title: Towards the rational design of antimicrobial proteins: single point mutations can switch on bactericidal and agglutinating activities on the RNase A superfamily lineage.
abstract: The ribonuclease (RNase) A superfamily lineage includes distant members with antimicrobial properties, suggesting a common ancestral host-defense role. In an effort to identify the minimal requirements for the eosinophil cationic protein (ECP or RNase 3) antimicrobial properties we applied site-directed mutagenesis on its closest family homolog, the eosinophil-derived neurotoxin (EDN or RNase 2). Both eosinophil secretion proteins are involved in human immune defense, and are reported as being among the most rapidly evolving coding sequences in primates. Previous studies in our laboratory defined two regions at the N-terminus involved in the protein antimicrobial action, encompassing residues 8-16 and 34-36. Here, we demonstrate that switching two single residues is enough to provide EDN with ECP antipathogen properties. That is, the EDN double-mutant Q34R/R35W displays enhanced bactericidal activity, particularly towards Gram-negative bacteria, and a significant increase in its affinity towards the bacterial outer membrane lipopolysaccharides. Moreover, we confirmed the direct contribution of residue W35 in lipopolysaccharide binding, membrane interaction and permeabilization processes. Furthermore, additional T13 to I substitution provides EDN with an exposed hydrophobic patch required for protein self-aggregation and triggers bacterial agglutination, thereby increasing the final antimicrobial activity by up to 20-fold. Our results highlight how single selected mutations can reshape the entire protein function. This study provides an example of how structure-guided protein engineering can successfully reproduce an evolution selection process towards the emergence of new physiological roles.
authors.affiliation: Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Pulido
authors.forename: Mohammed
authors.initials: M
authors.lastname: Moussaoui
authors.forename: M Victòria
authors.initials: MV
authors.lastname: Nogués
authors.forename: Marc
authors.initials: M
authors.lastname: Torrent
authors.forename: Ester
authors.initials: E
authors.lastname: Boix
mesh_terms: D000373:Agglutinins / Q000235:genetics / Q000276:immunology; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D047150:Eosinophil Cationic Protein / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
chemical_list: D000373:Agglutinins; D023181:Antimicrobial Cationic Peptides; D011994:Recombinant Proteins; D004722:Endoribonucleases; D047150:Eosinophil Cationic Protein; C029494:ribonuclease U; D012259:Ribonuclease, Pancreatic
keywords: antimicrobial peptides; eosinophil cationic protein; eosinophil-derived neurotoxin; protein evolution; ribonuclease