Structural Analysis of the Laetiporus sulphureus Hemolytic Pore-forming Lectin in Complex with Sugars

Abstract

LSL is a lectin produced by the parasitic mushroom Laetiporus sulphureus, which exhibits hemolytic and hemagglutinating activities. Here, we report the crystal structure of LSL refined to 2.6-Å resolution determined by the single isomorphous replacement method with the anomalous scatter (SIRAS) signal of a platinum derivative. The structure reveals that LSL is hexameric, which was also shown by analytical ultracentrifugation. The monomeric protein (35 kDa) consists of two distinct modules: an N-terminal lectin module and a pore-forming module. The lectin module has a ␤-trefoil scaffold that bears structural similarities to those present in toxins known to interact with galactose-related carbohydrates such as the hemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum, abrin, and ricin. On the other hand, the C-terminal pore-forming module (composed of domains 2 and 3) exhibits three-dimensional structural resemblances with domains 3 and 4 of the ␤-pore-forming toxin aerolysin from the Gram-negative bacterium Aeromonas hydrophila, and domains 2 and 3 from the ⑀-toxin from Clostridium perfringens. This finding reveals the existence of common structural elements within the aerolysin-like family of toxins that could be directly involved in membrane-pore formation. The crystal structures of the complexes of LSL with lactose and N-acetyllactosamine reveal two dissacharide-binding sites per subunit and permits the identification of critical residues involved in sugar binding.

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…These proteins are analogous to bacterial and plant AB toxins including the previously mentioned plant toxin ricin. The best characterized examples of this lectin family are MOA and LSL from the saprophytic mushrooms Marasmius oreades (Cordara et al 2011;Grahn et al 2007;Grahn et al 2009;Wohlschlager et al 2011) and Laetiporus sulphureus (Mancheno et al 2005;Mancheno et al 2010;Tateno and Goldstein 2003). These proteins consist of a single N-terminal β-trefoil-type lectin domain fused to a cysteine protease (MOA) and to an aerolysin-type pore-forming domain (LSL).…”

“…These proteins consist of a single N-terminal β-trefoil-type lectin domain fused to a cysteine protease (MOA) and to an aerolysin-type pore-forming domain (LSL). The numbers and specificities of the carbohydrate-binding sites of the β-trefoil domains differ in that all three canonical binding sites of MOA are functional and bind to glycans carrying terminal Gal-α1,3-Gal/ GalNAc-β epitopes (Grahn et al 2007;Grahn et al 2009;Wohlschlager et al 2011) while only two of the three canonical binding sites of LSL appear to be functional and specific for β-galactosides including lactose and LacNAc (Angulo et al 2011;Mancheno et al 2005). Homologs of both proteins from other dikaryotic fungi have been partially characterized (Chumkhunthod et al 2006;Kadirvelraj et al 2011;Plaza et al 2014;Wohlschlager et al 2011).…”

“…LSL was shown to associate into hexamers in solution and crystals (Mancheno et al 2005). By analogy to other aerolysin-like pore-forming toxins, it has been hypothesized that LSL undergoes a conformational change in its aerolysin domain on insertion into the target membrane, thus forming a pore (Mancheno et al 2010).…”

Structural classification of insecticidal proteins – Towards an in silico characterisation of novel toxins

“…In contrast, in the Ext/Mtx2 family, the head lacks the beta trefoil and is composed of residues from the N-terminal region and from a further stretch of amino acids much closer to the C-terminus (before the C-terminal sequence completes the last long beta strand of the tail domain). In these features, Toxin_10 proteins resemble toxins such as the haemolytic lectin from the parasitic mushroom Laetiporus sulphureus (Mancheno et al, 2005), while the Etx/Mtx2 family resembles mammalian toxins such as aerolysin (Figure 3) and epsilon toxin from Clostridium perfringens.…”

“…shown along with the structures of the haemolytic toxin from L. sulphureus (Lsulph: PDB 1W3A (Mancheno et al, 2005) and proaerolysin (PDB 1PRE (Parker et al, 1994)). Head regions are coloured cyan, tails blue and the extra domain in proaerolysin in green.…”