to elicit cell death, demonstrating the potential utility of these cells as models for better understanding Tpp49/Cry48 mechanism of action. Bioassays identified activity of the protein pair against three previously unrecognized mosquito targets. These data will help inform the future design and use of biological mosquitocides.


Figure S1 .
Figure S1.Transmission electron microscopy on negative stained Tpp49Aa1 nanocrystals.(A) Tpp49Aa1 nanocrystals and remaining parasporal bodies from the crystal purification process.(B) Visualization of crystal lattice of Tpp49Aa1 nanocrystals allows assessment of the quality of different purification batches.(C) Confirmation of crystal quality is obtained by selected area electron diffraction imaging (SAED).

Figure S2 .
Figure S2.Electron density map and model of the XFEL structure of Tpp49Aa1.A slice through the centre of the electron density of a monomer of Tpp49Aa1 is shown.Protein backbone is coloured yellow, nitrogen atoms are coloured blue, and oxygen atoms are coloured pink.(A) Tpp49Aa1, pH 7 (B) Tpp49Aa1, pH 3 (C) Tpp49Aa1, pH 11

Figure S3 .
Figure S3.Tpp49Aa1 forms a homodimer similar to the Tpp1Aa2/Tpp2Aa2 heterodimer.(A) Tpp49Aa1 homodimer (magenta and light pink).(B) Tpp1Aa2 (blue)/Tpp2Aa2 (light blue) heterodimer.Equivalent disulphide bonds in Tpp49Aa1 and Tpp2Aa2 monomers are shown as spheres (cyan).In the Tpp49Aa1 homodimer, the interface between the two monomers involves 41 residues from monomer A and 42 residues from monomer B, making 16 hydrogen bonds.Interface analysis by PISA estimates the Tpp49Aa1 interface area at 1329.1 Å 2 and the binding energy at -11.1 kcal mol -1 .In the Tpp1Aa2/Tpp2Aa2 heterodimer, the interface between the Tpp1Aa2 and Tpp2Aa2 monomers involves 49 residues from Tpp1Aa2 and 63 residues from Tpp2Aa2, making 19 hydrogen bonds and 2 salt bridges.Consistent with this, interface analysis by PISA estimates the interface area at 1833.1 Å 2 and the binding energy at -22.5 kcal mol -1 , indicating a more stable complex for Tpp1Aa2/Tpp2Aa2 heterodimers than for Tpp49Aa1 homodimers.The RMSD of the aligned atoms between the Tpp49Aa1 homodimer and Tpp1Aa2/Tpp2Aa2 heterodimer was estimated by PyMOL at 9.023 Å. (C) Alignment of Tpp49Aa1 (light pink) and Tpp2Aa2 (light blue).The equivalent Tpp49Aa1 Cys91-Cys183, and Tpp2Aa2, Cys67-Cys161 disulphide bonds are shown as sticks.

Figure S5 .
Figure S5.Tpp49Aa1 is predominantly monomeric in solution.A combination of size exclusion chromatography (SEC) and static light scattering (RALS) was used to determine if Tpp49Aa1 is dimeric or monomeric in solution.(A) Crystalline protein solubilised in Na2CO3 overnight showing Cry48Aa1 at ~ 70 kDa and Tpp49Aa1 as two bands at ~ 49 and ~55 kDa.(B) SEC shows three absorbance peaks (UV 280 nm) representing 766, 121, and 51 kDa, respectively.SDS-page resolution of the fractions taken from those peaks shows no product at 1 (*column void volume, presumably non-protein contaminants), and bands of approximately the expected sizes in 2 and 3. (C) Protein-containing peaks (2 & 3) were concentrated and used to decipher molecular weight via Right Angle Light Scattering (RALS, blue) and Refractive Index (RI, pink) measurements.From these data the molecular weight (Da, black) for the protein was calculated to be 52.1 kDa when calibrated to BSA (1mg/ mL) using OmniSEC software.

Figure S6 .
Figure S6.Tpp49Aa1 dimer highlighting the regions proposed to interact with Cry48Aa1.Tpp49Aa1 regions proposed to interact with Cry48Aa1 (cyan) are partially buried within the dimer interface.Tpp49Aa1 monomers shown in magenta and pink.

Figure
Figure S7.Solvent channels in Tpp49Aa1 crystals Figure S8.SF9 cells show no reduction in viability after 48-hour exposure toTpp49Aa1/Cry48Aa1.Resazurin was used to quantify the effect of these proteins on cell viability.SF9 cells were treated with a range of concentrations of either Cry48Aa1 ("48"), Tpp49Aa1 ("49"), or equimolar amounts in combination (48/49).Resazurin was added to the cells 48 h post toxin exposure.No significant difference was observed.All data are presented as percentage of control conditions (buffer only) with the mean ± SD and all statistical analysis was performed using a oneway ANOVA.