comparative study of protein-protein interaction observed in polygalacturonase-inhibiting proteins...

Comparative study of protein-protein interaction observed inPolyGalacturonase-Inhibiting Proteins from

P. vulgaris and G. max and

PolyGalacturonase from Fusarium moniliforme

Soumalee Basu

Department of BioinformaticsSchool of Biotechnology & Biological Sciences

West Bengal University of Technology Kolkata, India

International Conference on Bioinformatics (InCoB2009), September 7- 11, 2009

Singapore

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

PolyGalacturonase-Inhibiting Protein (PGIP) are believed to be proteins involved in plant defence

PGIP – the plant protein

Phaseolus vulgaris (bean)

Glycine max (soya bean)

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 moniliforme

Infected corn

Cell wall degradingCell wall degrading

Polygalacturonase(PGPolygalacturonase(PG))

Elicitor-activeElicitor-active

oligogalacturonoidesoligogalacturonoides

(OGAs) (OGAs)

Inactive

fragmentsC source

receptor

signal

cascade

Defense-related genes

Defense Defense

responseresponse

Cell Wall

Plasma

membrane

PGPG

HH

GG

AAPolygalacturonase Inhibiting ProteinPolygalacturonase Inhibiting Protein

(PGIP)(PGIP)

Fungal Fungal

pathogenpathogen

Model depicting the role

of PGIP-PG in plant

defence response

Nucleus

P

e

c

t

i

n

Interplay of PGIP and PG

PvPGIP1

PvPGIP2

GmPGIP3

FmPG

FmPG

does not

inhibit98%

inhibit

Bean

plant

Soya bean

plant

Fusarium

moniliforme

Recognition Specificity

88%

LRRNT_2 R1 R2 R3 R4 R5 R6 R7 R8 R9

60 152 178 224 271 291

297

311

LRRNT_2 R1 R2 R3 R4 R5 R6 R7 R8 R9

LRRNT_2 R5 R6 R7 R8 R9

311

297

290273

279266

232

227219

220192

160

145

136 143

116 140

10492

9590

84-88

82

72

70

67

59

54

42

29

R4R2 R3R1

PS PS PS PS

PvPGIP1

PvPGIP2

GmPGIP3

PS PS PS PS

PS PS PS PS

Domain architecture (DA) of the three PGIP molecules

Multiple sequence alignments of the

nine repeats of the PGIP

molecules

Ribbon representation of Crystal structure of PvPGIP2 (1OGQ)

PvPGIP2 PvPGIP1 GmPGIP3

Structure Already Solved

Structure not yet solved

Structure not yet solved

Structure Already Solved

FmPG

FmPG PvPGIP2 FmPG PvPGIP1 FmPG GmPGIP3

Docking

Energy minimization followed by Molecular Dynamics Simulation

GROMACS

GRAMM-X

MODELLER

Homology modeled

Homology modeled

PvPGIP1

GmPGIP3

Homology Models

Docked complexes of PGIP and FmPG

A. PvPGIP2 hinders the substrate binding site and blocks the active site cleft of FmPG

C. GmPGIP3 hinders the substrate binding site and blocks the active site cleft of FmPG

B. The only model of PvPGIP1-FmPG complex where PvPGIP1 docks near the active site of FmPG although not blocking it

Electrostatic surface potential

PvPGIP1 PvPGIP2

GmPGIP3 FmPG

Electrostatic surface potential of the

three complexes

PvPGIP1-FmPG PvPGIP2-FmPG

GmPGIP3-FmPG

Active site residues Change in SASA due to complex formation

PvPGIP1 PvPGIP2 GmPGIP3

D(167) No Yes Yes

D(188) No Yes Yes

D(189) No Yes Yes

R(243) No Yes Yes

K(245) No Yes Yes

Change in Solvent Accessible Surface Area in FmPG

Interacting residues as found through mutational studies

with PvPGIP2

Change in SASA

PvPGIP2 PvPGIP1 (residues are

different)

GmPGIP3

V(152) Yes No Yes

S(178) Yes No Yes

Q(224) Yes No Yes

H(271) Yes No Yes

Change in Solvent Accessible Surface Area in PGIPs

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

Electrostatic surface potential of PvPGIP2 with

a single mutation at 224

2.47Å

5.36Å

Wild type docked complex(PvPGIP2-FmPG)

Q224K

Q224

K300

K300

N C

C

C

Q224K mutant of the docked complex

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

Acknowledgment

Hiren GhoshAditi Maulik

Thank you!