1. Comparative study of protein-protein interaction observed in PolyGalacturonase-Inhibiting Proteins from P. vulgaris and G. max and PolyGalacturonase from Fusarium moniliforme Soumalee Basu Department of Bioinformatics School of Biotechnology & Biological Sciences West Bengal University of Technology Kolkata, India International Conference on Bioinformatics (InCoB2009), September 7- 11, 2009 Singapore

2. It is thus an interaction of two proteins one from plant PolyGalacturonase-Inhibiting Protein (PGIP) is the protein of plant origin and is believed to have involvement in plant defence. an enzyme from the fungus infecting the plant PolyGalacturonase (PG) is the enzyme from the fungus. another

3. PolyGalacturonase-Inhibiting Protein (PGIP) are believed to be proteins involved in plant defence PGIP – the plant protein Phaseolus vulgaris (bean) Glycine max (soya bean)

4. Fusarium moniliforme is responsible for root rot, stem rot, foot rot, wilting etc Pineapple –fusariose disease Fig- endosepsis PG –the fungal enzyme Fungicides- Chlorothalonil, mancozeb, drenches of thiophanate methyl Fusarium moniliformeInfected corn

5. Cell wall degrading Polygalacturonase(PG)‏ Elicitor-active oligogalacturonoides (OGAs) (OGAs) Inactive fragments C source receptor signal cascade Defense-related genes Defense response Cell Wall Plasma membrane PG H H G G A AH H G G A A G A Polygalacturonase Inhibiting Protein (PGIP)‏ Fungalpathogen Model depicting the role of PGIP-PG in plant defence response Nucleus PectinPectin Interplay of PGIP and PG

6. PvPGIP1 PvPGIP2 GmPGIP3 FmPG does not inhibit 98% inhibit Bean plant Soya bean plant Fusarium moniliforme Recognition Specificity 88%

7. LRRNT_2 R1 R2 R3 R4R5R6R7R8R9 60152178224271 291 297 311 LRRNT_2 R1 R2 R3 R4R5R6R7R8R9 LRRNT_2 R5R6R7R8R9 311 297 290273 279266 232 227219 220192 160 145 136143 116140 10492 9590 84-88 82 72 70 67 59 54 42 29 R4R2 R3 R1 PS PvPGIP1 PvPGIP2 GmPGIP3 PS Domain architecture (DA) of the three PGIP molecules

8. Multiple sequence alignments of the nine repeats of the PGIP molecules

9. Ribbon representation of Crystal structure of PvPGIP2 (1OGQ)

10. PvPGIP2PvPGIP1GmPGIP3 Structure Already Solved Structure not yet solved Structure Already Solved FmPG PvPGIP2 FmPG PvPGIP1 FmPG GmPGIP3 Docking Energy minimization followed by Molecular Dynamics Simulation GROMACS GRAMM-X MODELLER Homology modeled Homology modeled

11. PvPGIP1 GmPGIP3 Homology Models

12. Docked complexes of PGIP and FmPG A. Pv PGIP2 hinders the substrate binding site and blocks the active site cleft of Fm PG C. Gm PGIP3 hinders the substrate binding site and blocks the active site cleft of Fm PG B. The only model of Pv PGIP1- Fm PG complex where PvPGIP1 docks near the active site of Fm PG although not blocking it

13. Electrostatic surface potential PvPGIP1PvPGIP2 GmPGIP3FmPG

14. Electrostatic surface potential of the three complexes PvPGIP1-FmPG PvPGIP2-FmPG GmPGIP3-FmPG

15. Active site residuesChange in SASA due to complex formation PvPGIP1PvPGIP2GmPGIP3 D(167)NoYes D(188)NoYes D(189)NoYes R(243)NoYes K(245)NoYes Change in Solvent Accessible Surface Area in FmPG

16. Interacting residues as found through mutational studies with PvPGIP2 Change in SASA PvPGIP2PvPGIP1 (residues are different) GmPGIP3 V(152)YesNoYes S(178)YesNoYes Q(224)YesNoYes H(271)YesNoYes Change in Solvent Accessible Surface Area in PGIPs

17. Studies on ionic interaction of the complexes reveal the interaction to play important role in the PvPGIP2-FmPG and GmPGIP3-FmPG complexes only Q(224)K mutation that was found to be responsible for 70% reduction in inhibition properties was next studied for using in silico mutation

18. Electrostatic surface potential of PvPGIP2 with a single mutation at 224

19. 2.47Å 5.36Å Wild type docked complex ( Pv PGIP2- Fm PG) Q224K Q224 K300 N C C C Q224K mutant of the docked complex

20. Conclusion Model three-dimensional structure of PvPGIP1 and GmPGIP3 show an rmsd of 1.45Aº and 1.66Aº respectively with the template Docking techniques suggest the mode of binding of the fungal enzyme FmPG by PGIP2 from Phaseolus vulgaris to be similar to that of its homologue PGIP3 from Glycine max. In each case of binding, PGIPs hinder the substrate binding site and block the active site cleft of FmPG PGIP1 from the same plant Phaseolus vulgaris which is incapable of inhibiting FmPG, binds to FmPG in an evidently different mode Electrostatic and van der Waals interactions may play a significant role in PGIPs for proper recognition and discrimination of PGs

21. Acknowledgment Hiren GhoshAditi Maulik

22. Thank you!