In silico docking analysis of CCL28 (C-C motif chemokine ligand 28) and astragalin as the potential inhibitor of rheumatoid arthritis

Target protein structure

The three-dimensional crystal structure of the target protein CCL28 (PDB ID: 6CWS)¹¹ was downloaded from the Protein Data Bank¹² in PDB format using a similar method to that carried out by Esther et al.¹³

Preparation of ligand

The two-dimensional chemical structure of astragalin was accessed from the PubChem database (PubChem CID: 5282102) and drawn using Accelrys Draw 4.1 software¹⁴ with default parameters. The resulting MOL file of the ligand was converted into PDB format by Accelrys Discovery Studio 4.1 software¹⁵ using default parameters. The chemical structure of astragalin is shown in Figure 1. We used naproxen as standard drug which is being used against arthritis. The chemical structure of this molecule was taken from the PubChem database (PubChem CID: 156391).

Figure 1. Chemical structure of astragalin accessed from PubChem (ID: 5282102).

Optimization of target and ligand

PDB coordinates of target and ligand proteins were optimized by energy minimization, 3D protonation and stable conformation parameters of Molecular Operating Environment (MOE) software¹⁶ (version 2014.0901) and files were saved in MDB format.

Quality estimation of target protein

The quality of the CCL28 predicted protein structure was estimated using the Phyre² (Protein Homology/analogY Recognition Engine, version 2.0) online web server¹⁷. The ProQ2 quality assessment and Ramachandran plot was applied for identifying the structural conformation of the proteins using the default parameters of the tool.

Binding site analysis

We computed and analysed the binding sites of target protein using the default parameters of the MOE software¹⁶.

Molecular docking analysis

A computational ligand-target docking approach was used to determine structural complexes of CCL28 with astragalin in order to understand the structural basis of these protein targets specificity. Finally, docking was carried out by MOE based on RMSD and E-score. The energy of the interaction of these derivatives with the protein targets is assigned “grid point”. At each step of the simulation, the energy of the interaction of ligand and protein was evaluated using atomic affinity potentials computed on a grid. Molecular docking was performed to analyse the binding of the astragalin ligand with the active sites of the CCL28 target protein. The optimized ligand was docked with the target using MOE software¹⁶ with default parameters as described by Esther et al.¹³

Quality estimation of predicted CCL28 protein structure

The quality of the CCL28 predicted protein structure was estimated by the Phyre² web server¹⁷. Phyre² provided a 3D structure of the target protein and the dimensions of the 3D structure in Å were X=48.506, Y=53.506 and Z=38.552. Analysis of the secondary structure of the target protein provided by Phyre² revealed high confidence in the predicted structure, indicated by red regions in Figure 2, suggesting that the protein structure quality is high. The sequence profile graph shows that M residues are highly favourable, L and V residues are moderately favourable and remaining residues are less favourable. The ProQ2 quality assessment showed the quality of protein target and could be considered as "good" model indicating less mutation sites in the protein structure (Figure 3). The Ramachandran plot shows that approximately 98% amino acid residues of the target protein model exist in the most favoured region whereas <2% exist in the disallowed region. This Ramachandran plot representing the backbone conformation of CCL28 amino acid residues is shown in Figure 4.

Figure 2. The CCL28 was modelled using the Phyre2 server.

The sequence alignment of protein molecule shows high confidence, indicated by red colour, while blue indicates low confidence. 49% of the structure is in alpha helices, 11% is in beta-strands and 13% is in transmembrane helices.

Figure 3. The quality of the CCL28 model was estimated by ProQ2 of the Phyre2 server.

Red colour indicates “Good” regions while blue represents “Bad” regions of the structure. The quality of model indicated the minimal disordered region (shown by broken lines).

Figure 4. The Ramachandran plot of the CCL28 protein target constructed by ProQ2 indicated whether the amino acid residues occur in the “favoured region” or “disallowed region” of the plot.

For a “good” protein model there must be ≥90% amino acid residues in the most favoured region or <2% in the disallowed region of the plot.

Binding site analysis and molecular docking

Binding sites of the target protein were analysed using MOE software¹⁶. Analysis revealed that His 99 and Thr 101 residues were present in a pocket of the target protein. The spatial orientation of the astragalin ligand in the binding pocket of the target is shown in Figure 5. In silico molecular docking using MOE software¹⁶ resulted in the successful docking of target CCL28 with the astragalin ligand. The binding energy of astragalin with CCL28 is -5.40 kcal/mol, as compared to standard naproxen which has a binding energy of -4.87 kcal/mol. A comparison of the binding energies and root-mean-square deviation (RMSD) values of astragalin and standard naproxen with the CCL28 target is shown in Table 1 and Table 2 respectively. Results show that astragalin has strong binding affinity for the CCL28 target molecule as compared to naproxen. The ligand interaction module of MOE¹⁶ was used to view ligand interactions. Figure 6 shows astragalin interacting with CCL28 via hydrogen bonding with the His 99 and Thr 101 residues.

Figure 5. Confirmation and pose of molecular docking interaction of astragalin as ligand molecule with CCL28 as target protein.

Figure 6. Binding site atoms of CCL28 residues around ligand showed interaction of His99 and Thr101 amino acid residues of CCL28 with ligand atoms (carbon and oxygen atoms).

Underlying data

Structure of CCL28 from Protein Data Bank, Accession number 6CWS: https://identifiers.org/pdb/6CWS.

Structure of naproxen from PubChem Database, Accession number 156391: https://identifiers.org/pubchem.compound/156391

Structure of astragalin from PubChem Database, Accession number 5282102: https://identifiers.org/pubchem.compound/5282102