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Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation.

Surya W, Chooduang S, Choong YK, Torres J, Boonserm P - PLoS ONE (2016)

Bottom Line: Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution.This oligomeric state did not change after incubation of these heterodimers with detergent.Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

No MeSH data available.


Related in: MedlinePlus

SLS molecular weight determination of BinA and BinB in aqueous solution.Debye plots of scattered intensity (■) and KC/RoP (○) plotted against protein concentration. (A) act-A; (B) act-B; (C) 1:1 mixture of pro-A and pro-B; (D) 1:1 mixture of act-A and act-B; (E) Calculated weight-average molecular weight, where bars represent 95% confidence interval as shown in panels A-D (greyed area, with limit values indicated.). The molecular weight was obtained from the inverse of the Y-intercept of the linear fit of KC/RoP. Experiments were done in triplicate (each data point represents the mean ± SD).
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pone.0158356.g003: SLS molecular weight determination of BinA and BinB in aqueous solution.Debye plots of scattered intensity (■) and KC/RoP (○) plotted against protein concentration. (A) act-A; (B) act-B; (C) 1:1 mixture of pro-A and pro-B; (D) 1:1 mixture of act-A and act-B; (E) Calculated weight-average molecular weight, where bars represent 95% confidence interval as shown in panels A-D (greyed area, with limit values indicated.). The molecular weight was obtained from the inverse of the Y-intercept of the linear fit of KC/RoP. Experiments were done in triplicate (each data point represents the mean ± SD).

Mentions: Light scattering was used to determine the molecular weight and size of the species in these samples. The activated toxins act-A and act-B, independently, produced similar molecular weight values (~47 kDa), consistent with monomers (Fig 3A and 3B). The equimolar mixture of the protoxins, pro-A and pro-B produced a weight-average molecular weight of 41 ± 1.3 kDa (Fig 3C). This is close to the average ~45 kDa expected for protoxin monomers and far from the 93 kDa expected for a heterodimer, therefore this is consistent with the gel filtration results shown in Fig 2A. In contrast, mixing the two activated subunits, act-A and act-B, in a 1:1 molar ratio produced a molecular weight of ~70 kDa (Fig 3D), consistent with heterodimer formation. This confirms the conclusions from the gel filtration experiments shown in Fig 2B, and the SLS results are summarized in Fig 3E.


Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation.

Surya W, Chooduang S, Choong YK, Torres J, Boonserm P - PLoS ONE (2016)

SLS molecular weight determination of BinA and BinB in aqueous solution.Debye plots of scattered intensity (■) and KC/RoP (○) plotted against protein concentration. (A) act-A; (B) act-B; (C) 1:1 mixture of pro-A and pro-B; (D) 1:1 mixture of act-A and act-B; (E) Calculated weight-average molecular weight, where bars represent 95% confidence interval as shown in panels A-D (greyed area, with limit values indicated.). The molecular weight was obtained from the inverse of the Y-intercept of the linear fit of KC/RoP. Experiments were done in triplicate (each data point represents the mean ± SD).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920411&req=5

pone.0158356.g003: SLS molecular weight determination of BinA and BinB in aqueous solution.Debye plots of scattered intensity (■) and KC/RoP (○) plotted against protein concentration. (A) act-A; (B) act-B; (C) 1:1 mixture of pro-A and pro-B; (D) 1:1 mixture of act-A and act-B; (E) Calculated weight-average molecular weight, where bars represent 95% confidence interval as shown in panels A-D (greyed area, with limit values indicated.). The molecular weight was obtained from the inverse of the Y-intercept of the linear fit of KC/RoP. Experiments were done in triplicate (each data point represents the mean ± SD).
Mentions: Light scattering was used to determine the molecular weight and size of the species in these samples. The activated toxins act-A and act-B, independently, produced similar molecular weight values (~47 kDa), consistent with monomers (Fig 3A and 3B). The equimolar mixture of the protoxins, pro-A and pro-B produced a weight-average molecular weight of 41 ± 1.3 kDa (Fig 3C). This is close to the average ~45 kDa expected for protoxin monomers and far from the 93 kDa expected for a heterodimer, therefore this is consistent with the gel filtration results shown in Fig 2A. In contrast, mixing the two activated subunits, act-A and act-B, in a 1:1 molar ratio produced a molecular weight of ~70 kDa (Fig 3D), consistent with heterodimer formation. This confirms the conclusions from the gel filtration experiments shown in Fig 2B, and the SLS results are summarized in Fig 3E.

Bottom Line: Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution.This oligomeric state did not change after incubation of these heterodimers with detergent.Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

No MeSH data available.


Related in: MedlinePlus