Extended Data Fig. 3: Interactions of S89 with MFN fragments.
From: Small molecule agonist of mitochondrial fusion repairs mitochondrial dysfunction
(a) Analytical ultracentrifugation shows the size of wild-type (WT) MFN1-MGD (theoretical MW 47 kD, concentration: 0.4 mM) in the presence of the indicated nucleotides (2 mM) or S89 (20 µM). The estimated molecular masses are given above the peaks in kilodaltons. Data are representative of three independent experiments with similar results. (b) SDS-PAGE and Coomassie blue staining of purified MFN1 and MFN2. (c) Structural comparison of MFN1 (PDB code 5GOF for the MGD) and IniA (PDB code 6J72). The domains of the two proteins are colored in the same pattern. Predicted secondary structural elements of MFN1-HB2 are shown in the inlet with key regions highlighted. (d, e) Biolayer interferometry (BLI) to test S89 binding to MFN1 peptides L1, L2, or other alpha helices (α7, α8, α9, α10, α11a). All peptides were biotinylated in vitro. Data are representative of three independent experiments. (f) BLI of S89 binding to purified MFN1-MGD. (g) HPLC of small molecule S89 or S3 binding to MFN1 or its FF/AA mutant-expressing lysate from HEK293T cells. Left: Western blot using anti-HA antibody confirms equal loading of the indicated proteins.
