## 3 Structure and Function of the _B. thuringiensis_ Binary Toxins
### Vpb4  
Great strides have been made to discern the structure and function of Vpb4. X-ray crystallography was used to resolve the structure of Vpb4 Da2 at atomic resolution (3.22 A), which elucidated six structural domains (Figure 3) [4]. Domain 1 (residues 1-263) encompasses a PA14 domain, contains a ten-stranded \(\beta\)-sandwich conformation similar in structure to bacterial carbohydrate-binding modules, six small \(\alpha\)-helices, and features two proximal Ca\({}^{2+}\)-binding sites that are highly conserved among binary toxins [4; 34]. The region directly encompassing this dual Ca\({}^{2+}\) ion site is referred to as the \"Ca-edge\" in anthrax toxin PA and binary toxin B subunits and is situated at the N-terminus of active Vpb4 Da2 (Figure 2) [14; 15; 34]. Domain 1 also contains the trypsin cleavage site necessary for Vpb4 Da2 oligomerization, as revealed by fluorescence-based SDS-PAGE analyses of Vpb4 Da2 and Vpb4 Da2 variants [4]. Domain 2 (residues 264-483) contains a \(\beta\)-hairpin motif, seven anti-parallel \(\beta\)-strands, and an additional three \(\alpha\)-helices [4]. A long stem region/pore-forming loop (residues 280-354) is present in Domain 2, which is thought to undergo a conformational change in an acidic pH to form a \(\beta\)-barrel pore. Domain 3 (residues 484-593) contains a seven-stranded \(\beta\)-sandwich and four \(\alpha\)-helices [4]. Domain 4 (residues 594-730) contains a \(\beta\)-jelly roll fold and a Ca\({}^{2+}\)-binding site [4]. Interestingly, Domain 4 resembles the receptor-binding domain 1 (RBD1) from CDTb, which also contains a Ca\({}^{2+}\)-binding site and whose \(\beta\)-sheet-rich structure is intrinsically dependent on the presence of Ca\({}^{2+}\) ions [11; 12]. Domain 5 (residues 731-820) has six \(\beta\)-strands oriented inan immunoglobulin-like fold and one \(\alpha\)-helix, whereas Domain 6 (residues 821-937) has seven \(\beta\)-strands oriented in an immunoglobulin-like fold and two \(\alpha\)-helices [4]. Using the knowledge that domain 4 from the PA of anthrax toxin, the receptor binding domains (residues 616-876) in CDTb, and Domain 4 (residues 616-875) in Ib are involved in receptor binding, domains 4-6 of Vpb4 Da2 were assessed for receptor recognition [4; 11; 14]. To verify this, the authors conducted a carboxyl terminal domain swap analysis using Vpb4C.6693, a nonlethal Vpb4 with 77% protein sequence homology to Vpb4 Da2, generating two Vpb4 chimeras, Vpb4 Da2-Vpb4C.6693 (chimera-1) and Vpb4C.6693-Vpb4 Da2  
Figure 3: Crystal structure of _Bacillus thuringiensis_ Vpb4 Da2. This structure identifies some of the key regions of Vpb4 Da2 in its “prepore” state that are required for its host cell receptor binding and pore-forming activities. (A) Crystal structure of pro-Vpb4 Da2 colored by its domains (D1–D6). The predicted pore-forming loop in the prepore conformation is highlighted in red. The putative receptor-binding domains and the Ca-edge are also indicated. Ca2+ ions are represented by green spheres. A zoomed-in view of D4 bound to a Ca2+ ion is show in the inset. This structure was modified from PDB: 7 MJR [4]. This figure was created with the program BioRender. (chimera-2) [4]. Chimera-1 was found to be non-larvicidal against WCR. Alternatively, Chimera-2, containing domains 1-3 of Vpb4C.6693 and domains 4-6 of Vpb4 Da2, displayed WCR larval susceptibility on par with wild-type Vpb4 Da2 [4]. These results show the significance of domains 4-6 in Vpb4 Da2 toxicity and given the structural homology to binary toxin receptor-binding domains; these observations suggest Vpb4 Da2 receptor binding is conferred by unknown solvent-exposed residues within these domains [4]. Ultimately, these findings present a major development in understanding the function of Vpb4 and other _Bt_-expressed insecticidal binary toxins. To continue this progress, the structure of heptameric Vpb4 Da2 and the receptor it binds to must be examined further.