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Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis.

Panwar D, Rawal L, Sehgal N, Ali S - PLoS ONE (2015)

Bottom Line: The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface.Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex.This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.

ABSTRACT
Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

No MeSH data available.


KGF-KITLG interacting interface and binding residues.(A) Structural overview of KGF-KITLG interacting interface predicted by PISA, the interacting residues are shown in spheres (KGF: yellow and KITLG: blue). (B) A close view of KGF-KITLG binding interface showing the interacting residues corresponding to KGF and KITLG proteins in yellow and blue, respectively. Dotted lines (red) represent atomic distances between hydrogen bonds formed by binding residues.
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pone.0127993.g006: KGF-KITLG interacting interface and binding residues.(A) Structural overview of KGF-KITLG interacting interface predicted by PISA, the interacting residues are shown in spheres (KGF: yellow and KITLG: blue). (B) A close view of KGF-KITLG binding interface showing the interacting residues corresponding to KGF and KITLG proteins in yellow and blue, respectively. Dotted lines (red) represent atomic distances between hydrogen bonds formed by binding residues.

Mentions: Intermolecular protein-protein interactions and surface interface areas of the docked complexes were determined using the PISA server (Table 4). KGF-KITLG complex showed interaction having an interface area of 737.6 Å2 and solvation free energy (ΔiG) as -7.5 kcal/mol. Analysis of the docked complex (KGF-KITLG) revealed the presence of residues involved in extensive H-bonding and salt bridges (Table 5). Molecular docking showed the amino acids involved in KGF-KITLG binding namely Lys123, Glu135, Lys140, Lys155, and Trp156 corresponding to KGF protein, while KITLG specific ones included Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 (Fig 6A and 6B and Table 5). Subsequent analysis of KGF-KITLG binding interface showed the KGF interacting residues belonged to tyrosine receptor interaction site, suggesting its crucial role in regulating major activities of KGF protein through kinase activity.


Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis.

Panwar D, Rawal L, Sehgal N, Ali S - PLoS ONE (2015)

KGF-KITLG interacting interface and binding residues.(A) Structural overview of KGF-KITLG interacting interface predicted by PISA, the interacting residues are shown in spheres (KGF: yellow and KITLG: blue). (B) A close view of KGF-KITLG binding interface showing the interacting residues corresponding to KGF and KITLG proteins in yellow and blue, respectively. Dotted lines (red) represent atomic distances between hydrogen bonds formed by binding residues.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470682&req=5

pone.0127993.g006: KGF-KITLG interacting interface and binding residues.(A) Structural overview of KGF-KITLG interacting interface predicted by PISA, the interacting residues are shown in spheres (KGF: yellow and KITLG: blue). (B) A close view of KGF-KITLG binding interface showing the interacting residues corresponding to KGF and KITLG proteins in yellow and blue, respectively. Dotted lines (red) represent atomic distances between hydrogen bonds formed by binding residues.
Mentions: Intermolecular protein-protein interactions and surface interface areas of the docked complexes were determined using the PISA server (Table 4). KGF-KITLG complex showed interaction having an interface area of 737.6 Å2 and solvation free energy (ΔiG) as -7.5 kcal/mol. Analysis of the docked complex (KGF-KITLG) revealed the presence of residues involved in extensive H-bonding and salt bridges (Table 5). Molecular docking showed the amino acids involved in KGF-KITLG binding namely Lys123, Glu135, Lys140, Lys155, and Trp156 corresponding to KGF protein, while KITLG specific ones included Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 (Fig 6A and 6B and Table 5). Subsequent analysis of KGF-KITLG binding interface showed the KGF interacting residues belonged to tyrosine receptor interaction site, suggesting its crucial role in regulating major activities of KGF protein through kinase activity.

Bottom Line: The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface.Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex.This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.

ABSTRACT
Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

No MeSH data available.