Fig 1: Surface plasmon resonance (SPR) with biotin-conjugated GRASP shown in dose–response sensorgrams with overlaid TraceDrawer kinetic evaluation curves generated using a 1:1 local Bmax fit. Results from increasing concentrations of GFRAL (20, 100, 200, and 600 nM) are displayed in different shades of blue with GDF15 (1 μM) in red and RET (0.29 μM) in orange.
Fig 2: (A) In rats, anorexia induced by exogenous GDF15 (20 μg/kg, IP) was not affected by intraventricular administration (4th ICV) of GRASP (n = 10 per group). (B) GRASP 4th ICV administration elicited dose-dependent attenuation of GDF15-induced kaolin consumption, a well-validated proxy for emesis and nausea in rats (GDF15 at 20 μg/kg, n = 10 per group). (C) Systemic administration of GRASP failed to attenuate GDF15-induced anorexia in rats (GDF15 at 20 μg/kg IP, n = 12 per group). (D) The highest dose of GRASP tested (100 nmol/kg, i.e., 328 μg/kg) significantly attenuated kaolin intake resulting from systemic delivery of GDF15 (20 μg/kg IP, n = 12/group). Data were analyzed with repeated measure 2 × 3 ANOVAs followed by Tukey post hoc tests. All data are expressed as means ± standard error of the mean (SEM). Means with different letters are significantly different from each other (P < 0.05).
Fig 3: Solution-state structure of GRASP [T(K-azido)EELIHAHADPMVLIQKTDTGVSLQTYD; 300 μM, pH 6.8 in 50 mM PBS buffer spiked with 10% D2O, 800 MHz NMR with a cryoprobe at 25 °C] as a snapshot based on MD simulations, which reveals a secondary hairpin-like structure with a GDF15-like loop. This structure was the result of a 15 ns implicit solvent AMBER18 run25,26 in which NMR nuclear Overhauser enhancement (NOE)-based distance restraints and TALOS+-based PHI and PSI angle ranges were incorporated.27 Structure is deposited in the Biological Magnetic Resonance Databank (BMRB) as accession number 51672.
Fig 4: GRASP docking to GFRAL and GFRAL–GDF15. (A) The CryoEM structure of GFRAL–GDF15-RET complex (PDB 6Q2J) was used to generate an in silico prediction of GRASP docking (green; described herein as NMR solution structure (BMRB 51672); see also Figure 6); shown are GFRAL (red), GDF15 (gray), and RET (cyan).28 (B) GFRAL extracellular domain (ECD) (Trp129–Asn318; red, PDB 5VZ4) with the GRASP NMR structure in bound configuration displaying the top three MOE calculations and corresponding docking scores. Gray-colored GFRAL residues are those directly involved in GDF15 binding; gold-colored residues are those directly involved in RET recruitment to the GFRAL–RET interface. (C) GDF15 (gray) bound to GFRAL (red) (PDB 5VZ4) displaying the preferential docking domain of GRASP to the GFRAL–GDF15 complex; gold-colored residues are those directly involved in RET recruitment to the GDF15–RET interface. Docking model PDB is supplied as Supporting Information.
Supplier Page from Abcam for Recombinant Human GDF15 protein (Active)