Analysis of Polygalacturonase sequence
The protein was analyzed by means of ProtParam server. A polygalacturonase protein has 58.47 kDa as its theoretical pI value and 529 amino acids as its molecular weight. The residue composition of this enzyme was found as Ala (6%), Arg (3.4%), Asn (8.7%), Asp (7.6%), Cys (1.9%), Gln (3.8%), Glu (3.6%), Gly (9.1%), His (2.5%), 33 (6.2%), Leu (6.2%), Lys (3.2%), Met (2.3%), Phe (3.4%), Pro (4.5%), Ser (6.6%), Thr (6.4%), Trp (2.6%), Tyr (4.2%), and Val (7.8%). The total count of negatively charged residues (comprising Aspartic Acid and Glutamic Acid) and positively charged residues (consisting of Arginine and Lysine) was determined 59 and 35, correspondingly. A total number of atoms present in the target protein were 8038 with the chemical formula C2585H3920N706O805S22. Indicating the light absorption capacity, the extinction coefficient is computed at 280 nm and expressed in M-1 cm-1 [J. Yang et.al.2013]. Estimated half-life when M (Met) is considered as the N-terminal of the sequence. The projected half-life of the polygalacturonase protein was predicted 30 hours for mammalian reticulocytes in vitro model. Instability index of the polygalacturonase protein was found 37.97 that indicate a stable form of protein. Aliphatic index (AI) of a protein has a direct relation with the volume engaged by the surface chains of amino acids (alanine, leucine, valine, and isoleucine).
Thermal stability of the globular proteins increases with an increase in AI value [A. Roy et.al.2010]. Aimed at protein, the aliphatic- index was computed to be (73.18). The GRAVY index for this protein was -0.331, which indicates its better transportation through the water (medium) solvent.
Secondary and tertiary structural analysis
SOPMA, a software tool was used for Secondary structure analysis of protein. Polygalacturonase protein is consisting of 18.90 % alpha-helices, 39.51% haphazard coils, 34.40%, extended beta strands and 7.18% beta turns (Fig. 1). The sub cellular localization by Cello indicates that this is an extracellular protein. The 3-D structure of Polygalacturonase was modeled using I-TASSER which is based on threading approach (Fig.2) [Nakamura et.al.2019]. Quality of tertiary model was assessed with the help of PROCHECK tool and related Ramachandran plot result indicates that 38.5% residues belongs to favored regions while 42.2% residues resides in allowed regions. Liberally allowed region contains 12.3% residues whereas 7% residues prohibited or outlier regions (Fig.3).
Protein preparation, binding site analysis and construction of grid box
The structure of polygalacturonase was constructed by I-TASSER. This structure was analyzed by AutoDock tool and converted into. pdbqt file format after the addition of polar hydrogen. I- TASSER also determined the binding site residues of the protein, Gln205, Gln261, Tyr262, Asn270, Ile271, Val273, and Asn320 were found to be present at active site.
The grid map centred at the active site pocket of protein (www.scfbio.iitd.res.in) lies in the centre x 66.361, centre y 66.444, centre z 68.583 with size 46, 44, 42.
Recovery and groundwork of Ligand molecules
Structures of phytoalexins were downloaded from
The PubChem database. 3-dimensional coordinates of phytoalexins were organized by using Marvin sketch in .pdb format and these files were used to prepare. pdbqt files by autodock [R.K. Pathak et.al.2017] for molecular docking studies.
Molecular docking, Visualization and analysis of protein ligand complex
The studies of molecular docking were made by AutoDock vina by means of the prepared 3D structure of different phytoalexin viz. nimbolide, nimbolin, Azadiradione, Quercetin and Azadirone with polygalacturonase as molecular target whereas the visualization and analysis of protein ligand complex was done using ligplot. nimbolide, nimbolin, Azadiradione, Quercetin and Azadirone docked with polygalacturonase with docking energy -8.0, -8.0,-7.8,-7.6,-7.4 kcal/mol respectively. Hydrogen bonds between phytoalexis and amino acid residues of pathogenic protein are showed in Fig. 4.
Table 1. Binding- free energy of all phytoalexins with target protein obtained through molecular docking studies.
Sno.
|
Name of compound
|
Free Binding energy (Kcal/mol)
|
No. of
Hydrogen bond
|
Interacting amino acid residue through Hbond
|
1
|
nimbolide
|
-8.0
|
4
|
Gln205, Gln261, Tyr262
|
2
|
nimbolin
|
-8.0
|
3
|
Lys235, Ser258
|
3
|
Azadiradione
|
-7.8
|
3
|
Asn176, Asn179, Tyr262
|
4
|
Quercetin
|
-7.6
|
7
|
Gln205, Asp229, Asp230, Ser258, Tyr290, lys292
|
5
|
Azadirone
|
-7.4
|
3
|
Ser100, Gln205, Lys235
|
6
|
Oleuropein
|
-7.4
|
13
|
Gln205, Asn206, Asp229, lys292,
Asp230,lys235 ,Gln261, Tyr262
|
7
|
Nimbin
|
-7.3
|
7
|
Trp173, Asn206, Ser258, Gln261, Tyr262
|
8
|
Salannin
|
-7.3
|
2
|
Arg237, Gln261
|
9
|
Nimbinene
|
-7.1
|
3
|
Asn206, Lys235, Ser258
|
10
|
Desacetylnimb
in
|
-7.0
|
3
|
Trp155, Asn176, Tyr262
|
11
|
Beta sitasterol
|
-6.9
|
2
|
Gln205, Asp229
|
12
|
caryophyllene
|
-6.1
|
-
|
No significant interaction
|
13
|
Caffeic acid
|
-6.0
|
7
|
Gln64, lys86, Thr88, Asn134, Asp169, Gln471
|
14
|
Carvacrol
|
-5.9
|
7
|
Ile45, Met46, Phe49, Ala48, Phe49, Glu50, Glu51, Cys52, Gly53
|
15
|
Protocatechoic acid
|
-5.8
|
5
|
Val204, Gln205, Gly228, Asn252, Tyr281
|
16
|
4-hydroxy benzoic acid
|
-5.6
|
4
|
Gly228, Asn252, Tyr281, Asp284
|
17
|
Syringic acid
|
-5.6
|
6
|
Trp173, Asp208, Ser258, lys292
|
18
|
Vanillic acid
|
-5.5
|
3
|
Trp203, val204, Tyr281
|
19
|
Tsibulin2
|
-5.4
|
2
|
Asn252, Tyr281
|
20
|
linalool
|
-5.1
|
2
|
Trp203, val204
|
21
|
Pyrocatechol
|
-4.8
|
5
|
Asn134, Lys168, Asp169, Gln471
|
22
|
Allicin
|
-4.2
|
1
|
Asn252
|
Molecular Dynamics Simulation of protein and protein-ligand complex
The stability of the projected protein model was assessed during 10 ns MD simulation and the binding mode of the protein-ligand complex was also analyzed using 10 ns MD simulation. MDS applying solvent, pressure and set temperature predicted the mechanism of precise binding of the complex. We computed the root mean square volatility and deviation from the trajectory.
Root Mean Square Deviation (RMSD)
RMSD serves as an indicator of system stability. The RMSD of both the protein and the protein-ligand complex was observed to increase between 1 and 4 ns, indicating that both structures remained stable as they dissolved in the cubic box solution, and any internal repulsion was eliminated over time. After 5 ns, both systems reached equilibrium and maintained a steady trajectory for analysis. The protein and protein-ligand complex exhibited average RMSDs of 0.43 nm and 0.38 nm, respectively (as shown in Fig. 5). The RMSD values suggest that the protein-ligand complex was more stable compared to the protein alone(Sapundzhi et.al.2022).
Root mean square fluctuation (RMSF)
We computed RMSF values to compare the flexibility of every amino acid residue in the complex and the protein. RMSF gives light on the structural differences of every residue. Lower RMSF values indicate well-structured regions, while higher values suggest more flexible or disordered areas, such as loops or lethal domains.
In the study, it was calculated the value of RMSF for the 10 ns. The peak of RMSF protein- ligand complex was to some extent higher than protein; however the average RMSF value was 0.15 nm and 0.14 nm for protein and protein-ligand complex (Fig. 6). The complex projected less variation as comparison to protein.
Radius of gyration (Rg)
Utilising Rg, the conformational variations and hardness of the protein-ligand complexes and the apo-protein were determined. The Rg value was determined for all the complexes with the apo-protein using the 10 ns trajectories. For the apo-protein and protein-ligand complex, the average Rg values were determined to be 2.38 and 2.33 nm, respectively. (Fig. 7). Protein-ligand complex showed lesser Rg value in comparison to apo-protein. The result suggested that protein ligand complex is more stable than the apo-protein.
Hydrogen bonds
Protein-ligand complexes are stabilised by many interactions, including hydrophobic, electrostatic, and hydrogen bonds. Highly definite and transitory interactions, hydrogen bonds are a crucial component of protein-ligand stabilisation. The many hydrogen bonds vs. time are explained in Figure 8. For the protein-ligand complex, the hydrogen bonds were counted normally between 0 and 1. It shows how the ligand keeps creating hydrogen bonds right up until the simulation is over.
Prospect of phytoalexins as antifungal molecule in plant protection
Phytoalexins acts as a significant role in plant fighting in opposition to plant pathogens, it not only in dicot species but in monocots as well [I. Ahuja et.al.2012],. It has been lately depicted that assault of maize stem by Rhizopus microspores and Collect otrichumgraminicola induces the gathering of 6 ent- kauranne pertaining to diterpenoids, communally termed kauralexins which hamper the expansion of the pathogens [E.A. Schmelz et.al.2011].
The outcomes of the current study noticeably shows, phytoalexin nimbolide can perform a direct molecule for the protection against fungal- diseases. Nimbolide is tiny hydrophobic molecule which can do cross cell membranes because of its perfect logP value and lower molecular weight that can maintain diffusion of the hydrophobic molecule in the course of the membrane. It has been ascertained that nimbolide have showed highest affinity towards pathogenic proteins of aspergilus niger. Therefore, nimbolide could be positive for safeguarding the Allium cepa in opposition to fungal diseases together with black mold (Fig 9).