Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Ac{alpha}2-6Gal{beta}1-4GlcNAc human-type influenza receptor

Abstract

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7 Å) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding.

Fig. 1.

(Left panel) Quaternary structure of PSL. The two protomers are shown in cartoon mode and the bound ligand (6′-SLN) is shown in ball-and-stick mode. (Right panel) The ricin-type β-trefoil lectins structurally related to PSL and their respective carbohydrate-binding sites. The figure shows that the N-terminal domain of PSL bound to Neu5Acα2-6Galβ1-4GlcNAc and its secondary structural overlay against SRC bound to Neu5Acα2-6Galβ1-4Glc (6′-SL, PDBID: 2DS0) and the two trefoil domains of SNA-II bound to Galβ1-4GlcNAc (PDBID: 3CA4).

Fig. 2.

Stereo view of the PSL β subdomain carbohydrate-binding site with bound ligand shown in ball-and-stick mode. The F_o − F_c difference density map is contoured at the 2.5 σ level. It was calculated after structure solution using MR and an initial maximum-likelihood-based refinement of the protein model but prior to the fitting of the ligands into the map.

Fig. 3.

Secondary structure-based sequence alignment and manual motif identification of the β-trefoil fold (residues 1–153) of PSL1a and MOA with bound Galα(1,3)[Fucα(1,2)]Gal (PDBID 3EF2) using Sequoia (Bruns et al. 1999) and Coot (Emsley and Cowtan 2004) . RMSD: 2.34 Å. Light grey: rPSL residues within 4 Å of the central Gal of the ligand from the MOA structure. Dark Grey: residues within 4 Å of the central Gal of the ligand in MOA. Bold: conserved tryptophans from the ricin B-type (Q/N-x-W) motifs. In capital letters: residues whose atomic coordinates are structurally equivalent in the alignment.

Fig. 4.

(Top panel) Stereo view of the hydrogen-bonding network in the 6'-SLN–PSL complex. (Bottom panel) Stereo view of the hydrogen-bonding network in the Neu5Acα2-6Galβ1-4Glc (6'-SL)–SRC complex. For a detailed interaction analysis of the hydrogen-bonding network in the 6'-SLN–PSL complex refer to Table I and Supplementary Table S1.

Fig. 5.

Superimposition of the ligand from PSL with that from influenza A H1 HA (PDBID:1RVZ) and SRC (Gamblin et al. 2004). Although some disorder in the Neu5Ac residues is present, all the interglycosidic torsion angles populate the same canonical rotamers (Table II).

Fig. 6.

(A–C) Models of the Neu5Acα2-3Galβ-1-4GlcNAc trisaccharide bound to PSL, indicating that no rotamers of the 2-3 linkage can be adopted that avoid collisions with the PSL surface. (D) Orientation of a GalNAc-containing trisaccharide in the binding site of PSL, indicating a collision with His76 (magenta).