Extended Data Figure 3. Trefoil domains of BinA and BinB.

a, Structural relationships among trefoil domains illustrated by a phylogenetic tree plot. Four of the structures used for comparison were identified from a structural similarity search through the Protein Data Bank conducted by the Dali server (using BinA residues 6-156 as the probe). The top 4 hits occupy the top half of the plot (3AH1, 3VT2, 2E4M, and 4JP0). The remaining structures chosen for comparison (1W3G and 3ZXG) were selected based on their membership in the aerolysin family of toxins, of which BinA and BinB are members. That is, these are trefoils covalently linked to aerolysin type pore-forming domains. These are highlighted in blue text and include another insecticidal protein from B. thuringiensis, Cry35Ab1 (4JP0). Note that BinA and BinB are nearly as distant from each other as they are from the closest homologs, hemagglutinin, ricin, and Cry35Ab1. Carbohydrate molecules are shown in sticks where coordinates are available. Notable loop insertions in BinA and BinB are coloured in orange and magenta, respectively. b, Carbohydrate binding modules of BinA and BinB display different levels of structural integrity. No carbohydrates were included or observed in the crystals structure of BinAB. To investigate the structural integrity of the putative carbohydrate binding pockets of BinAB we superimposed coordinates of lectin (1W3G) and hemagglutinin (3AH1). These crystal structures illustrated in the left column are carbohydrate complexes chosen for their structural similarity to BinAB. Some modules appear competent for carbohydrate binding, such as the β and γ modules of BinA and the β module of BinB. Others show steric clash (yellow starburst) such as the α module of BinA and β module of BinB, which could be overcome by allowing adjustments in torsion angles. Notably, the α module of BinB is completely occluded by the insertion in its sequence (magenta) and stapled shut by a disulphide bond. In addition to the canonical α, β, and γ binding modules, 3AH1 displays another weakly bound carbohydrate marked site IIIA (bottom panel). This site is illustrated here because its superimposed coordinates lie adjacent to Y150 in BinB. The Y150A mutation causes complete loss of receptor binding ⁵⁴.