NMR investigation of random coil regions: clues to assess the different biological activity of two cerato platanin family members

Plant pathogenic fungi secrete several non-catalytic proteins involved in various aspects of the pathogenesis process. Amongst these, cerato-platanin (CP) was first identified and characterized as a PAMP (pathogen-associated molecular pattern) [1,2]. A sequence homology search revealed a set of fungal Cys-rich secreted proteins that have been grouped in the CP family. They induce synthesis of phytoalexins, overexpression of defense-related genes, H2O2 and NO production, markers of defense activation [3]. The core member of this family, CP (from C. platani) shows a double beta barrel fold [2]. Here we present the results of a CP orthologue with 73% of similarity, cerato-populin (Pop1), produced by C. populicola. Though both CP and Pop1 are host defense inducers, Pop1 shows a slower and weaker defense induction capacity than CP [4]. The aim of the present investigation is to define the basis of the different biological activity of the two proteins at a molecular level. Pop1 15N, 13C and 1H resonances have been assigned [5]. The analysis of Pop1 structure obtained by homology modelling, in comparison with the CP NMR structure, will be presented. A detailed analysis of the NMR-derived protein dynamics (fast and slow regime) and NOE data indicated differences between the two proteins, mainly located in the random coil region. Interestingly, this region was proposed to have an important role in oligosaccharides binding and in necrosis induction of leaves’ cells [6]; therefore, the different pattern of residues’ interactions might be the leading cause of their diverse biological activity. To address this hypothesis we have performed NMR experiments in the course of a titration of both Pop1 and CP with oligosaccharides. The high dependence on chemical pesticides in Europe poses large risks to both the environmental and human health; thus, reducing the use of those chemicals in crop production is one of the major objectives in sustainable agriculture. We expect that our results, besides providing new hints on the molecular mechanisms operating in plants induced resistance, will contribute to reach the major goal of environment protection. Besides that, from a basic science perspective, this work illustrates a time saving approach for the investigation of a protein structure-function relationship when the high-resolution structure of an orthologous protein is already available. [1] Pazzagli et al., Cell Biochem Biophys., 44 (2006), 512. [2] de Oliveira et al., J. Biol. Chem., 286 (2011), 17560. [3] Comparini et al., Appl. Microbiol. Biotechnol., 84 (2009), 309. [4] Lombardi et al., Phys. Plant. 149 (2013), 408. [5] Baroni et al., Biomol NMR Assign., (2013), epub. [6] Frias et al., Mol. Plant Pathology (2013), epub.