Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering  

Abstract

We report here the low-resolution structure of the complex formed by the endo-polygalacturonase from Fusarium phyllophilum and one of the polygalacturonase-inhibiting protein from Phaseolus vulgaris after chemical cross-linking as determined by small-angle x-ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor, accounting for the competitive mechanism of inhibition observed. The structure is in agreement with previous site-directed mutagenesis data and has been further validated with structure-guided mutations and subsequent assay of the inhibitory activity. The structure of the complex may help the design of inhibitors with improved or new recognition capabilities to be used for crop protection.

Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns

“…S1). Previous work demonstrated that FpPG and PvPGIP2 can be specifically cross-linked by formaldehyde in vitro as a heterodimeric complex (33,35). In vitro cross-linking of the purified PGIP-PG chimera resulted in complexes ranging from homodimers (∼160 kDa) to homotetramers (∼320 kDa), supporting the conclusion that the fusion protein is able to establish intermolecular interactions (Fig.…”

“…To circumvent this problem, a chimeric protein in which PvPGIP2 and FpPG were linked by three alanine residues was engineered. Although this linker is too short to permit intramolecular enzyme-inhibitor interactions (33), it should allow intermolecular interactions between the PGIP and PG moieties (Fig. 1A).…”

“…To realize this goal, we constructed a chimeric protein by fusing PG from the fungal pathogen Fusarium phyllophilum (FpPG) to PvPGIP2, a PGIP from common bean (Phaseolus vulgaris). PvPGIP2 is the only known effective inhibitor of FpPG (33). The rationale for constructing the chimeric PGIP-PG was that the stoichiometric and simultaneous accumulation of the two proteins in the same plant cannot be easily obtained, either by crossing transgenic plants separately expressing either PG or PGIP, or by using a construct that independently expresses PG and PGIP as individual genes/proteins, because their rate of transcription, translation, posttranslational modification, and turnover would likely be different.…”

SPR and differential proteolysis/MS provide further insight into the interaction between PGIP2 and EPGs

“…This noncompetitive inhibition was also observed for PGIP2 and AnPGII (Spinelli et al 2009). In contrast, for PGIP2/FmPG combination, a competitive inhibition was observed and binding of PGIP2 takes place at the active site of the FmPG (Federici et al 2001;Maulik et al 2009;Benedetti et al 2011). While this makes a general consensus more difficult, it emphasizes the complex nature of these systems and the need for independent evaluation of each enzymeeinhibitor pair.…”

“…A number of specific amino acids of PGIP have been proposed to be involved in the binding to EPGs (Stotz et al 1999;Leckie et al 1999;Di Matteo et al 2003). Conflicting models of the architecture of the EPG/PGIP complex have been proposed, and there has been a significant amount of confusion surrounding what type of inhibitor PGIP is, a competitive, a noncompetitive or a mixed inhibitor (Abu-Goukh & Labavitch 1983;Lafitte et al 1984;Barmore & Nguyen 1985;Johnston et al 1993;Stotz et al 1999;Federici et al 2001;James & Drubery 2001;King et al 2002;Barnes 2004;Sicilia et al 2005;Spinelli et al 2009;Maulik et al 2009;Benedetti et al 2011). Previous studies in our laboratory have shown PGIP2 to be resistant to hydrolysis by pepsin under conditions that readily digest EPGs (King et al 2002).…”