The Bacterial Exotoxin B is a family of translocating pore-forming proteins that function as the binding (B) component in the AB Toxin system. The proteins bind to the cell surface and perforate the cells, allowing the transport of the enzymatically active (A) component into the cell to confer its toxicity. While most family members are toxic to humans and livestock, one class of proteins called the Vpb proteins has been discovered to be active against invertebrates including beetle and aphid species. The Vpb class is further divided into two subclasses: Vpb1 and Vpb4. The Vpb1 subclass adheres to the canonical AB Toxin system, where the presence of an A component is integral to confer toxicity. In contrast, the Vpb4 subclass is unique among the related protein groups and can function independently without an additional component. While the two subclasses shared a high degree of sequence and structural identity, the knowledge of their precise molecular mechanisms remains limited.
Here, a structural characterisation using cryo-electron microscopy revealed contrasting oligomeric states of the two subclasses at neutral basic pH, where Vpb1 forms an incomplete oligomeric pre-pore and Vpb4 forms a fully inserted pore. For Vpb1, functional characterisations using a liposome-based approach showed the ability to form pores at low pH, while SPR showed the ability to bind to its secondary component as binary toxins. For Vpb4, functional characterisations using electrophysiology displayed the ability to transport ions bidirectionally like pore-forming proteins with a direct killing mechanism. Collectively, these results demonstrate key differences alluding to diverging molecular mechanisms of the Vpb proteins. Given the increasing global demand for alternatives to currently available insecticides against beetle and aphid pests, understanding the molecular mechanisms of these bioinsecticide proteins may serve as a key to future applications in the agricultural industry.