Table 1.
Details of primers used for amplification of full length coding sequence of KGF and KITLG.
Fig 1.
Bubalus bubalis KGF and KITLG genes amplification.
Representative gel picture showing the amplicons corresponding to KGF and KITLG genes. For size marker, 100 bp ladder was used.
Fig 2.
SDS-PAGE and validation of buffalo KGF and KITLG proteins.
SDS-PAGE profiles of buffalo KGF (A) and KITLG (B) proteins showing their resolved chromatographic fractions. M, Un, In and P denote marker, uninduced, induced and purified protein samples. The purified KGF and KITLG proteins were validated using western blot (WB) with anti-His (panel A) and anti-GST antibodies (panel B), respectively. The 23 and 57 kDa bands correspond to the purified KGF (His-tagged) and KITLG (GST tagged) proteins. (C and D) Co-immunoprecipitation of KGF and KITLG proteins. (C) The buffalo KGF and KITLG protein interactions were confirmed by immunoprecipitation of the tissue (ovary) lysate with anti-KGF antibody followed by immunoblotting with anti-KITLG IgG and detected a specific band of 31 kDa corresponding to KITLG protein. (D) In the reciprocal assay, a band of 22 kDa was detected on immunoprecipitation with anti-KITLG antibody followed by western blotting with anti-KGF IgG. The tissue lysate and resin in both C and D panels denote the positive and negative controls, respectively.
Fig 3.
Modeling of the buffalo KGF and KITLG structures.
(A) The 3D KGF protein structure generated by the MODELLER (V9.14). The N-terminus and C-terminus loops are marked. Magenta, teal and orange colors represent helix, beta sheets and loops, respectively. (B) Predicted model of KITLG by I-TASSER showing helix, beta sheets and loops in yellow, magenta and teal colors, respectively.
Table 2.
CASTp predictions for buffalo KGF and KITLG binding site residues.
Table 3.
Statistical analysis for HADDOCK generated KGF-KITLG docked complexes.
Fig 4.
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.
Fig 5.
(A) Pie-chart showing the distribution of Haddock clusters with cluster size, cluster 1 (KGF-KITLG docked complex) occupies 51% (Size—94) out of 183 complexes generated by HADDOCK (B) The HADDOCK scores of docked models were plotted against their i-RMSDs. The color codes represent the i-RMSD values of all 9 HADDOCK clusters. Wherein, cluster 1 (green) with the lowest i-RMSD value of 1.1 +/- 0.7 Å represents the best docked complex. (C) Diagrammatic illustration of selected KGF-KITLG docked complex, where KGF and KITLG are shown in red and green, respectively.
Table 4.
PISA predicted KGF- KITLG interacting interface.
Table 5.
PISA analysis of the H-bonding and salt-bridge interactions among the residues participating in KGF-KITLG binding interface,
Fig 6.
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.
Fig 7.
KGF-KITLG hydrophobic residual interactions.
(A) KGF-KITLG docked complex with spheres representing their binding interface. (B and C) DIMPLOT program generated two-dimensional plots representing hydrogen (B) and hydrophobic (C) interactions between KGF and KITLG proteins. Green and black (dashed) lines indicate hydrogen bonds and hydrophobic interactions in KGF-KITLG complex, respectively.
Table 6.
Interaction energies (ΔiG) of native and mutant forms of KFG-KITLG docked complex.