Structural/Functional Properties of Human NFU1, an Intermediate [4Fe-4S] Carrier in Human Mitochondrial Iron-Sulfur Cluster Biogenesis

Abstract

SUMMARY Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is associated with a fatal mitochondrial disease. We determined three-dimensional structures of the N-and C-terminal domains of human NFU1 by nuclear magnetic resonance spectroscopy and used these structures along with small-angle X-ray scattering (SAXS) data to derive structural models for full-length monomeric apo-NFU1, dimeric apo-NFU1 (an artifact of intermolecular disulfide bond formation), and holo-NFUI (the [4Fe-4S] cluster-containing form of the protein). Apo-NFU1 contains two cysteine residues in its C-terminal domain, and two apo-NFU1 subunits coordinate one [4Fe-4S] cluster to form a cluster-linked dimer. Holo-NFU1 consists of a complex of three of these dimers as shown by molecular weight estimates from SAXS and size-exclusion chromatography. The SAXS-derived structural model indicates that one N-terminal region from each of the three dimers forms a tripartite interface. The activity of the holo-NFU1 preparation was verified by demonstrating its ability to activate apo-aconitase.

Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)—Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway

“…Inhibition most likely arises from stabilization of the cluster on holo IscU [56] that slows its ability to transfer the cluster. In the case of transfer to human NFU1 in the presence of chaperones, we postulate that no transfer is observed, at least on the time scale considered, due to possible steric interaction of NFU1 with the chaperone proteins [5, 57] in conjunction with the stabilization of cluster on IscU.…”

“…S3B), in contrast to the native protein, which demonstrated slightly more tetramer than dimer. For both the native and the mutant, no clear additional peaks were observed in the AUC trace [1, 5]. The remaining percentages correspond to protein aggregation and crashing out.…”

“…This pattern is altered in the mutant protein with the mutation of a nearby glycine residue at position 208 to cysteine, which gives a CXCXXC motif. ( B ) Solution NMR structure of human C-terminal domain of the NFU1 protein (PDB ID: 2M5O) with the cluster binding cysteines shown in yellow [5]. The glycine at position 208 in the full length protein (58 above), which is mutated to a cysteine in MMDS1, is colored red.…”

“…The thermodynamic stability of NFU1 has been studied in depth to reveal that the isolated C-terminal domain of human NFU1 exhibits characteristics of a molten globular state [4, 30], while the isolated N-terminal domain demonstrates a highly ordered structure. However, when the two domains come together the overall protein maintains a relatively well-folded structure, suggesting a requirement for the unique N-terminal domain of the human protein to provide structural stabilization as well as serving a potential functional role [4, 30] that may include a protein oligomerization surface or a binding site for chaperones, such as Hsc20 [5, 37]. Structure-function characteristics of human NFU1 remain unclear, because this protein has been implicated in a variety of cellular roles.…”

“…NFU1 is also known as the HIRA-interacting protein, where HIRA is the histone cell cycle regulation homologue A [38]. Furthermore, it exhibits thioredoxin-like activity in the apo form [29, 30], assembles and transfers a [2Fe-2S] cluster [1], assembles a [4Fe-4S] cluster [31, 39], and may transfer cluster to apo aconitase [5]. Homologs of human NFU1 have been implicated in similar roles, with [2Fe-2S] [33, 34, 36, 40] and [4Fe-4S] [32, 33, 35, 41] cluster transfer, suggesting that both functionalities are possible and potentially physiologically relevant.…”