Structure and glycolipid binding properties of the nematicidal protein Cry5B

Abstract

Crystal (Cry) proteins are globally used in agriculture as proteinaceous insecticides. They have also been recently recognized to have great potential as anthelmintic agents in targeting parasitic roundworms (e.g., hookworms). The most extensively characterized of the anthelmintic Cry proteins is Cry5B. We report here the 2.3 Å resolution structure of the proteolytically activated form of Cry5B. This structure, which is the first for a nematicidal Cry protein, shows the familiar three-domain arrangement seen in insecticidal Cry proteins. However, domain II is unusual in that it more closely resembles a banana lectin than it does other Cry proteins. This result is consistent with the fact that the receptor for Cry5B consists of a set of invertebrate-specific glycans (attached to lipids) and also suggests that domain II is important for receptor binding. We found that not only galactose but also N-acetylgalactosamine (GalNAc) is an efficient competitor for binding between Cry5B and glycolipids. GalNAc is one of the core arthroseries tetrasaccharides of the Cry5B receptor and galactose an antennary sugar that emanates from this core. These and prior data suggest that the minimal binding determinant for Cry5B consists of a core GalNAc and two antennary galactoses. Lastly, the protoxin form of Cry5B was found to bind nematode glycolipids with a specificity equal to that of activated Cry5B, but with lower affinity. This suggests that the initial binding of Cry5B protoxin to glycolipids can be stabilized at the nematode cell surface by proteolysis. These results lay the groundwork for the design of effective Cry5B-based anthelmintics.

Figure 1. Structure of Cry5B

Cry5B is shown in a ribbon diagram. Domain I (red) is a five α-helix bundle, domain II (green) a β-prism domain, and domain III (blue) a β-sandwich domain.

Figure 2. Domains of Cry5B

a. Left, domain I in ribbon representation. Right, superposition of Cry5B domain I (red) with domain I from Cry1 Aa (green), Cry2Aa (blue), Cry3Aa (yellow), Cry3Bb (orange), Cry4Aa (grey), Cry4Ba (magenta), and Cry8Ea1 (cyan), all in chain representation. b. Left, domain II in ribbon representation, and right, superposition with domain II from other Cry proteins, as in panel a. c. Left, Banana lectin in ribbon representation with bound glycans (yellow) boxed. Right, superposition of banana lectin (yellow), including glycans (laminaribiose), with Cry5B domain 2 (green), in chain representation. d. Left, domain III in ribbon representation, and right, superposition with domain III from other Cry proteins, as in panel a.

Figure 3. Sequence alignment

Structure-based sequence alignment of Cry5B and other structurally characterized Cry proteins was carried out with Expresso (Di Tommaso et al., 2011), and annotated with the secondary structure of Cry5B, indicated at top, with ESPript (Gouet et al., 2003). Horizontal red boxes indicate the GXXXE and GG sequences in domain II.

Figure 3. Sequence alignment

Structure-based sequence alignment of Cry5B and other structurally characterized Cry proteins was carried out with Expresso (Di Tommaso et al., 2011), and annotated with the secondary structure of Cry5B, indicated at top, with ESPript (Gouet et al., 2003). Horizontal red boxes indicate the GXXXE and GG sequences in domain II.

Figure 4. Binding of activated Cry5B and Cry5B protoxin to upper phase glycolipids

Upper phase glycolipids of C. elegans (C.e.) and P. pacificus (P.p.) were resolved by thin layer chromatography and stained with orcinol (a and g), and overlaid with elastase-activated Cry5B (b–f) or Cry5B protoxin (h–l) in the presence of 100 mM galactose (Gal, c, i), 100 mM glucose (Glc, d, j), 100 mM N-acetylglucosamine (GlcNAc, e, k), or 100 mM N-acetylgalactosamine (GalNAc, f, 1). The glycolipid band species are indicated to the left of panels b and h. This is a representative set from several experiments carried out.

Figure 5. Glycan-binding motifs

a. The β1β2 loop of BanLec containing the GG motif (red) and the β11β12 loop containing the GXXD motif (blue) in ribbon representation. The structure of bound laminaribiose is shown in bonds representation with carbons in cyan and oxygens in red. b. The β1β2 loop of Cry5B domain II containing the GXXE motif (blue), and the β11β12 loop containing the GG motif (red) in ribbon representation.