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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

Gel filtration of pro- and act-Bin subunits.(A) Elution profile of pro-A, pro-B, and their mixture (1:1 molar ratio, *); (B) the same as (A) for act-A and act-B. Protein concentration before and after chromatography was typically 60 μM and 18 μM (total monomer), respectively. The SDS PAGE gel for the elution band of the 1:1 mixture is shown in the insert; (C) MALDI-TOF mass spectrum of the elution band shown in B. Four peaks are observed, associated with masses of approximately 40 kDa (mass to charge ratios (m/z) of 40,800 and 20,402 Da) and 45 kDa (m/z of 22,500 and 45,026 Da).
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pone.0158356.g002: Gel filtration of pro- and act-Bin subunits.(A) Elution profile of pro-A, pro-B, and their mixture (1:1 molar ratio, *); (B) the same as (A) for act-A and act-B. Protein concentration before and after chromatography was typically 60 μM and 18 μM (total monomer), respectively. The SDS PAGE gel for the elution band of the 1:1 mixture is shown in the insert; (C) MALDI-TOF mass spectrum of the elution band shown in B. Four peaks are observed, associated with masses of approximately 40 kDa (mass to charge ratios (m/z) of 40,800 and 20,402 Da) and 45 kDa (m/z of 22,500 and 45,026 Da).

Mentions: To study the oligomerization behavior of Bin toxin subunits without detergent, their elution profile was monitored using gel filtration. The pro-toxins, pro-A and pro-B, and their 1:1 mixture, eluted with almost overlapped elution volumes, between 14.5 and 14.8 mL (this volume corresponds to globular proteins of ~ 60 kDa) (Fig 2A). The fact that the bands overlap, and that the single elution band observed for the 1:1 mixture contained both pro-toxin subunits in a 1:1 molar ratio (Fig 2A, insert), suggests that there is no oligomerization in any of these samples.


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)

Gel filtration of pro- and act-Bin subunits.(A) Elution profile of pro-A, pro-B, and their mixture (1:1 molar ratio, *); (B) the same as (A) for act-A and act-B. Protein concentration before and after chromatography was typically 60 μM and 18 μM (total monomer), respectively. The SDS PAGE gel for the elution band of the 1:1 mixture is shown in the insert; (C) MALDI-TOF mass spectrum of the elution band shown in B. Four peaks are observed, associated with masses of approximately 40 kDa (mass to charge ratios (m/z) of 40,800 and 20,402 Da) and 45 kDa (m/z of 22,500 and 45,026 Da).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0158356.g002: Gel filtration of pro- and act-Bin subunits.(A) Elution profile of pro-A, pro-B, and their mixture (1:1 molar ratio, *); (B) the same as (A) for act-A and act-B. Protein concentration before and after chromatography was typically 60 μM and 18 μM (total monomer), respectively. The SDS PAGE gel for the elution band of the 1:1 mixture is shown in the insert; (C) MALDI-TOF mass spectrum of the elution band shown in B. Four peaks are observed, associated with masses of approximately 40 kDa (mass to charge ratios (m/z) of 40,800 and 20,402 Da) and 45 kDa (m/z of 22,500 and 45,026 Da).
Mentions: To study the oligomerization behavior of Bin toxin subunits without detergent, their elution profile was monitored using gel filtration. The pro-toxins, pro-A and pro-B, and their 1:1 mixture, eluted with almost overlapped elution volumes, between 14.5 and 14.8 mL (this volume corresponds to globular proteins of ~ 60 kDa) (Fig 2A). The fact that the bands overlap, and that the single elution band observed for the 1:1 mixture contained both pro-toxin subunits in a 1:1 molar ratio (Fig 2A, insert), suggests that there is no oligomerization in any of these samples.

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