The Structure of the Elicitor Cerato-platanin (CP), the First Member of the CP Fungal Protein Family, Reveals a Double ψβ-Barrel Fold and Carbohydrate Binding

Abstract

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ␤-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the ␤-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ␤-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.

Cerato-platanin shows expansin-like activity on cellulosic materials

“…As expected on the basis of previously obtained results with CP on carboxymethylcellulose (de Oliveira et al 2011), during the action of CP and Pop1 on filter paper, no detectable amount of reducing sugars was produced (data not shown). It has recently been shown that glycosyl hydrolases GH61 can internally cleave cellulose molecules in a direct oxidative way that does not lead to the generation of new reducing ends (Westereng et al 2011).…”

“…CP, like the orthologous protein Pop1, weakened filter paper in a concentration-dependent manner. No detectable amount of reducing sugars was released by CP and Pop1 during the action on filter paper, thus confirming a lack of β-1,4-glucanase activity that had already been reported for CP on carboxymethylcellulose (de Oliveira et al 2011). At the same time, we also excluded the possibility that the weakening activity was the result of an oxidative mechanism like that recently found for the glycosyl hydrolases GH61 that does not lead to the generation of new reducing ends (Westereng et al 2011;Žifčáková and Baldrian 2012).…”

“…Most notably Asp-82, an amino acid considered essential for this activity and also well conserved among expansins. Cerato-platanins have the residue Asp-77 well conserved among members of the family (de Oliveira et al 2011), and given the results presented in this paper, its role will be the object of further investigation.…”

“…CP is a component of the cell wall of C. platani, but is also released during growth (Bernardi et al 2011;Boddi et al 2004;Scala et al 2004). The three-dimensional structure of this protein has been defined: CP has a double ψβ-barrel fold similar to that occurring in endoglucanases, in the plant defence protein barwin and in domain I of expansins (de Oliveira et al 2011). However, CP does not show hydrolytic activity and is known to the scientific community for its eliciting activity on plants (Fontana et al 2008;de Oliveira et al 2011;Pazzagli et al 1999;Scala et al 2004).…”

Self-assembly at Air/Water Interfaces and Carbohydrate Binding Properties of the Small Secreted Protein EPL1 from the fungus Trichoderma atroviride

“…However, hydropathy plots of cerato-platanin proteins show that they have fewer hydrophobic regions than hydrophobins, suggesting that these proteins have at least slightly different properties from "traditional" hydrophobins (14). Recently, an NMR-based structural study of CP showed that this protein has structural features that are similar to Barwin-like endoglucanases and plant expansins, which are involved in plant cell wall extension (19). Furthermore, based on structural NMR data, an N-acetylglucosamine binding pocket was found in CP; this is interesting in view of the fact that chitin, which is composed of N-acetylglucosamine monomers, is a structural component of the fungal cell wall.…”