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In silico screening, genotyping, molecular dynamics simulation and activity studies of SNPs in pyruvate kinase M2.

Kalaiarasan P, Kumar B, Chopra R, Gupta V, Subbarao N, Bamezai RN - PLoS ONE (2015)

Bottom Line: SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important.The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2.We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India.

ABSTRACT
Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

No MeSH data available.


Related in: MedlinePlus

3D structure of wild PKM2 monomer.All three substitutions are highlighted in stick model, C31F in green color, Q310P in red color, S437Y in purple color and FBP highlighted in orange stick. Four domains of PKM2 are highlighted in the cartoon model with different colors.
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pone.0120469.g002: 3D structure of wild PKM2 monomer.All three substitutions are highlighted in stick model, C31F in green color, Q310P in red color, S437Y in purple color and FBP highlighted in orange stick. Four domains of PKM2 are highlighted in the cartoon model with different colors.

Mentions: The details of in-silico sequence and structure based analysis of 29 nsSNP and 2 mutations are provided in Tables 1 and 2; and the results of the combined analysis of SIFT, PolyPhen and PANTHER tools reflected in Table 1. Seven out of 31 nsSNPs were predicted as damaging by all these programs. SNP&GO, PhD-SNP, SNAP and SNPeffect tools predicted 9 nsSNPs as damaging (Table 2). We selected nsSNPs for further analysis which were predicted as damaging by at least 5 prediction programs with RI > 2 for SNAP and 3 for PhD-SNP and SNP&GO. The combined analysis of all tools predicted 5 nsSNPs as damaging in PKM2 (Table 3). Further structural analysis of 5 variant proteins (C31F, G204V, Q310P, R339P & S437Y) was carried out using HOPE (Have yOur Protein Explained) (http://www.cmbi.ru.nl/hope/home) [36], which predicted only 3 of these as highly damaging by all prediction tools. Q310P, located in an α-helix very close to the active site (Fig. 2), was predicted to disrupt the α-helix with replaced proline and affecting the structure of the protein severely (S1 Fig). S437Y was located in the part of the allosteric site, the binding site of FBP (Fig. 2). It was predicted that differences in amino acid properties could disturb this region and its function by causing the loss in interaction with FBP. It was also predicted that the variant tyrosine could loose hydrogen bond with threonine at position 522. C31F, located in the inter subunit contact domain (Fig. 2), was predicted to disturb the monomer or dimer interaction, by affecting the local stability, which in turn could affect the ligand contacts made by one of the neighboring residues. The 3 nsSNPs (C31F, Q310P & S437Y) chosen on the basis of HOPE (considering tetrameric PKM2) results were subjected to molecular dynamics simulations. The stability analysis of PKM2 tetramer using FOLDX predicted three of the nsSVPs (C31F, Q310P and S437Y) with significantly decreased stability in compared to the wild type (Table 4) is based on total energy. Though the entropy was not significant, the analysis has taken into consideration all the factors, including Van der Waals clashes, Electrostatics and Van der Waals, showing a significant total energy. A comparison of the dimeric form of wild and nsSVPs followed a similar pattern (S1 Table). Further, in-vitro protein activity of all the three nsSVPs were compared with the wild PKM2 (described later).


In silico screening, genotyping, molecular dynamics simulation and activity studies of SNPs in pyruvate kinase M2.

Kalaiarasan P, Kumar B, Chopra R, Gupta V, Subbarao N, Bamezai RN - PLoS ONE (2015)

3D structure of wild PKM2 monomer.All three substitutions are highlighted in stick model, C31F in green color, Q310P in red color, S437Y in purple color and FBP highlighted in orange stick. Four domains of PKM2 are highlighted in the cartoon model with different colors.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359060&req=5

pone.0120469.g002: 3D structure of wild PKM2 monomer.All three substitutions are highlighted in stick model, C31F in green color, Q310P in red color, S437Y in purple color and FBP highlighted in orange stick. Four domains of PKM2 are highlighted in the cartoon model with different colors.
Mentions: The details of in-silico sequence and structure based analysis of 29 nsSNP and 2 mutations are provided in Tables 1 and 2; and the results of the combined analysis of SIFT, PolyPhen and PANTHER tools reflected in Table 1. Seven out of 31 nsSNPs were predicted as damaging by all these programs. SNP&GO, PhD-SNP, SNAP and SNPeffect tools predicted 9 nsSNPs as damaging (Table 2). We selected nsSNPs for further analysis which were predicted as damaging by at least 5 prediction programs with RI > 2 for SNAP and 3 for PhD-SNP and SNP&GO. The combined analysis of all tools predicted 5 nsSNPs as damaging in PKM2 (Table 3). Further structural analysis of 5 variant proteins (C31F, G204V, Q310P, R339P & S437Y) was carried out using HOPE (Have yOur Protein Explained) (http://www.cmbi.ru.nl/hope/home) [36], which predicted only 3 of these as highly damaging by all prediction tools. Q310P, located in an α-helix very close to the active site (Fig. 2), was predicted to disrupt the α-helix with replaced proline and affecting the structure of the protein severely (S1 Fig). S437Y was located in the part of the allosteric site, the binding site of FBP (Fig. 2). It was predicted that differences in amino acid properties could disturb this region and its function by causing the loss in interaction with FBP. It was also predicted that the variant tyrosine could loose hydrogen bond with threonine at position 522. C31F, located in the inter subunit contact domain (Fig. 2), was predicted to disturb the monomer or dimer interaction, by affecting the local stability, which in turn could affect the ligand contacts made by one of the neighboring residues. The 3 nsSNPs (C31F, Q310P & S437Y) chosen on the basis of HOPE (considering tetrameric PKM2) results were subjected to molecular dynamics simulations. The stability analysis of PKM2 tetramer using FOLDX predicted three of the nsSVPs (C31F, Q310P and S437Y) with significantly decreased stability in compared to the wild type (Table 4) is based on total energy. Though the entropy was not significant, the analysis has taken into consideration all the factors, including Van der Waals clashes, Electrostatics and Van der Waals, showing a significant total energy. A comparison of the dimeric form of wild and nsSVPs followed a similar pattern (S1 Table). Further, in-vitro protein activity of all the three nsSVPs were compared with the wild PKM2 (described later).

Bottom Line: SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important.The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2.We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India.

ABSTRACT
Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

No MeSH data available.


Related in: MedlinePlus