title: SPR and differential proteolysis/MS provide further insight into the interaction between PGIP2 and EPGs.
abstract: By using surface plasmon resonance (SPR) technology, the kinetics of the interaction of various fungal endopolygalacturonases (EPGs) (13 EPGs) with Phaseolus vulgaris (bean) PGIP2 was carried out to determine whether or not there is any interaction between polygalacturonases-inhibiting protein (PGIP) and EPG. The effect of polygalacturonic acid (PGA) on these interactions was also evaluated. The results show that all EPGs evaluated bind to PGIP2, except for AnPGb and the strength of the interaction depends on the EPG/PGIP2 pairing. Further, the presence of PGA has a moderate to strong effect on the EPG/PGIP2 interaction and the strength of the effect is dependent on the exact EPG/PGIP2 pairing. The differences in affinity in the absence and presence of PGA, suggest a certain level of cooperativity. These results indicate a three-component complex similar to that observed for the heparin-ATIII-thrombin, the FGF-FGFR-heparin, or the hedgehog-interference hedgehog-heparan complexes. This data points to an architecture in which the inhibitor binds at a location distant from the substrate binding site. Furthermore, we applied differential proteolysis mass spectrometry (DPMS) to study the location of the binding site between EPG and PGIP2. DPMS studies indicate that PGIP2 does not bind AnPGII, AnPGa, and AnPGc directly over the active site but instead binds on the face opposite to the active site, creating an allosteric interaction.
authors: Complex Carbohydrate Research Center, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-4712, USA. gerardo@ccrc.uga.edu
authors: Gerardo
authors: G
authors: Gutierrez-Sanchez
authors: Daniel
authors: D
authors: King
authors: Gabre
authors: G
authors: Kemp
authors: Carl
authors: C
authors: Bergmann
mesh_terms: D005658:Fungi / Q000201:enzymology*; D007700:Kinetics; D013058:Mass Spectrometry; D010368:Pectins / Q000378:metabolism; D027805:Phaseolus / Q000382:microbiology*; D010940:Plant Proteins / Q000378:metabolism*; D011096:Polygalacturonase / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D059748:Proteolysis; D020349:Surface Plasmon Resonance
chemical_list: C081479:PGIP protein, plant; D010940:Plant Proteins; D010368:Pectins; D011096:Polygalacturonase; C003181:polygalacturonic acid