Figure 1.
The predicted 3-D model and functional mapping of Fortilin from Penaeus monodon.
(A) The predicted structure model of PmFortilin contains six beta hairpins, eleven strands and three helices: The helical domain (helix-helix interaction) along the residues 77–126 and a flexible loop at residues 38–62. (B) A graphical representation of the functional mapping of PmFortilin protein, was analyzed by SMART and the Motif Scan server. The following elements are shown: TCTP signatures at residues 45–55 on the flexible loop and 123–145 on the C-terminal, Ca2+-binding domain at residues 76–110 and DNA binding domain at residues 80–81, 83–84 and 88 also on the helical domains, serine phosphorylation sites at the residues 50, 62 and 64, threonine phosphorylation sites at the residues 17, 60 and 103, tyrosine phosphorylation sites at the residues 18, 20, 28 and 92. The ten amino acid residues at the N-terminal show high conservation with the protein transduction domains (PTDs) and may allow trans-membrane transport.
Figure 2.
The predicted 3-D model and functional mapping of Fortilin Binding Protein 1.
(A) The FBP1 contains two helical domains located at residues 8 to 21 and residues 23 to 25, forming a disulphide bond between the Cys59 and Cys76. (B) A graphical representation of the functional mapping of FBP1. The following elements are shown: Two helix domains, 16 beta turns and 12 gamma turns. A signal peptide at residues 1–24, a cleavage site at residues 22–25, with cleavage between Ala24 and Thr25. A protein kinase C phosphorylation site “[ST]–x–[RK]” at residues 4–6 at the N-terminal. An amidation pattern “x–G–[RK]–[RK]” at residues 90–93 at the C-terminal end. Two segments of compositionally biased regions or Low Complexity Regions (LCRs) at 27–48 and 59–87 and “x–P–P–x” signature sequences of antiviral peptide signatures at 59–76, serine phosphorylation site at the residue 26, threonine phosphorylation site at the residue 25, tyrosine phosphorylation site at the residue 55.
Figure 3.
Transmembrane localization of FBP1-GFP in Sf9 cell.
The cellular localization of FBP1-GFP expressed in Sf9 cell was observed using confocal laser scanning microscopy at 48 h post-transfection. The plasma membrane was stained with DiI (Invitrogen) and shows co-localization of both fluorescent (green for GFP and red for DiI) after merging. Bright field is shown in the last panel. The scale bars indicate 10 µm.
Figure 4.
Live-cell and confocal imaging of the subcellular co-localization of GFP-FBP1 and PmFortilin.
After co-transfection (48 h), the sf9 cells were fixed with paraformaldehyde and stained with rabbit anti-PmFortilin antibody, followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (Invitrogen). The cells were observed with a confocal laser scanning microscope. The scale bars indicate 10 µm.
Figure 5.
Molecular interaction models of PmFortilin/FBP1.
(A top) A space-filling model representing the combination of two possible interactions of FBP1 (grey), at the opposite sides of PmFortilin (blue). (A bottom) A cartoon of the space-filling model showing the two conformations, A and B and the binding of PmFortilin to the C-terminus of FBP1. The predictions were performed with four separate modes: Balance, Electrostatic, Hydrophobic and VdW+Elec mode. (B–E) The lowest energy conformations of each of the four docking modes. The PmFortilin molecule (blue) and FBP1 (pink, yellow, magenta and green).
Figure 6.
Transmembrane topology and PmFortilin/FBP1 interaction complex.
(A left) The FBP1 protein integrated with the cell membrane, bound with PmFortilin. The residues 1–6 are intracellular, the residues 7–26 include the transmembrane segment and the residues 27–93 are extracellular, binding to PmFortilin. Ca2+-binding domain is shown in the orange color, the TCTP_1 on the flexible loop is in green, the TCTP_2 on the C-terminal is in light blue and other residues are in blue. (A right) A cartoon of the PmFortilin interaction complex, the FBP1 binding to PmFortilin and integration with the cell membrane; the PmFortilin (blue oval), the amidation site (light green), the cleavage site (light blue hexagon), the phosphorylation site (yellow), and the blue regions denote the binding regions of FBP1 at amino acid residues Pro44–Ala51 and Asn77–Ala80, Tyr88. This is according to the best of the interaction with the balance mode docking (probability is 72.00%). (B) The distances of amino acids in TCTP_1 interacting with the C-terminus of FBP1: Ala47 of PmFortilin and Asn77, Try88 of FBP1 and, Asn48 of PmFortilin and Asn77, Cys78 of FBP1, and Ala51 of PmFortilin and Pro79 of FBP1. (C) The distances in Angstroms between adjoining amino acids between the PmFortilin Ca2+-binding domain and FBP1: Thr77 of PmFortilin and Pro48 of FBP1, Gly78 of PmFortilin and Pro48, Ala49 of FBP1. (D) The distances of amino acids in the TCTP_2 interaction with the C-terminus of FBP1: Gln127 of PmFortilin and Ala51 of FBP1, Phe128–129 of PmFortilin and Pro48 of FBP1, Met134 of PmFortilin and Pro44 of FBP1.
Figure 7.
The PmFortilin/FBP1 trimer (FBP1-PmFortilin-FBP1) interaction complex.
The Ca2+-binding domain (orange) and the TCTP_1 of the flexible loop (green). The trimer docking simulation based on the balance mode yielded the lowest docking energy score of −1,162.00 Kcal/mol, the highest −888.70 Kcal/mol and the average docking energy of −986.43 Kcal/mol.
Figure 8.
The yeast two-hybrid interaction between PmFortilin fragments and FBP1 in S.cerevisiae AH109 cells. (A)YPDA control to show yeast growth. (B) β-galactosidase activity of the yeast cultures in selective medium determined by a colony-lift filter assay.
Table 1.
Summary of the growth of yeast harboring recombinant plasmids.