Fig. 4: Identification of FDX2 residues interacting with NFS1 that have a mechanistic involvement in [2Fe–2S] synthesis.
From: Cross-regulation of [2Fe–2S] cluster synthesis by ferredoxin-2 and frataxin
a, AlphaFold snapshot of interacting residues at the FDX2–(NIAU)2 interface. Cofactors (PLP, [2Fe–2S] and Fe) were inserted using AlphaFill42. FDX2 is shown in magenta, the first NFS1 subunit in dark green (left), the second NFS1 subunit in light green (centre top) and ISCU2 in salmon. Blue dashes represent hydrogen bonding, yellow dashes represent a salt bridge and orange dashes represents a π-cation interaction (H181FDX2–R393NFS1). FDX2 residues selected for mutation are annotated on FDX2 helix F (E134, D137, D138, D141 and E148) and the C terminus (Y177, D179 and H181). Predicted hotspot residues selected by SpotOn are marked with an asterisk (D137*, D138*, D141* and Y177*). b, Overview of the FDX2–(NIAU)2 complex predicted by AlphaFold with subunit colouring as in a. c, FIDA-performed competition assays of the FDX2 mutants titrated into a preformed complex of FXNALC–(NIAU)2. d, Plot of the Kd value of the wild-type (WT) FDX2 and mutant constructs interacting with the (NIAU)2 complex as determined by FIDA in c, and with the (NIA)2 complex as determined using ITC. e–h, Effect of FDX2 mutants on the rate of [2Fe–2S] synthesis measured under conditions of excess FXN (10 eq. FXN and 1 eq. FDX2) (e), equal FXN and FDX2 (1 eq. FDX2 and 1 eq. FDX2) (f) and excess FDX2 (10 eq. FDX2 and 1 eq. FXN) (g) and in the absence of FXN (0 eq. FXN) and in the presence of FDX2 (1 eq. FDX2) (h). In c–h, data for helix F and C terminus mutant constructs are highlighted in blue and green, respectively. In c, WT FDX2, mean ± s.d., n = 3 independent experiments, FDX2 mutant constructs n = 1. In d, FIDA data: Kd as determined from c; ITC data: WT FDX2, Kd determined using representative experiment from n = 3 independent experiments with similar results; FDX2 mutant constructs, Kd determined using n = 1 experiment per construct shown in Extended Data Fig. 5a. Data in e–h are mean ± s.d., n = 3 independent experiments.
