Fig 1.
Crystal Structure of the B. subtilis cysteine desulfurase SufS at 1.7 Å.
(A) Overall structure of the BsSufS homodimer. The dimer forms a two-fold symmetry axis which covers the active site pocket. One monomer is colored light green and the other, cyan. (B) Monomeric BsSufS consists of two domains: The larger, N-terminal domain harbors the PLP binding pocket and the smaller, C-terminal domain contains the flexible Cys361-loop (on which the catalytic Cys361 resides). (C) Magnified view of the active site pocket of BsSufS, showing the products alanine and Cys361-persulfide. Pyrodoxal-5ʹ-phosphate is bound to Lys224 as an aldimine. A 2Fo-Fc map density (grey mesh) is shown, contoured at 1.0 r.m.s.d. N-terminus (NT), C-terminus (CT).
Table 1.
Data collection and refinement statistics for BsSufS.
Fig 2.
The dynamics of BsSufS were analyzed in solution by incubation in deuterated buffer. The relative amount of deuterium incorporated after 15 s, indicated by a color code ranging from blue (low; stable region) to red (high; flexible region), is mapped on the detected peptic peptides of (A) BsSufS or mapped onto the surface of the BsSufS (B) monomer and (C) homodimer. N-terminus (NT), C-terminus (CT).
Fig 3.
BsSufU alters the H/D exchange of BsSufS upon binding.
Changes in relative fractional deuterium uptake of BsSufS after incubation with BsSufU for 15 s in D2O buffer compared to BsSufS alone were mapped onto the surface of the BsSufS (A) monomer and (B) homodimer. A decrease (blue) in deuterium uptake signals protection (i.e., a binding event), whereas an increase (red) signals a structural rearrangement. Black regions were not detected. Binding of BsSufU to (C) the C-terminus and (D) the α-hinge of BsSufS as a function of deuterium uptake over time. Color code: BsSufS alone (green), BsSufS + BsSufU (red), BsSufS + BsFra (blue), and BsSufS + BsSufU/BsFra (violet). N-terminus (NT) and C-terminus (CT).
Fig 4.
BsSufS alters the H/D exchange of BsSufU upon binding.
(A) Detected peptic peptides of BsSufU with the relative fractional uptake after 15 s of incubation in deuterated buffer. (B) Changes in the relative fractional deuterium uptake of BsSufU after incubation with BsSufS for 15 s in D2O buffer compared to BsSufU alone were mapped onto the surface of BsSufU (PDB ID 2AZH). The heat map represents the differences in deuterium uptake compared to BsSufU alone. A decrease (blue) in deuterium uptake signals protection (i.e., a binding event), whereas an increase (red) signals a structural rearrangement. Black regions were not detected. Binding of BsSufU to (C) the α/β-linker and (D) the Cys128-loop of BsSufS as a function of deuterium uptake over time. Color code: BsSufU alone (red), BsSufU + BsSufS (green), BsSufU + BsFra (blue), and BsSufU + BsSufS/BsFra (violet). N-terminus (NT) and C-terminus (CT).
Fig 5.
Characterization of the affinity of BsFra for BsSufS, BsSufU, and BsSufS/BsSufU using microscale thermophoresis.
MST binding curve from the interaction of fluorophore-labeled BsFra with (A) BsSufS, (B) BsSufS/BsSufU, and (C) BsSufU. A Hill model was applied for Kd determination. Fra* indicates fluorophore-tagged frataxin.
Fig 6.
Peptic peptides of BsFra detected from H/DX measurements.
The relative amount of deuterium incorporated after 15 s, indicated by a color code ranging from blue (low; stable region) to red (high; flexible region), is mapped on the detected peptic peptides of BsFra.
Fig 7.
H/DX analysis of BsFra upon binding BsSufU/BsSufS.
(A) Changes in the relative fractional deuterium uptake of BsFra after incubation with BsSufS/BsSufU for 15 s in D2O buffer compared to BsFra alone were mapped onto the surface of BsFra (PDB ID 2OC6). The heat map represents the differences in deuterium uptake compared to BsFra alone. A decrease (blue) in deuterium uptake signals protection (i.e., a binding event), whereas an increase (red) signals a structural rearrangement. Black regions were not detected. Binding of BsSufS/BsSufS to (B) the ‘acidic ridge’ and (C) the KWN-loop of BsFra as a function of deuterium uptake over time. Color code: BsFra alone (blue), BsFra + BsSufS (green), BsFra + BsSufU (red), and BsFra + BsSufU/BsSufS (violet). N-terminus (NT) and C-terminus (CT).
Fig 8.
BsFra alters the H/D exchange of BsSufS and BsSufU upon binding the BsSufS/BsSufU complex.
Changes in the relative fractional deuterium uptake of BsSufS/BsSufU after incubation with BsFra for 15 s in D2O buffer compared to BsSufS and BsSufU alone were mapped onto the surface of the BsSufS (A) monomer and (B) homodimer as well as (C) BsSufU (PBD ID 2AZH). The heat map represents the differences in deuterium uptake compared to the solo incubation. A decrease (blue) in the uptake signals protection (i.e., a binding event), whereas an increase (red) signals a structural rearrangement. Black regions were not detected. Changes in (D) the SufS Cys361-loop and (E) the β-hook as a function of deuterium uptake over time. Color code: BsSufS alone (green), BsSufS + BsFra (blue), BsSufS + BsSufU (red), and BsSufS + BsSufU/BsFra (violet). Changes in (F) the SufU α5-helix and (G) the Cys41-loop as a function of deuterium uptake over time. Color code: BsSufU alone (red), BsSufU + BsFra (blue), BsSufU + BsSufS (green), and BsSufU + BsSufS/BsFra (violet). N-terminus (NT) and C-terminus (CT).
Fig 9.
Sulfur transfer and Fe-S cluster biosynthesis assays of BsSufS and BsSufU in the presence of BsFra.
(A) The specific activity of BsSufS was measured in vitro by the release of sulfide from cysteine. The activity of BsSufS (green) is unaffected by BsFra (cyan), but is greatly increased by the presence of BsSufU (red). The addition of BsFra to BsSufS/BsSufU does not affect the activity of BsSufS. (B) In vitro biogenesis of Fe-S clusters by BsSufS/BsSufU over time (red). Addition of BsFra (blue) results in a very slight increase in cluster yield.
Fig 10.
Proposed model for the interaction of BsFra/BsSufU/BsSufS.
(A) Based on the H/DX results, we identified the binding sites of BsSufU (PDB ID 2AZH, shown in salmon), BsFra (PDB ID 2OC6, shown in sky blue) and the BsSufS homodimer (light green and cyan) in the BsFra/BsSufS/BsSufU complex. The binding epitopes are highlighted according to the color of the respective interaction partner (i.e., residues protected by BsFra are shown in blue, those by BsSufS in green, and those by BsSufU in red). (B) Proposed binding of BsFra and BsSufU to BsSufS. The binding of BsSufU to BsSufS brings the SufS residue Cys361 and the SufU residue Cys41 into close proximity for persulfide transfer. While binding of BsFra to BsSufS does not affect the transfer of persulfide from BsSufS to BsSufU, it brings the putative iron binding site into close proximity to the SufS/SufU active site.