Iron-sulfur cluster biogenesis in mammalian cells: New insights into the molecular mechanisms of cluster delivery

Abstract

Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i.e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein-protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Keywords: HSC20; HSPA9; ISCU; LYR motif protein; Mammalian Fe-S cluster assembly; SDHB.

Figure 1. Fe-S cluster biogenesis in mammalian cells: a general scheme of the main steps

(A) Assembly of a nascent Fe-S cluster upon the scaffold protein ISCU. A cysteine desulfurase NFS1 forms a dimer to which monomers of the primary scaffold ISCU bind at either end. ISD11 is a structural component of the core complex in eukaryotes, required for the activity of NFS1, which, aided by its cofactor pyridoxal phosphate, provides sulfur, removed from cysteine, for the nascent cluster. Frataxin (FXN) is part of the core complex, potentially binding in a pocket- like region between NFS1 and ISCU. The cluster assembles upon ISCU when iron is provided together with the reducing equivalents that allow achievement of the final electronic configuration of the cluster. (B) Cluster transfer to recipient apoproteins. A dedicated chaperone/cochaperone (HSPA9/HSC20) system facilitates cluster transfer from the primary scaffold ISCU to recipient apoproteins or to intermediate carriers, which then target specific recipients. The cochaperone HSC20 interacts with ISCU through a patch of hydrophobic amino acid residues in the C- terminus, and with the chaperone HSPA9 through the N-terminal domain (J-domain). The J-domain of HSC20 contains a conserved His (H), Pro (P), Asp (D) tripeptide essential for activation of the ATPase activity of HSPA9. The energy derived from activation of HSPA9 drives a conformational rearrangement in the substrate- binding domain of the chaperone (see text for further details), which is proposed to propagate a conformational change to its binding partner, ISCU (arrow 1). The conformational change is thought to facilitate release of the cluster from the main scaffold, and allow protected transfer directly or via intermediate carriers to specific subsets of Fe-S recipient proteins. A nucleotide exchange factor (NEF), known as Mge1 in yeast and SIL1 (or BAP) in humans exchanges ADP with ATP and completes the ATPase cycle of the chaperone (arrow 2).

Figure 2. 3D- structures of cochaperones dedicated to Fe-S cluster biogenesis in H. sapiens (HSC20, panel A), S. cerevisiae (Jac1, panel B), and E. coli (HscB, panel C)

Conserved amino acid residues which interact with the main scaffold ISCU on the surface of the C-terminal domains of the cochaperones dedicated to Fe-S cluster biogenesis in humans (A), yeast (B), and bacteria (C): in blue are the hydrophobic amino acids (Leu, Met, and Phe), and in green the polar residues (Tyr). The His (H), Pro (P), Asp (D) tripeptide in the N-terminal domains (J- domains) of the cochaperones is indicated by arrows and colored in yellow. Two CxxC modules, which coordinate zinc in the crystal structure of human HSC20, are shown in red.

Figure 3. 3D- structural comparison between the substrate- binding domains of bacterial HscA and human HSPA9, chaperones involved in Fe-S cluster biogenesis

Three- dimensional comparison of the crystal structures of the substratebinding domain of HscA (residues 390– 540, in cyan) in complex with a peptide containing the IscU recognition region (LPPVK in blue), and the region 439– 590 of human HSPA9 (in violet). Panels A and B. Ribbon representation (A) and equivalent surface representation (B) of the two overlapped structures of the substrate binding domains of HscA and HSPA9. (C) Primary sequences of the regions of the two HSP70s showed in (A) and (B).

Figure 4. Schematic representation of Fe-S cluster delivery to specific recipients mediated by binding of HSC20 to LYR motifs

Fe-S cluster incorporation into succinate dehydrogenase B (SDHB): in SDHB, two LYR motifs (IYR and LYR) engage the HSC20- HSPA9- ISCU complex, which assists incorporation of three Fe-S clusters within the final structure of complex II. An important aspect of the model is that the first motif may enter the mitochondrial matrix first, as the SDHB polypeptide translocates from the site of its synthesis in the cytosol, to its destination, the mitochondria. The accessibility of the first motif would not be yet affected by C- terminal sequences and secondary structure formation. Indeed, donation of a [Fe₂-S₂] cluster could determine secondary structure by driving folding around the Fe-S cluster, to which cysteines 93, 98, 101, and 113 bind with high affinity. SDHAF1 (LYRM8), an assembly factor for complex II and an annotated member of the LYR motif family, associates with SDHB through a non-LYR binding site that is not yet fully defined, but that resides between the two LYR motifs. In our working model, SDHAF1 binds to SDHB through a non-LYR portion of its sequence and utilizes its own LYR motif to position an ISCU-HSC20- HSPA9 complex near to Fe-S transfer complexes directly associated with the second LYR binding site of SDHB. The propensity of HSC20 to dimerize may allow two holo- ISCU molecules at neighboring binding sites to reorganize their adjacent [Fe₂-S₂] centers, thus coalescing into the [Fe₄-S₄] and [Fe₃-S₄] clusters of mature SDHB. Again, the primary peptide would be driven to fold around the newly incorporated Fe-S clusters, potentially explaining how some Fe-S centers can be deeply buried in proteins. The structures reported are relative to porcine succinate dehydrogenase complex (96% identical to human. PDB ID: 3SFD).