Volume 10 Supplement 3
Comparative study of protein-protein interaction observed in PolyGalacturonase-Inhibiting Proteins from Phaseolus vulgaris and Glycine max and PolyGalacturonase from Fusarium moniliforme
© Maulik et al; licensee BioMed Central Ltd. 2009
Published: 3 December 2009
The PolyGalacturonase-Inhibiting Proteins (PGIP) of plant cell wall limit the invasion of phytopathogenic organisms by interacting with the enzyme PolyGalacturonase (PG) they secrete to degrade pectin present in the cell walls. PGIPs from different or same plant differ in their inhibitory activity towards the same PG. PGIP2 from Phaseolus vulgaris (Pv) inhibits the PG from Fusarium moniliforme (Fm) although PGIP1, another member of the multigene family from the same plant sharing 99% sequence similarity, cannot. Interestingly, PGIP3 from Glycine max (Gm) which is a homologue of PGIP2 is capable of inhibiting the same PG although the extent of similarity is lower and is 88%. It therefore appears that subtle changes in the sequence of plant PGIPs give rise to different specificity for inhibiting pathogenic PGs and there exists no direct dependence of function on the extent of sequence similarity.
Structural information for any PGIP-PG complex being absent, we resorted to molecular modelling to gain insight into the mechanism of recognition and discrimination of PGs by PGIPs. We have built homology models of Pv PGIP1 and Gm PGIP3 using the crystal structure of Pv PGIP2 (1OGQ) as template. These PGIPs were then docked individually to Fm PG to elucidate the characteristics of their interactions. The mode of binding for Pv PGIP1 to Fm PG considerably differs from the mode observed for Pv PGIP2-Fm PG complex, regardless of the high sequence similarity the two PGIPs share. Both Pv PGIP2 and Gm PGIP3 despite being relatively less similar, interact with residues of Fm PG that are known from mutational studies to constitute the active site of the enzyme. Pv PGIP1 tends to interact with residues not located at the active site of Fm PG. Looking into the electrostatic potential surface for individual PGIPs, it was evident that a portion of the interacting surface for Pv PGIP1 differs from the corresponding region of Pv PGIP2 or Gm PGIP3.
van der Waals and eletrostatic interactions play an active role in PGIPs for proper recognition and discrimination of PGs. Docking studies reveal that Pv PGIP2 and Gm PGIP3 interact with the residues constituting the active site of Fm PG with implications that the proteins bind/block Fm PG at its active site and thereby inhibit the enzyme.
Plants are under constant threat of infections caused by pathogens that range from viruses, bacteria, fungi to nematodes and insects. The efficacy of plant defense depends on its ability to recognize a pathogen and mount the appropriate defense response. Plants often employ cell surface and intracellular receptors to detect a pathogen associated molecular pattern (PAMP) and trigger immune response against the invader. PolyGalacturonase-Inhibiting Protein (PGIP) is one among the pathogenesis-related (PR) proteins that is found at the cell surface of plant cells. It binds and inhibits the enzyme PolyGalacturonase (PG) from the invading pathogen which could be fungus, insect or bacterium, thus preventing its colonization in the host cell and hence the progress of the disease [1, 2]. PolyGalacturonases (PG) are a class of pectinolytic enzyme secreted by the pathogen at the early stages of infection to depolymerize homogalacturonan (HGA), the main component of pectin in the plant cell wall . HGA is the 1, 4 linked alpha-D-galactosyluronic acid polymer found in the plant cell which forms the first line of barrier and thereby plays a critical role in controlling pathogen invasion [4–7].
The interaction of the plant protein PGIP with the fungal or insect PG limits the destructive potential of the PG and leads to the accumulation of elicitor active oligogalacturonides as shown by in vitro studies . Hence PGIP seems to exert a dual role during fungal attack. It limits pathogen penetration and tissue colonization by inhibiting PG activity, this in turn, favours the accumulation of oligogalacturonides, which activate a prompt defense response .
PGIPs function as very specific inhibitors . PGIP from a particular plant that is able to inhibit the PG from a certain pathogen might not be able to inhibit PGs from other pathogens. Besides, for cases when a PGIP is able to inhibit PGs from different pathogens, the percent inhibition differs. Thus in plants, small gene families encode PGIP isoforms that differ in affinity and specificity for PGs secreted by different pathogens . Recognition specificity results from a few variations in the amino acid sequence of the PGIPs . Plants successfully defend themselves from the attack of a wide range of PGs from pathogenic microorganisms through the role played by protein-protein interaction involving these PGIP proteins.
Significant contributions have been made with experimental studies on the different isoforms of PGIP from a variety of plants. It has been seen that the overexpression of two PGIP genes, Atpgip1 and Atpgip2 genes from Arabidopsis thaliana limits the colonization by B. cinerea and also reduces the disease symptoms . In transgenic tomato and grapevine plants, a significant increase of PG-inhibitory activity and a decrease in susceptibility to B. cinerea have been observed. Plant resistance have been successfully enhanced by the overexpression of pear PGIP in tomato  and Vitis vinifera , and also bean PGIP in tobacco . Accumulation of information on several of the properties exhibited by PGIPs have generated an interest in exploiting them as tools for enhancing plant resistance. In this regard, PGIPs of bean plant (Phaseolus vulgaris) needs special mention.
Bean PGIPs are encoded by a family of genes , the products of which are Pv PGIP1, Pv PGIP2, Pv PGIP3 and Pv PGIP4. All of these show distinct regulation and specificity exhibiting different inhibitory capabilities against the PGs of Botrytis cinerea, Colletotrichum gloeosporioides, Stenocarpella maydis, Fusarium moniliforme and Aspergillus niger . The four mature products of the pgip genes have a very high extent of sequence similarity differing between 8 to 81 amino acids. Phylogenetic analysis indicates that Pvpgip1-Pvpgip2 groups with Gmpgip3 of soybean (Glycine max), a species of the Phaseoleae tribe close to bean, which suggests that these two genes are probably closer to the ancestral gene than are Pvpgip3 and Pvpgip4 . Interestingly, although PGIP1 and PGIP2 from Phaseolus vulgaris share 99% sequence similarity, Pv PGIP2 has been reported to inhibit PG from Fusarium moniliforme (Fm) while Pv PGIP1 does not . Strikingly, Gm PGIP3 of soybean (Glycine max), shares a much lesser similarity of 88% with Pv PGIP2 and yet has the ability to inhibit FmPG with an inhibition kinetics similar to that of Pv PGIP2 . It is thus evident that the ability of PGIP molecules to defend plants against infection is not buried in the extent of similarity shared between the sequences alone. Probable implications could be the presence of key structural features important for the necessary protein-protein interaction that need to be conserved to preserve the function.
It would be interesting to study the molecular interaction of different PGIPs sharing different range of sequence variation, with the same PG molecule to elucidate the relation between the extent of sequence similarity and the corresponding ability/inability to inhibit PG. Three PGIPs, Pv PGIP1 and Pv PGIP2 from Phaseolus vulgaris and Gm PGIP3 from Glycine max have been chosen for this purpose keeping in mind that they share a high level of sequence similarity and yet show distinct difference in their function as expressed through their ability/inability to inhibit the same PG from Fusarium moniliforme (Fm PG). In this study, homology models of two PGIP molecules namely Pv PGIP1 and Gm PGIP3 have been built using the crystal structure of Pv PGIP2 as the template. The crystal structure of Fm PG being available, the three PGIPs have been subsequently docked to Fm PG. In order to elucidate the mechanism underlying and the strategy undertaken by the plants in utilizing the different isoforms of PGIP, a comparative study of the interaction between these three pairs namely Pv PGIP1 and Fm PG, Pv PGIP2 and Fm PG and Gm PGIP3 and Fm PG have been accomplished. A comparison of the binding modes, the interacting residues and the electrostatic potential surface along the interface of the PGIP-PG complex have been investigated. Change in intermolecular distances, hydrogen bond formation and electrostatic surface potential upon in silico mutation of Pv PGIP2 have also been explored.
Results and discussion
Pairwise alignment carried out in order to identify similarities between the PGIP molecules at the sequence level, shows only eight varying residue positions between Pv PGIP1 and Pv PGIP2 (shown by the vertical bars in Figure 2A) and 34 varying positions between Pv PGIP2 and Gm PGIP3 (shown in Figure 2C). Four out of the eight variations between Pv PGIP1 and Pv PGIP2 fall in the repeat regions and the rest reside in the non-LRR (intervening region between two repeats) region of the proteins. Two (R5 and R9) out of the four PS-LRRs and the first (R1) and the sixth (R6) repeat harbour these four variations mentioned above. The 34 variations between Pv PGIP2 and Gm PGIP3 on the other hand, are divided amongst all domains including LRRNT_2 and all the plant specific LRRs (Figure 2C).
Distribution of the variations in the domains
The crystal structure of Pv PGIP2 (1OGQ) being available and also since Pv PGIP1 and Pv PGIP2 as well as Pv PGIP2 and Gm PGIP3 share high percentage of sequence similarity, homology models of Pv PGIP1 and Gm PGIP3 were built using Pv PGIP2 as template. One model each for Pv PGIP1 and Gm PGIP3 were selected using PROCHECK . In each case, the model that was chosen supported the following criteria:
(i) more number of residues in the core region
(ii) no residues in the disallowed region
These models of Pv PGIP1 and Gm PGIP3 were solvated in water and were subjected to energy minimization followed by molecular dynamics simulation of 1 ns. A similar scheme of equilibration was taken up for the crystal structure of Pv PGIP2. RMSD of the equilibrated structures of Pv PGIP1 and Gm PGIP3 were calculated with respect to the equilibrated structure of Pv PGIP2 and was found to be 1.45Å and 1.66Å respectively.
We used GRAMM-X  for docking three pairs of proteins in order to unveil the mode of interaction within and across each pair. This docking program executes a rigid-body search using Fast Fourier Transform (FFT) correlation with simplified geometry employing shape complementarity and hydrophobicity in the scoring function. In this study we have used GRAMM-X as a protein-protein docking program as it extends the GRAMM Fast Fourier Transformation methodology by applying smoothed Lennard-Jones potential, refinement stage and knowledge-based scoring, thus giving rise to best surface match. The output of GRAMM-X is a PDB file containing the structures of ten models ranked as the most probable prediction candidate, according to the scoring function used. The cell-wall degrading enzyme PG, from the fungus Fusarium moniliforme was common in all the pairs and the other three molecules include Pv PGIP1, Pv PGIP2 and Gm PGIP3. The crystal structure of Fm PG (1HG8) used in this study had also been solvated in water and was then subjected to energy minimization followed by molecular dynamics (MD) of 1 ns.
Docking studies of Fm PG-Pv PGIP2
Change in Solvent Accessible Surface Area (SASA) in PGIP and PG upon complexation: The table summarizes the change in solvent accessible surface area of some biologically important residues of Pv PGIP2-Fm PG and Gm PGIP3-Fm PG before and after complexation.
Change in SASA (in nm2) upon complexation of Pv PGIP2 with Fm PG
Residues in Pv PGIP2
SASA in Pv PGIP2
SASA in complex
Residues in Fm PG
SASA in Pv PGIP2
SASA in complex
Change in SASA (in nm 2 ) upon complexation of Gm PGIP3 with Fm PG
Residues in Gm PGIP3
SASA in Gm PGIP3
SASA in complex
Residues in Fm PG
SASA in Gm PGIP3
SASA in complex
In this model structure, the residues of Pv PGIP2 that is known to be functionally important, have shown considerable change in SASA (Table 1), indicating that they play a key role in interaction. Four residues [V(152), S(178), Q(224) and H(271)] out of five that were identified by Leckie et al.  as interacting residues of Pv PGIP2 are expected to reside in the interacting surface of Pv PGIP2 in the Pv PGIP2-Fm PG complex, inferred on the basis of the considerable change in SASA (Table 1).
Docking studies of Fm PG-Pv PGIP1
Out of the ten potential candidates for models of Pv PGIP1-Fm PG complex obtained from GRAMM-X, only in one model Pv PGIP1 docked to the same face of Fm PG as was found in the model for the complex of Pv PGIP2-Fm PG (Figure 4B). In the rest of the nine models, the PGIP molecule docked to a different face of Fm PG indicating a gross inability of these models of Pv PGIP1 to block either the substrate binding site or the active site of Fm PG. In the selected model, whereas the portion marked with the yellow ellipse is evidently seen to be blocked in the other two complexes, the same is not the case with Pv PGIP1. This is in complete agreement with the experimental observation of Pv PGIP1 being incapable of inhibiting the PG from Fusarium moniliforme. This model in which Fm PG docks to Pv PGIP1 at the face similar to the one in Pv PGIP2 (Figure 4B) of the Pv PGIP2-Fm PG complex, is considered for further studies to confirm the inability of the protein in inhibition towards the enzyme.
Docking studies of Fm PG-Gm PGIP3
It has been reported that Gm PGIP3 possesses an inhibiting activity towards the PG of Fusarium moniliforme like Pv PGIP2. No mutational studies being available for Gm PGIP3, the docked structure of the Gm PGIP3-Fm PG complex was therefore selected from the top scoring ten models reported by GRAMM-X using the knowledge obtained from experimental studies carried out for Pv PGIP2. In the selected model of the complex (Figure 4C), Gm PGIP3 has blocked the active site cleft of Fm PG. The docked model therefore is in agreement with the experimental observation of the ability of Gm PGIP3 in inhibiting the enzymatic activity of Fm PG. The important residues V(152), S(178), Q(224) and H(271) of Gm PGIP3 that are also present in Pv PGIP2 show considerable decrease in solvent accessible surface area (Table 1) indicating their active involvement in protein-protein interaction, an identical observation that holds true for Pv PGIP2. Thus like Pv PGIP2, Gm PGIP3 too blocks the active site cleft and hinders the substrate binding site of Fm PG bearing a similar mode of competitive inhibition.
Comparative study of protein-protein interactions in the three complexes
Ionic interaction in Pv PGIP2-Fm PG, Pv PGIP1-Fm PG and Gm PGIP3-FmPG complexes: The ionic interaction as obtained from Protein Interaction Calculator, showing the positions of the residue pairs in Pv PGIP2-Fm PG, Pv PGIP1-Fm PG and Gm PGIP3-Fm PG docked complexes.
Ionic interaction showing the positions of residue pairs in the Pv PGIP2-Fm PG complex for
Ionic interaction showing the positions of residue pairs in the Pv PGIP1-Fm PG complex for
Ionic interaction showing the positions of residue pairs in the Gm PGIP3-Fm PG complex for
Electrostatic surface potential of individual PGIPs and the complexes
In silico mutation of Pv PGIP2 and Pv PGIP2-Fm PG complex
Pv PGIP2 was mutated at six positions, with the amino acid present in Pv PGIP1 at that position, using WHAT IF . Electrostatic surface potential was generated for all the six mutated Pv PGIP2 molecules. The surface drawn with the Q224K mutation showed considerable change from the wild type protein (Additional file 2). The other single mutations did not show much of a significant change in the electrostatic surface potential. It may be mentioned that when these mutated Pv PGIP2 molecules were docked to Fm PG, none of the complexes showed any resemblance to the conformation found for the Pv PGIP1-Fm PG complex, shown in Figure 6A.
When in the Pv PGIP2-Fm PG complex, Q (224) of Pv PGIP2 was mutated to K, using WHAT IF, the resultant complex showed quite a few of the following deviations from the complex with the wild type protein. Hard sphere contact distances  between heavy atoms revealed an existence of steric clash between the Nζ of Q224K of the mutated Pv PGIP2 and Cε of K(300) of Fm PG. Whereas the contact distance between the atoms should be more than 2.52 Å, the distance in the complex with mutation was 2.47 Å. The complex bearing the mutation also showed the absence of the predicted hydrogen bond involving Q(224) of Pv PGIP2 as obtained from PIC results. Even the ionic interaction that prevails in the wild type complex of Pv PGIP2-Fm PG vanishes with this single mutation. The in silico mutation study, confirms the importance of the Q(224) residue which has been experimentally found to give rise to a 70% reduction in the inhibition ability of the protein upon single mutation. The study also suggests probable reasons like a different electrostatic potential surface, steric clash or lack of hydrogen bonds that might explain why Pv PGIP1 with only eight residues has a different mode of binding to Fm PG.
The above in silico study clearly portrays how a single mutation in Pv PGIP2 results in disallowed hard sphere contact distances between the two protein molecules thus indicating the importance of van der Waals interaction in plant defense. Q at the 224th position which is also involved in forming hydrogen bonds, plays a substantial role in this interaction. Since GRAMM-X shows that the mode of binding of Pv PGIP1 with Fm PG is different from Pv PGIP2 and Gm PGIP3 and further more since GRAMM-X has a major involvement of smoothed Lennard-Jones potential in scoring, we infer that the discrimination in recognition has a significant contribution from van der Waals interaction. Role of van der Waals forces have already been shown to be important in offering stability to several protein-ligand and in some cases DNA-ligand complexes prevailing in biological systems. Molecular Dynamics studies have shown van der Waals interaction to play a vital role in groove binding of the repressor fragment of a bacteriophage to its operator oligonucleotide . In a study with proteins, it has been shown that the mouse major urinary protein (MUP) and its ligand show a binding thermodynamics that is dominated by dispersion interactions . The involvement of the van der Waals forces in plant defense although not in specific recognition has also been reported. Many plants respond to pathogenic attack by producing defense proteins that bind to chitin, a polysaccharide present in the cell wall of the invading fungus or exoskeleton of infecting insects. NMR studies show the involvement of van der Waals interactions between the methyl acetamide group of the GlcNAc unit and a hydrophobic patch of the plant protein . Studies on the characteristics of the sugar-binding sites in plant and animal lectin show van der Waals interaction and hydrogen bonds to contribute significantly. Selectivity for different sugars result from hydrogen bonds and unwanted recognition is prevented by steric exclusion . Here in the case of plant defense through PGIP, the influence of van der Waals interaction therefore does not seem to be atypical.
In the present study, we have failed to address pertinent components like the change in the binding free energy change (ΔΔG) between the wild type and the mutated PGIPs. In order to obtain experimentally relevant information concerning the thermodynamic properties of binding, we wish to work in future, with advanced simulation methods such as free energy perturbation or thermodynamic integration to assess the binding free energy changes upon mutation of the important residues in PGIP.
It has been noted from our study on sequence analysis that 50% of the sequence variation between Pv PGIP1 and Pv PGIP2 fall in the non-LRR region whereas only 15% of the variation in Gm PGIP3 with Pv PGIP2 fall in the non-LRR region. This observation suggests specific regions in PGIPs favourable for the introduction of variation in recognition of the pathogen molecules. Structural studies involving docking techniques suggest the mode of binding of the fungal enzyme Fm PG by PGIP2 from Phaseolus vulgaris to be similar to that of its homologue PGIP3 from Glycine max. In each case of binding, the active site of the enzyme from Fusarium moniliforme is being blocked by the PGIP molecule, supporting the experimentally observed phenomenon of these PGIP molecules being able to inhibit the fungal enzyme upon invasion. PGIP1 from the same plant Phaseolus vulgaris which is incapable of inhibiting Fm PG, binds to Fm PG in an evidently different mode, although Pv PGIP1 and Pv PGIP2 share a higher degree of sequence similarity compared to Pv PGIP2 and Gm PGIP3. Electrostatic surface potential reveals considerable difference in the interacting surface of Pv PGIP1 when compared to Pv PGIP2 or Gm PGIP3. Thus, electrostatic and van der Waals interactions may play a significant role in PGIPs for proper recognition and discrimination of PGs.
The sequences of the three PGIPs, namely Pv PGIP1, Pv PGIP2 and Gm PGIP3 were retrieved from NCBI. We used the Pfam domain search program  to determine the domain architecture of these proteins. The plant specific Leucine-rich Repeats (PS_LRR) in the proteins were identified by the program PATMATDB of EMBOSS  using the pattern [ILVF]xx[ILVF]xx[ILVF]x [ILVF]xx[NTSC]x [ILVF] [TS]GxIPxx[ILVF]Gx as a regular expression. Pairwise alignments, one between Pv PGIP1 and Pv PGIP2 and another between Gm PGIP3 and Pv PGIP2 were carried out by the program NEEDLE of EMBOSS. All the nine repeats identified by Pfam were aligned by CLUSTALW  for the three proteins, individually.
Homology models of Pv PGIP1 and Gm PGIP3 were generated using MODELLER 9v4  by satisfying the spatial restraints, based on the homology they share with the protein Pv PGIP2 (PDB Id: 1OGQ). The best model was selected using PROCHECK  based on steric correctness. Energy minimization was performed for all the structures that were involved in our studies, with the help of GROMACS 3.3.3 [38, 39].
The four protein molecules namely, the models of Pv PGIP1 and Gm PGIP3 and the crystal structure of Pv PGIP2 and the fungal enzyme Fm PG (PDB Id: 1HG8) were embedded in a box of simple point charge water molecules with 1 nm separation between the box boundary and the solute. Energy minimization of solvent with the solute fixed was carried out with 500 steps of steepest descent followed by 2000 steps of conjugate gradient algorithm. After that the constraint on the solute was released and energy minimization of the whole system was carried out again with 500 steps of steepest descent followed by 2000 steps of conjugate gradient algorithm. The resulting structures were subjected to 1 ns of molecular dynamics (MD) simulations in GROMACS at 300 K. For all the simulations GROMOS96 43a1 force field was used.
The model structures of Pv PGIP1 and Gm PGIP3 have been deposited to PMDB Protein Model Database http://mi.caspur.it/PMDB/ with the PMDB identifier of PM0075783 and PM0075784 respectively.
We have used GRAMM-X Protein Docking Web Server v.1.2.0  for docking the three PGIP molecules to PG taken one at a time. The output PDB file that GRAMM-X produces contains 10 models ranked as the most probable prediction candidates according to the scoring function used by the program. The PGIPs acted as receptors while the PG acted as ligand in this study of protein-protein interaction. To select a model out of the ten top scoring docked complexes that GRAMM-X reported, we studied the associated change in Solvent Accessible Surface Area (SASA) of the amino acid residues in the individual molecules and when they are in the complex. We used the g_sas program in GROMACS to estimate the change in SASA for the individual residues of the proteins.
Mutation prediction studies
We have used mutation prediction tool  of the WHAT IF  web interface to mutate Q (224) of Pv PGIP2 to K in the Pv PGIP2-Fm PG complex. The same mutation (Q224K) and five others that differ in Pv PGIP1 were carried out on Pv PGIP2 by using the same server.
Determination of ionic interaction and hydrogen bonding patterns in the complexes
The Protein Interaction Calculator (PIC)  was used to detect the ionic interaction between the proteins in the three complexes.
Electrostatic surface potential
Adaptive Poisson-Boltzmann Solver (APBS) is a software package for the numerical solution of the Poisson-Boltzmann equation (PBE), one of the most popular continuum models for describing electrostatic interactions between molecular solutes in salty, aqueous media. The colour coded electrostatic surface potential was drawn using the APBS (Adaptive Poisson-Boltzmann Solver) package  within PYMOL 0.99rc6  for the three PGIP molecules, the PG molecule and the three complexes. Electrostatic surface potential was also generated for a model where a single mutation was carried out on Pv PGIP2 (Q224K) by WHAT IF [30, 40] using the APBS package .
Other papers from the meeting have been published as part of BMC Bioinformatics Volume 10 Supplement 15, 2009: Eighth International Conference on Bioinformatics (InCoB2009): Bioinformatics, available online at http://www.biomedcentral.com/1471-2105/10?issue=S15.
List of abbreviations used
PolyGalacturonase Inhibiting Protein
PolyGalacturonase Inhibiting Protein coding gene
- Pv PGIP Phaseolus vulgaris:
PolyGalacturonase Inhibiting Protein
- Gm PGIP Glycine max:
PolyGalacturonase Inhibiting Protein
- Fm PG Fusarium moniliforme:
pathogen associated molecular pattern
plant specific Leucine-rich Repeats
Solvent Accessible Surface Area
Adaptive Poisson-Boltzmann Solver
Protein Interaction Calculator.
AM is grateful to Department of Biotechnology, Government of India for providing her with fellowship. The authors deeply acknowledge Dr. Ansuman Lahiri and Dr. Anindita Seal for their critical reading of the manuscript and useful discussion.
This article has been published as part of BMC Genomics Volume 10 Supplement 3, 2009: Eighth International Conference on Bioinformatics (InCoB2009): Computational Biology. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2164/10?issue=S3.
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