Structure of the lectin regulatory domain of the cholesterol-dependent cytolysin lectinolysin reveals the basis for its lewis antigen specificity

Abstract

The cholesterol-dependent cytolysins (CDCs) punch holes in target cell membranes through a highly regulated process. Streptococcus mitis lectinolysin (LLY) exhibits another layer of regulation with a lectin domain that enhances the pore-forming activity of the toxin. We have determined the crystal structures of the lectin domain by itself and in complex with various glycans that reveal the molecular basis for the Lewis antigen specificity of LLY. A small-angle X-ray scattering study of intact LLY reveals the molecule is flat and elongated with the lectin domain oriented so that the Lewis antigen-binding site is exposed. We suggest that the lectin domain enhances the pore-forming activity of LLY by concentrating toxin molecules at fucose-rich sites on membranes, thus promoting the formation of prepore oligomers on the surface of susceptible cells.

Figure 1. Structure of LLY^lec

(See also Figure S1, which shows how LLY^lec is oriented with respect to the rest of the toxin molecule.) The structure is represented in colors, from the N terminus in magenta to the C terminus in red. Glycerol is shown as ball and stick, the calcium ion is shown as a red sphere, and the Ni ion is shown as a magenta sphere. Secondary structural elements and CDR loops are labeled as described in the text. This and all following structural figures were drawn with PyMol (Delano, 2002).

Figure 2. Close-up Stereo Views of the Ligand Binding Pocket of LLY^lec

LLY^lec is complexed to (A) glycerol, (B) α-L-fucose, (C) Le^y antigen, and (D) Le^b antigen. Residues that create the binding pocket are shown as blue and magenta sticks and the ligands as yellow sticks. Water molecules are shown as green spheres, and hydrogen bonds by dashed lines.

Figure 3. Superposition of the Dimers of LLY^lec

The dimers are created by crystallographic symmetry. The dimers of LLY^lec, LLY^lec-fucose, LLY^lec-Le^y, and LLY^lec-Le^b are shown in red, yellow, blue, and green, respectively. The Ca²⁺ ion is shown in blue.

Figure 4. Structural Similarities of LLY^lecto Other Proteins

(A) Superposition of the LLY^lec domain (in blue) on to the structure of the fucolection of AAA (in red). Both structures are complexed with fucose. The Ca²⁺ atom, which is located in the same positionin both structures, is shown in green. The CDR loops that are different in AAA, compared to the LLY^Lec domain, are shown in yellow. (B) Superposition of the fucose complex structures of LLY^lec domain (in blue) and the fucolectin module of SpX-1 (in orange). (C) Superposition of the Le^y antigen complex structures of LLY^lec domain (in blue) and the fucolectin module of SpX-1 (orange).

Figure 5. Protein Recognition of Lewis Antigens

All superpositions are based on the Lewis antigen, and LLY^lec is shown in orange. (A) Griffonia simplifolia legume lectin (PDB code: 1LED) is shown in blue with Lewis b antigens superimposed. (B) Monoclonal antibody hu3S193 (PDB code: 1SK3) heavy and light chains are shown in different shades of blue, with Lewis y antigens superimposed.

Figure 6. Comparison of the Ab Initio Shape Envelope with Computationally Docked and SAXS-Derived LLY Models

Each panel shows orthogonal views of the fits. The blue spheres represent missing C-terminal residues and loops between secondary structural elements, including the linker peptide between the lectin domain and the rest of the toxin molecule, which were missing in the ALO structure that was used in the modeling. See also Figure S2 for the SAXS data. (A) Rigid-body refinement of the position of the LLY^lec domain with BUNCH (Petoukhov et al., 2005) generated an ensemble of models that were in good agreement with the experimental scattering profile. In all these models, the position of the LLY^lec domain corresponded well to the unoccupied density proximal to the N terminus of the LLY^CDC domains in the shape envelope. The BUNCH model closest to the average position is shown. (B) As an alternate method of identifying likely LLY^lec domain positions, a large pool of models with randomized LLY^lec and LLY^CDC orientations, constrained by the intervening linker sequence, were generated and interrogated with the EOM (Bernadó et al., 2007). The individual EOM models in the pool with theoretical scattering profiles that best matched the experimental curve were identified. The EOM model closest to the average position is shown.