title: Polygalacturonase-inhibiting protein interacts with pectin through a binding site formed by four clustered residues of arginine and lysine.
abstract: Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein that inhibits fungal polygalacturonases (PGs) and retards the invasion of plant tissues by phytopathogenic fungi. Here, we report the interaction of two PGIP isoforms from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) with both polygalacturonic acid and cell wall fractions containing uronic acids. We identify in the three-dimensional structure of PvPGIP2 a motif of four clustered arginine and lysine residues (R183, R206, K230, and R252) responsible for this binding. The four residues were mutated and the protein variants were expressed in Pichia pastoris. The ability of both wild-type and mutated proteins to bind pectins was investigated by affinity chromatography. Single mutations impaired the binding and double mutations abolished the interaction, thus indicating that the four clustered residues form the pectin-binding site. Remarkably, the binding of PGIP to pectin is displaced in vitro by PGs, suggesting that PGIP interacts with pectin and PGs through overlapping although not identical regions. The specific interaction of PGIP with polygalacturonic acid may be strategic to protect pectins from the degrading activity of fungal PGs.
authors.affiliation: Dipartimento di Biologia Vegetale, Università di Roma La Sapienza, 00185 Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Spadoni
authors.forename: Olga
authors.initials: O
authors.lastname: Zabotina
authors.forename: Adele
authors.initials: A
authors.lastname: Di Matteo
authors.forename: Jørn Dalgaard
authors.initials: JD
authors.lastname: Mikkelsen
authors.forename: Felice
authors.initials: F
authors.lastname: Cervone
authors.forename: Giulia
authors.initials: G
authors.lastname: De Lorenzo
authors.forename: Benedetta
authors.initials: B
authors.lastname: Mattei
authors.forename: Daniela
authors.initials: D
authors.lastname: Bellincampi
mesh_terms: D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004951:Esterification; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D027805:Phaseolus / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011096:Polygalacturonase / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D010940:Plant Proteins; D001120:Arginine; D011096:Polygalacturonase; D008239:Lysine