Fig 1.
Structural analysis for ligand-binding regions of AdipoR1.
A. Adiponectin docking model. Two predicted ligand-binding regions were indicated by yellow colour. For docking analysis, PDB ID 3WXV was used. B. Sequence and structure of active region of adiponectin. C and D. Z-Dock (protein-protein docking) and refinement. Two binding regions (C, site 1 and D, site 2) were separately presented with adiponectin active peptide sequence.
Fig 2.
Preparation of active AdipoR1.
A. Structural diagram for Adiponectin receptor 1 and its deletion mutant, Δ88. Shaded regions indicate transmembrane domain. ECL1, ECL2, ECL3, and c-terminal domain form extracellular surface. N-terminal 88 residues were deleted in AdipoR1-Δ88. Both constructs have N-terminal flag tag for further purification. B. Over-expressed recombinant AdipoR1 and AdipoR1-Δ88 in insect cells were analyzed before and after anti-flag affinity purification by Western blot with anti-flag antibody. C. AdipoR1-Δ88 purified by anti-flag beads were further purified by size exclusion chromatography to remove other impurities and flag peptide and to replace buffer. Eluents were analyzed by Western blot with anti-flag and anti-AdipoR1 antibodies. D. Purified AdipoR1-Δ88 was analysed by non-reducing SDS-PAGE.
Fig 3.
Surface plasmon resonance analysis.
ADP355 (A), BHD1028 (B), BHD43 (C), and BHD44 (D) were injected into AdipoR1-Δ88 -immobilized CM5 chip. SPR signals from different concentrations were plotted against concentrations to calculate Kd value.
Table 1.
Comparison of selected peptides to adiponectin and ADP355.
Fig 4.
Docking simulation of selected peptides into AdipoR1.
BHD1028 (A), BHD43 (B), and BHD44 (C) were simulated to bind to AdipoR1. Four major binding pockets were indicated with arched lines. Pocket 1 and 2 formed ligand binding site 2 and pocket 3 and 4 formed ligand binding site 1. Expected hydrogen bonds were indicated by red dot lines and key residues were annotated with number.
Fig 5.
AMPK activation by selected peptides.
A. Differentiated C2C12 myotubes were treated with 0.8, 4, and 20 μM of BHD1028, BHD43, and BHD44. As a positive control, ADP355 and 2.5 μg/ml globular adiponectin were treated. AMPK activation was analyzed by Western blot with anti-pAMPKthr172, anti-total AMPK, and GAPDH. B. Western blot signals were quantified by densitometer and relative signal were calculated by dividing pAMPK by GAPDH signals.
Fig 6.
AMPK activation by PEGylated peptides.
A and B. Selected peptides and PEGylated peptides treated to differentiated C2C12 myotubes at 20 μM. C and D. PEGylated BHD1028 induced AMPK and ACC activation with concentration dependent manners. Activation of AMPK and ACC were assessed by Western blot analysis (A and C) and their signals were quantified by densitometer (B and D).