title: Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity.
abstract: Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.
authors.affiliation: F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, Heverlee, Belgium.
authors.forename: G W
authors.initials: GW
authors.lastname: De Samblanx
authors.forename: I J
authors.initials: IJ
authors.lastname: Goderis
authors.forename: K
authors.initials: K
authors.lastname: Thevissen
authors.forename: R
authors.initials: R
authors.lastname: Raemaekers
authors.forename: F
authors.initials: F
authors.lastname: Fant
authors.forename: F
authors.initials: F
authors.lastname: Borremans
authors.forename: D P
authors.initials: DP
authors.lastname: Acland
authors.forename: R W
authors.initials: RW
authors.lastname: Osborn
authors.forename: S
authors.initials: S
authors.lastname: Patel
authors.forename: W F
authors.initials: WF
authors.lastname: Broekaert
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry*; D001798:Blood Proteins / Q000235:genetics*; D002942:Circular Dichroism; D018744:DNA, Plant / Q000737:chemistry; D023082:Defensins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D000935:Antifungal Agents; D001798:Blood Proteins; D018744:DNA, Plant; D023082:Defensins