Hemolytic lectin CEL-III heptamerizes via a large structural transition from α-helices to a β-barrel during the transmembrane pore formation process

Abstract

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.

Keywords: Carbohydrate; Hemolysin; Lectin; Membrane; Oligomer; Pore-forming Toxin; Toxins; X-ray Crystallography; β-Barrel.

FIGURE 1.

Crystal structure of CEL-III heptamer. Side (A) and bottom (B) views of the CEL-III heptamer are shown in ribbon representation. Each subunit is shown in a different color. Lactulose molecules bound to CEL-III are shown in orange sticks. Ca²⁺ and Mg²⁺ ions are indicated in green and light blue spheres, respectively.

FIGURE 2.

Comparison of the structures of domains 1 and 2. A, superposition of domain 1 in the monomer (blue) (Protein Data Bank ID code 1VCL) and the protomer (red). B, superposition of domain 2 in the monomer (blue) and the protomer (red). Lactulose molecules bound to the protomer are shown as stick models. Ca²⁺ and Mg²⁺ ions are shown as spheres. Residues with a C_α deviation above 1.5 Å are indicated by arrows.

FIGURE 3.

Comparison of the structures of the protomer of the heptameric CEL-III with the monomeric CEL-III. Bottom (left) and side (right) views of a protomer of the heptameric CEL-III (A) are compared with the monomeric CEL-III/methyl-α-d-galactopyranoside complex (B) (Protein Data Bank ID code 2Z49) (22) in the same orientation with respect to their domain 2. Domains 1 and 2 are shown in blue and yellow, respectively. Bundle and stem regions derived from domain 3 are shown in green and red, respectively. Ca²⁺ and Mg²⁺ ions are shown as purple and green spheres, respectively. Lactulose in A and methyl-α-d-galactopyranoside in B are shown as orange sticks.

FIGURE 4.

Lactulose bound to heptameric CEL-III. A, F_o − F_c omit electron density map (blue) for lactulose bound to subdomain 1α of the domain 1. The lactulose was omitted in the calculation of the F_o − F_c omit map (blue). The omit map is contoured at 4σ. B and C, binding of lactulose to subdomains 2α (B) and 2β (C) induced local structural changes, especially of Glu-184 and Tyr-222. CEL-III monomer (Protein Data Bank ID code 1VCL) is shown in gray. A yellow dotted line between lactulose and Glu-184 designates a hydrogen bond formed upon binding of lactulose.

FIGURE 5.

Surface representations of the CEL-III heptamer. Electrostatic potential of the outer (A) and inner (B) surfaces was mapped from negative (red) to positive (blue). Strong negative potential in the undersurface facing the membrane surface indicated by arrows is partly due to acidic residues (Asp and Glu) coordinating Ca²⁺ ions. The inside of the pore shows strong negative and positive potentials in the upper and lower regions, respectively.

FIGURE 6.

Conformational change in domain 3 during the heptamerization process. Domain 3 of monomeric CEL-III (A) is compared with that of heptameric CEL-III (B) in the same orientation. The corresponding regions (terminal, scaffold, wrapping, and stem regions) are indicated in different colors.

FIGURE 7.

Structural changes in the stem and wrapping regions for the pore formation. The stem (A and B) and wrapping (C and D) regions in the monomer (A and C) and protomer (B and D) are shown in schematic representations. Intra- and interprotomer hydrogen bonds forming the secondary structures are shown as yellow and orange dashed lines, respectively.

FIGURE 8.

Interactions between the protomers in heptameric CEL-III. A, Arg-378 in the right molecule and Asp-373′ and Asn-369′ in the left molecule (light blue) form ionic and hydrogen bonds (yellow dashed lines) to stabilize the interactions between the protomers. B, a postulated heptameric assembly model of domain 3, shows seven molecules of domain 3 of monomeric CEL-III arranged in the positions corresponding to those of the CEL-III heptamer. Terminal, scaffold, wrapping, and stem regions are shown in yellow, gray, blue, and red, respectively.

FIGURE 9.

Proposed mechanism of pore formation. A and B, binding of CEL-III monomer to Gal- or GalNAc-containing carbohydrate chains on the cell membrane induces movement of domain 3, thereby exposing the hydrophobic face of the scaffold region. C, this exposure of the hydrophobic face of the scaffold region leads to the formation of the heptameric ring as a pre-pore structure. D, the 14-stranded β-barrel elongates, penetrating the lipid bilayer, which is stabilized by the ring composed of domains 1 and 2 and the bundle region. Terminal, scaffold, wrapping, and stem regions are shown in yellow, gray, blue, and red, respectively.