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 .
they are secreted from the outer epithelial cell layer of placenti of various ungulate species possessing a predominant role in the placentogenesis , placental modeling , and embryogenesis during pregnancy in domestic species .
many pag genes have been cloned and identified in many domestic animals such as cattle , sheep , goat , pig , and wild ruminants ' species [ 24 ] .
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 [ 5 , 6 ] . recently
buffalo pregnancy - associated glycoprotein-1 ( pag-1 ) gene sequence was deduced and in silico analysis revealed buffalo pag-1 to share more than 80% homology with other members of aspartic proteinase family .
moreover deduced buffalo pag-1 amino acid sequence also reveals key mutation at the active site which renders it proteolytically inactive . predicted structure of buffalo pregnancy - associated glycoprotein-1 revealed it to possesses aspartic acid residues near the active site . but structural modeling and its analysis of buffalo pag-1 protein is still lacking . considering the paucity of knowledge
, the present work was carried out to design and analyze the structural model of pregnancy - associated glycoprotein-1 buffalo ( bubalus bubalis ) .
secondary protein structure prediction from amino acid sequence was performed with phrye and consurf server [ 8 , 9 ] to predict the location of the conserved sites in buffalo pag-1 amino acid sequence .
the comparative modeling program modeler 9.9   in conjunction with pymol   was done to construct various models of buffalo pag-1 .
structural docking studies with aspartic proteinase inhibitor , namely , pepstatin a on buffalo pag-1 protein was carried out using patchdock   server to confirm the affinity of protease inhibitors on buffalo pag-1 protein .
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 , procheck , and whatif .
multiple alignments of similar structurally related proteins were aligned with their pdb identity using phyre software to confirm the chemical nature of buffalo pag-1 .
saps software was used to predict the potential sites for proteolysis of the pag-1 protein indirectly measuring the stability of the protein [ 1618 ] .
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 1 ) .
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 ( 8595 aa ) and ( 270279 aa ) when compared to helices in other regions .
the helix conformation was deduced in signal sequence as well as in residues 2545 , 191199 , and 278290 ( figure 2 ) .
moreover the neural network algorithm in consurf server revealed the conserved , buried , surface , and functional residues in buffalo pag-1 protein .
the buffalo pag-1 protein showed very high degrees of conserved regions in amino and carboxy terminals near the active aspartic regions 8595 and 268274 residues confirming its belonging to aspartic proteinase family .
moreover majority of the residues near the active sites were buried inside the protein model and the potential functional residues ' locations were predicted in pag-1 protein molecule ( figure 3 ) .
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 .
molecules having more than 40% identity and showing 100% confidence level were taken into consideration for comparison with buffalo pag-1 ( table 1 ) .
ten different molecules retrieved from the protein database showed biochemical similarity to members of aspartic proteinase from phyre database .
it is evident from the structural comparison that all these members of aspartic proteinase family retain the bilobed structure similar to buffalo pag-1 protein structure .
buffalo pag-1 protein on docking with specific aspartic proteinase inhibitor , pepstatin a ( a hexapeptide consisting of rare amino acids , namely , isovaline and statine : pdb : 1 pso ) revealed binding near the active sites in the cleft region between the bilobed structure similar to docking sites reported in bovine and porcine pag model .
it is also evident that buffalo pag-1 possesses binding sites for aspartic protease inhibitors ( pepstatin a ) ( figure 4 ) .
analysis with prosa software revealed the energy status of buffalo pag-1 in comparison to its various amino acid residues ( figure 5 ) .
moreover energy status analysis of the entire molecule revealed the presence of more low energy pockets in comparison to high energy pockets .
the active sites in the buffalo pag-1 protein ( 8595 ) and ( 275279 ) also revealed low energy status revealing the stable structure of the molecule .
the z score of the buffalo pag-1 protein was 7.01 on analyses in x - ray and nmr methods showing the good quality of deduced buffalo pag-1 protein model ( figure 6 ) .  
analysis of buffalo pag-1 protein using saps software revealed potential regions of cleavage by any proteolytic enzyme at residues 8100 , 140160 , 160180 , and 260280 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 7 ) .
analysis of ramachandran plots of buffalo pag-1 depicted that 89.4% of all residues were located in the favored regions and 96.6% of all residues were located in the allowed regions .
there were 13 amino acid residues which were lying outside the favoured or allowed regions ( figure 8) .
four glycine residues ( 33 , 149 , 243 , and 325 aa ) and 1 proline residue ( 310 aa ) were outliers in the predicted buffalo pag-1 protein model .
moreover the predicted centre of mass of buffalo pag-1 protein is shown in table 2 .
thus structural modeling and analysis of buffalo pag-1 protein was done and reported for the first time .
  in conclusion this study reports structural modeling and analysis of buffalo pregnancy - associated glycoprotein-1 .
moreover in silico analysis of buffalo - associated glycoprotein-1 revealed its bilobed structure with conserved active sites having the property of binding aspartic protease inhibitors thereby confirming its belonging to aspartic proteinase superfamily .
 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 in silico analysis revealed buffalo pag-1 protein retained bilobed structure with pepstatin - binding clefts near the active sites by docking studies with pepstatin a using patchdock server . 
 structural studies revealed that the amino and carboxy terminal containing aspartic residues are highly conserved and buried within the protein structure . 
 structural conformity studies showed that more than 90% of the residues lie inside favored and allowed regions . 
 it was also deduced that buffalo pag-1 possesses low and high energy zones with a very low threshold for proteolysis ascertaining the stableness of the buffalo pag-1 protein structure . 
 this study depicts the structural conformity and stability of buffalo pag-1 protein . 
1. Introduction
2. Material and Methods
3. Result and Discussion
4. Conclusion