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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Polygalacturonase-Inhibiting Protein Interacts with Pectin through a Binding Site Formed by Four Clustered Residues of Arginine and Lysine¹

Dipartimento di Biologia Vegetale, Università di Roma "La Sapienza," 00185 Rome, Italy (S.S., O.Z., A.D.M., F.C., G.D.L., B.M., D.B.); and Danisco Innovation, DK–1001 Copenhagen, Denmark (J.D.M.)

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.

² These authors contributed equally to the paper.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Benedetta Mattei (benedetta.mattei{at}uniroma1.it).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.076950.

^* Corresponding author; e-mail benedetta.mattei{at}uniroma1.it; fax 390649912446.

Received January 14, 2006; returned for revision April 13, 2006; accepted April 19, 2006.

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