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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.


HADDOCK based structural mapping of KGF-KITLG docked complexes.HADDOCK generated 9 clusters after refinement and clustering. KGF-KITLG complexes were aligned with their respective HADDOCK scores. A surface-based protein representation in different color is used for each complex. Best docked complex (encircled) had the lowest HADDOCK score of -81.0.
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pone.0127993.g004: HADDOCK based structural mapping of KGF-KITLG docked complexes.HADDOCK generated 9 clusters after refinement and clustering. KGF-KITLG complexes were aligned with their respective HADDOCK scores. A surface-based protein representation in different color is used for each complex. Best docked complex (encircled) had the lowest HADDOCK score of -81.0.

Mentions: CASTp detected binding residues corresponding to KGF and KITLG proteins were subjected to protein docking (Table 2). HADDOCK clustered 183 docked complexes in 9 clusters representing 91.5% of the water-refined models (Table 3 and Fig 4). From these 9 clusters, cluster 1 with HADDOCK score:- 81.0 +/- 5.8 Kcal/mol, cluster size: 94, electrostatic energy: -222.1 +/- 19.7 Kcal/mol and Z-Score: -1.3 was selected as the best KGF-KITLG docked complex for further study (Fig 5A–5C).


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)

HADDOCK based structural mapping of KGF-KITLG docked complexes.HADDOCK generated 9 clusters after refinement and clustering. KGF-KITLG complexes were aligned with their respective HADDOCK scores. A surface-based protein representation in different color is used for each complex. Best docked complex (encircled) had the lowest HADDOCK score of -81.0.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127993.g004: HADDOCK based structural mapping of KGF-KITLG docked complexes.HADDOCK generated 9 clusters after refinement and clustering. KGF-KITLG complexes were aligned with their respective HADDOCK scores. A surface-based protein representation in different color is used for each complex. Best docked complex (encircled) had the lowest HADDOCK score of -81.0.
Mentions: CASTp detected binding residues corresponding to KGF and KITLG proteins were subjected to protein docking (Table 2). HADDOCK clustered 183 docked complexes in 9 clusters representing 91.5% of the water-refined models (Table 3 and Fig 4). From these 9 clusters, cluster 1 with HADDOCK score:- 81.0 +/- 5.8 Kcal/mol, cluster size: 94, electrostatic energy: -222.1 +/- 19.7 Kcal/mol and Z-Score: -1.3 was selected as the best KGF-KITLG docked complex for further study (Fig 5A–5C).

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.