Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex

Abstract

Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.

Fig. 1

Crystal structures of (NFS1-ISD11-ACP)₂ and (NFS1-ISD11-ACP-ISCU)₂ complexes. Overview of the 3D structures of the a (NIA)₂, b (NIAU)₂, and c (NIAU-Zn)₂ complexes. NFS1 is depicted in orange, ISD11 in magenta or purple, ACP in green, and ISCU in blue. Pyridoxal phosphate (PLP) and the phosphopantetheine with its fatty acyl chain (PPA) are depicted in black as spheres. In part a the ~56 Å distance between the centers of NFS1 and ACP as well as the N and C termini are indicated

Fig. 2

Conformational changes of NFS1 upon ISCU binding. a The NFS1 dimer rotation visualized by superposition of NFS1 as present in (NIA)₂ (orange) or (NIAU)₂ (gray). The rotation pivot point is located close to Pro71 (green) in front of the PLP moiety (black). Regions of NFS1 disordered in (NIA)₂, yet structured in (NIAU)₂ complexes are shown by spheres. They connect the ISCU binding site and helices α3 and α4 in a “domino-like” fashion (red arrows). The tips of helices α4 in the (NIA)₂ complex are close together and provide new stabilizing interactions in place of disordered regions. As a result of ordering due to ISCU binding, NFS1 helices α3 and α4 re-orient by 8 Å and 9 Å, respectively and the ‘standard’ interface is recreated. The cartoon illustrates the movement of the NFS1 dimer. b Mode of ISCU binding to NFS1 in the (NIAU)₂ and (NIAU-Zn)₂ complexes. Upon Zn binding to the active-site Cys381^NFS1, the Cys-loop (red) becomes fully structured. Additionally, Zn is coordinated by Asp46, Cys70, and His112 of ISCU. The Ala-loop (Ala41-Cys44) of ISCU (cyan in (NIAU)₂ and green in (NIAU-Zn)₂) is rearranged upon Zn binding (inset)

Fig. 3

Structure of ISD11 alone and in complex with NFS1 or ACP. Structures of subunits and sub-complexes were taken from the (NIAU)₂ complex. a Structure of ISD11. All hydrophobic residues found in ISD11 are in gray. Helices α1 (red), α2 (green), and α3 (blue) are antiparallel (α2 and α3) or tilted by ~21° (α1). N and C termini are marked. The LYR motif is shown in khaki by spheres. b Surface representation of ISD11 shows a hydrophobic tunnel between the three α-helices and a canyon. Surface is colored in light-magenta except for positively and negatively charged residues in blue and red, respectively. c Hydrophobic and polar interactions between ISD11 (magenta) and NFS1 (orange). Boxes show details of the interaction interfaces. d Interaction of the ISD11 and NFS1 dimers. Insets show details of ISD11–ISD11 interaction. e Structure of the ISD11-ACP sub-complex. Phosphopantetheine with a fatty acyl chain (PPA) is colored in black, and the LYR motif in cyan. f Surface representation of ISD11 in the ISD11-ACP sub-complex shows that PPA (red balls) and part of the fatty acyl chain (gray balls) bind to a hydrophobic canyon of ISD11, while the rest of the fatty acyl chain penetrates the hydrophobic tunnel of ISD11

Fig. 4

Biochemical verification of the (NFS1-ISD11-ACP-ISCU)₂ structure in yeast. a Sequence alignment of ISD11 from S. cerevisiae (Saccer), H. sapiens (Human), and C. thermophilum (Caethom). Mutations are indicated by arrows. Locations of the mutations are indicated in the crystal structure of human ISD11 using the corresponding Saccer nomenclature. b–f Gal-ISD11 yeast cells expressing S. cerevisiae Acp1-HA were transformed with plasmids encoding either no protein (empty) or wild-type (WT) or the indicated FLAG-tagged Isd11 mutant proteins. Gal-ISD11 yeast cells were depleted of endogenous Isd11 on glucose medium for 40 h, and mitochondrial extracts were prepared. b Anti-FLAG co-immunoprecipitation. The immunoprecipitate (Co-IP) and an aliquot of the mitochondrial extract (input) were analyzed by immunostaining for the indicated ISC proteins and porin as a loading control using antibodies against the respective protein or tag (anti-FLAG: Sigma and anti-HA: Santa Cruz). c Blue-native gel electrophoresis (7.5–20% polyacrylamide). Immunostaining was against FLAG of Isd11-FLAG. d Cysteine desulfurase activity of 200 µg mitochondrial extracts following the in vitro sulfide production. e, f Enzyme activity assays of mitochondrial aconitase e and SDH f normalized to malate dehydrogenase (MDH). g Orbitrap mass spectrometry of trypsin-digested purified CtNfs1-CtIsd11 solution with and without bound ACP. For quantification the peak area was used. Black and gray bars represent the samples purified from cells growing in high or low energy media, respectively. No ACP could be detected in the latter (n.d.). h Desulfurase activity was measured with purified human or C. thermophilum NFS1-ISD11 with or without co-purified ACP. The error bars indicate the SD (n ≥ 3). i In vitro enzymatic Fe/S cluster synthesis on ISCU2/CtIsu1 using purified human or C. thermophilum (Ct) ISC proteins. NFS1-ISD11 with or without co-purified ACP as indicated were mixed anaerobically with ISCU, FXN, FDX2, and FdxR, and Fe/S cluster synthesis on ISCU2 was followed by circular dichroism monitoring ΔCD at 431 nm (left). After 20 min CD spectra were recorded to document successful reconstitution of ISCU2 (right). Control reactions were performed without NFS1-ISD11

Fig. 5

SAXS shapes and stability of human ISC complexes. a–c, Small angle X-ray scattering (SAXS) analyses were performed with the indicated ISC complexes. Crystal structures of the (NIA)₂ complex a, the NFS1 dimer part alone b or the NFS1-ISCU part of (NIAU)₂ c were fitted into the determined SAXS densities. d Scattering curves with respective fits in gray (left) and Kratky plots (middle) for above complexes show no signs of aggregation and suggest a homogeneous solution of the respective complexes. The pair distribution plots (right) show the respective D_max values. e Cysteine induces dissociation of CtNfs1 from CtIsd11-ACP. The CtNfs1-CtIsd11-His₆-ACP complex was incubated with cysteine (Cys), serine (Ser) or dithiothreitol (DTT) under anaerobic conditions and bound to Ni-NTA resin. Proteins eluting from the column (unbound) and resin-bound material (after elution with imidazole) was analyzed by PAGE and Coomassie staining. f Equilibrium constants for human NFS1 interaction with ISCU2 were measured by microscale thermophoresis in the absence (left) or presence (right) of cysteine. In part f ISCU was used either aerobically or anaerobically (-O₂). The error bars indicate the SD of the sigmoidal fit (see Supplementary Fig. 5e for original data) of three biological replicates

Fig. 6

SAXS shapes of various C. thermophilum ISC complexes. a–c CtNfs1 (orange) was incubated with CtIsu1 (blue), CtFdx2 (red) or both in 1:1 or 1:1:1 ratios. The NFS1-ISCU crystal structure was fitted to the obtained density a. Ab initio fitting of FDX2 together with NFS1 into the density resulted in a distinct complex b. Both complexes were combined to be fit into the density obtained for the hetero-hexameric complex c. d, e, Upon addition of CtYfh1 (slate gray) to the CtNfs1-CtIsu1 or CtNfs1-CtFdx2 complexes in a 1:1:1 ratio, complex formation could be observed from the respective SAXS density. The NFS1-ISCU crystal structure was used for ab initio fitting of the positions of NFS1, ISCU, and FXN in the obtained density d. To fit NFS1, FDX2, and FXN ab initio into the SAXS density, results from b and d were combined e. f Mixing of CtNfs1-CtIsu1-CtFdx2-CtYfh1 yielded an octameric complex. Ab initio fitting into the SAXS densities made use of the above complexes. g Scattering curves with respective fits in gray (left), Kratky plots (middle), and pair distribution plots (right) as in Fig. 5. h Model of the entire ISC complex based on the crystal and SAXS data. The inset shows the active sites of NFS1, ISCU, FDX2, and FXN. PLP of NFS1 is shown in black, oxygen atoms of negatively charged residues on the iron-binding helix α1 of FXN as red balls, functionally important cysteine sulfurs of NFS1 and ISCU as yellow spheres, and the [2Fe–2S] cluster of FDX2 as spheres. i Diagram showing the conformational rearrangement of the NFS1 dimer upon binding of ISCU to a ‘standard’ cysteine desulfurase dimer conformation