The present study was conducted to design and analyze the structural model of buffalo pregnancy-associated glycoprotein-1 (PAG-1) using bioinformatics. Structural modeling of the deduced buffalo PAG-1 protein was done using PHYRE, CONSURF servers and its structure was subsequently constructed using MODELLER 9.9 and PyMOL softwares Buffalo PAG-1 structural conformity was analyzed using PROSA, WHATIF, and 3D-PSSM servers. Designed buffalo PAG-1 protein structure on BLAST analysis retrieved protein structures belonging to aspartic proteinase family. Moreover
The pregnancy-associated glycoproteins (PAGs) constitute a large group of proteins belonging to the aspartic proteinase superfamily expressing in the placenta of eutherian mammals. PAGs are acidic glycoprotein sharing more than 50% amino acid sequence identities with Pepsin, Cathepsin D and E [
Structural modeling studies in bovine and porcine reveal their bilobed structure and proteolytically inactiveness due to key mutation at the active sites (alanine replaced by glycine: Gly34) residue whose presence would cause displacement of the catalytic water molecule from its normal position between the two catalytic aspartic residues (Asp32 and Asp215). Studies on bovine PAGs show that they have retained the peptide-binding cleft of aspartic proteinases to bind pepstatin and this property is an important factor to characterize members of aspartic proteinase. Moreover PAGs share their identity with other members of aspartic proteinase family, namely, rennin [
Recently buffalo pregnancy-associated glycoprotein-1 (PAG-1) gene sequence was deduced and
Secondary protein structure prediction from amino acid sequence was performed with PHRYE and CONSURF server [
Structural docking studies with aspartic proteinase inhibitor, namely, pepstatin A on buffalo PAG-1 protein was carried out using PatchDock [
Buffalo PAG-1 structural analysis was carried out with sequences sharing similarity (>25% with respect to the amino acid residue-long sequence of PAG-1) and having similar secondary structure matching with the predicted secondary structure of the target PAG-1 sequence. They were obtained from threading PAG-1 sequence onto known structure by using the PHYRE server. Energy nature, structural conformity, chemical nature, Ramachandran plots, and Z scores of the PAG-1 of the deduced model of buffalo PAG-1 were analyzed using software packages such as PROSA [
In the present study buffalo PAG-1 was subjected to structural analysis to study the protein model conformation and stability.
Buffalo PAG-1 protein model was designed using software, namely, MODELLER 9.9 and reconstructed using PyMOL. Various models of buffalo PAG-1 were constructed, namely, line, ball and stick, mesh, and space filling model. It is evident from deduced models that buffalo PAG-1 has retained bilobed structure with cleft for pepstatin A binding (Figure
Various models of buffalo PAG-1 protein. (a) Line; (b) ball and stick; (c) mesh; (d) space filling.
Secondary structure prediction of buffalo PAG-1 protein using PHYRE and CONSURF software revealed surface and buried regions of buffalo PAG-1 protein with their respective conformation. The entire buffalo PAG-1 molecule has distinct regions of helices and strands separated with coils. It was deduced that buffalo PAG-1 predicted secondary structure revealed strands and coil in their active sites (85–95 aa) and (270–279 aa) when compared to helices in other regions. The helix conformation was deduced in signal sequence as well as in residues 25–45, 191–199, and 278–290 (Figure
Predicted secondary structure of buffalo PGA-1 protein.
Predicted functional and structural residues in buffalo PAG-1 protein.
The predicted protein structure was analyzed with PHYRE server to retrieve similar protein molecules having similar structural conformity. Blast analysis retrieved 50 protein molecules from protein database out of which only 10 molecules which showed close similarity to buffalo PAG-1 were considered for structural analysis. Using PHYRE server many structural similar molecules were retrieved with their unique PDB identity. Molecules having more than 40% identity and showing 100% confidence level were taken into consideration for comparison with buffalo PAG-1 (Table
Structurally related molecules retrieved by PHYRE server.
S.no | PDB identity | Confidence | Identity % | Chemical nature |
---|---|---|---|---|
1 | d1psoe | 100 | 42 | Pepsin-like |
2 | c1lywE | 99.9 | 41 | Cathepsin D |
3 | d4pepa | 100 | 40 | Pepsin-like |
4 | d3psga | 100 | 40 | Pepsin-like |
5 | c1tzsA | 100 | 38 | Cathepsin E |
6 | c1avfJ | 100 | 36 | Gastricsin |
7 | c1qdmA | 100 | 34 | Prophytepsin |
8 | c2x0bC | 100 | 28 | Renin |
9 | c2bjuA | 100 | 25 | Plasmepsin |
10 | c3fnuA | 100 | 22 | Histo-aspartic protein |
Docking of buffalo PAG-1 with pepstatin A. (a, b) Structure of pepstatin A; (c, d) docking of pepstatin A on buffalo PAG-1 protein.
Analysis with PROSA software revealed the energy status of buffalo PAG-1 in comparison to its various amino acid residues (Figure
Energy levels corresponding to amino acid residues of buffalo PAG-1 protein.
Predicted energy and Z score of buffalo PAG-1 protein.
Analysis of buffalo PAG-1 protein using SAPS software revealed potential regions of cleavage by any proteolytic enzyme at residues 8–100, 140–160, 160–180, and 260–280 in the amino acid sequence but it is evident buffalo PAG-1 is stable molecule as the potential sites of proteolysis do not cross the threshold scores (Figure
Extremes and approximate center of gravity of buffalo PAG-1 protein model.
S.No | Co-ordinates | Minimum | Maximum | Centre of mass |
---|---|---|---|---|
1 | X | −3.206 | 54.936 | 26.632 |
2 | Y | −19.376 | 41.891 | 11.393 |
3 | Z | −30.298 | 14.615 | −7.661 |
Potential sites and thresholds of proteolysis of buffalo PAG-1 protein.
Ramachandran plot of pregnancy-associated glycoprotein-1 of buffalo.
In conclusion this study reports structural modeling and analysis of buffalo pregnancy-associated glycoprotein-1. Moreover
The author thank the Director and Head (Animal Reproduction Division) of Indian Veterinary Research Institute for providing the necessary facilities for conducting the experiment and analysis.