Fig. 4: The NTD trefoil domain of Cry78Aa may bind galactose.

a Views of the NTD of Cry78Aa from different perspectives. The side view (left diagram) of the NTD shows that it consists of two layers, the cap layer (in light blue) and the barrel layer (in blue), which have nearly identical lengths. The top view (middle diagram) is shown from the cap side. It can be seen clearly that the trefoil domain comprise three blades, which are indicated beside. The bottom view (right diagram) was observed from the barrel side. b Structural alignment of the trefoil domain of Cry78Aa and the galactose-bound ricin B lectin domain (PDB: 3VT1). The color representation of Cry78Aa was the same as in a. The ricin B protein moiety was shown in cyan, while the carbon and oxygen atoms of the galactose molecule are shown in green and red, respectively. The smaller frame in the left diagram marks the galactose binding pocket of ricin B, while the larger frame in the right diagram represents the close-up view. The conserved resides of Cry78Aa (on blade 2) and ricin B that may interact with galactose are marked and shown as sticks (color representations: carbon, light blue for Cry78Aa and cyan for ricin B; nitrogen, blue; oxygen, red). c Structural alignment of three blades of the trefoil domain and ricin B. d Sequence alignment of three blades of the Cry78Aa trefoil domain. Secondary structural units of blade 2 are shown on the top. The numbers flanking the sequence specify the start and end of each blade. Totally conserved residues in the three blades are marked by a red background. Blue solid circles below represent the residues of Cry78Aa that were predicted to be involved in galactose binding. e Effects of the mutants on the insecticidal activity of Cry78Aa proteins against L. striatellus nymphs. Experiments were performed as described in the methods. Each column represents the average value of two independent trials, and error bars represent the standard deviation. The results of mutants on each blade have the same color representation as that in c for clarity. Two irrelevant mutants of Cry78Aa, D47A, and N115A (see also Supplementary Fig. 5c), were tested as negative controls. f Effects of sugars on the insecticidal activity of Cry78Aa against L. striatellus nymphs. Assays were performed as described in the methods, and the results are presented as in e. The results of the assay after treatment with galactose, GalNAc, and lactose are shown in grass green. Bars represent the mean ± SD (n = 3 independent experiments) in e, f. Statistical significance was tested using One-way ANOVA, and is indicated in figures by letters (a, b, c, …, P < 0.05). The difference is not significant when it contains the same letters, and it is significant only when the letters are completely different.