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: 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.
Fig 3: Representative images of the AP and the medial NTS documenting high levels of colocalization of fluorescently tagged GRASP (GRASP555) with unlabeled GFRAL. GRASP555 (300 pmol in 1 μL) was injected into the lateral ventricle of wild-type rats 2 h before sacrifice. Brain tissues were then removed and processed for immunohistochemistry. AP, area postrema; NTS, nucleus tractus solitarius; CC, central canal. Scale bar: 100 μm.
Fig 4: Flow cytometric analysis of GFRAL–HEK293 cells probed with GRASPCy5. (A) Control (untransfected) HEK293 cells with 100 nM GRASPCy5, (B) GFRAL–HEK293 cells treated with 100 nM Cy5 (unconjugated), (C) GFRAL–HEK293 cells probed with buffer control, (D) GFRAL–HEK293 probed with 100 nM GRASPCy5, (E) GFRAL–HEK293 cells probed with 100 nM GRASPCy5 in the presence of 10 10 μM unlabeled GRASP, and (F) fraction bound vs GRASPCy5 concentration (M) at GRASPCy5 concentrations of 0.1, 1, 5, 10, 50, 100, 500, and 1000 nM; KD = 8.98 nM with a Hill coefficient of 1.055.
Fig 5: Representative GRASP binding pockets for the top three calculations from GFRAL docking studies are shown in Figure 5B. Conserved GFRAL residues involved in GRASP recognition are highlighted. It should be noted that the HAHA motif of GRASP is depicted in the green box, displaying the best docking score.
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