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Review
. 2022 Jul 21;12(7):1009.
doi: 10.3390/biom12071009.

Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins

Francesca Camponeschi  1   2 , Simone Ciofi-Baffoni  1   2   3 , Vito Calderone  1   2   3 , Lucia Banci  1   2   3
Affiliations
Review

Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins

Francesca Camponeschi et al. Biomolecules. .

Abstract

The importance of mitochondria in mammalian cells is widely known. Several biochemical reactions and pathways take place within mitochondria: among them, there are those involving the biogenesis of the iron-sulfur (Fe-S) clusters. The latter are evolutionarily conserved, ubiquitous inorganic cofactors, performing a variety of functions, such as electron transport, enzymatic catalysis, DNA maintenance, and gene expression regulation. The synthesis and distribution of Fe-S clusters are strictly controlled cellular processes that involve several mitochondrial proteins that specifically interact each other to form a complex machinery (Iron Sulfur Cluster assembly machinery, ISC machinery hereafter). This machinery ensures the correct assembly of both [2Fe-2S] and [4Fe-4S] clusters and their insertion in the mitochondrial target proteins. The present review provides a structural and molecular overview of the rare diseases associated with the genes encoding for the accessory proteins of the ISC machinery (i.e., GLRX5, ISCA1, ISCA2, IBA57, FDX2, BOLA3, IND1 and NFU1) involved in the assembly and insertion of [4Fe-4S] clusters in mitochondrial proteins. The disease-related missense mutations were mapped on the 3D structures of these accessory proteins or of their protein complexes, and the possible impact that these mutations have on their specific activity/function in the frame of the mitochondrial [4Fe-4S] protein biogenesis is described.

Keywords: iron–sulfur cluster; mitochondrial proteins; multiple mitochondrial dysfunction syndromes; rare diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The three steps of the mitochondrial iron–sulfur cluster assembly machinery required to mature mitochondrial [4Fe-4S] target proteins. In the first step, a [2Fe-2S] cluster is assembled de novo on the scaffold protein ISCU2. The biosynthesis involves six additional ISC proteins including the cysteine desulfurase complex NFS1-ISD11-ACP1 as a sulfur donor, frataxin (FXN), and the electron (e−) transfer chain from NADPH via ferredoxin reductase (FDXR) to ferredoxin FDX2. Only one half of the symmetric core ISC complex is depicted. In the second step, a dedicated chaperone system (HSPA9, HSC20, and GRPLE1/2) facilitates the transfer of the [2Fe-2S] cluster from the ISCU2 scaffold to the monothiol glutaredoxin GLRX5, which binds the cluster in glutathione (GS)-dependent fashion. A secondary route (indicated by dashed arrows) involves BOLA3, which forms an apo complex with GLRX5 able to receive a [2Fe-2S] cluster from ISCU2 to form the homodimeric [2Fe-2S] GLRX5 complex; or, alternatively, the latter complex can be formed via the interaction of BOLA3 with the homodimeric [2Fe-2S] GLRX5 complex. The third step involves [4Fe-4S] cluster synthesis and apoprotein insertion. GLRX5 delivers its [2Fe-2S] cluster to three late-acting ISC proteins (ISCA1, ISCA2, and IBA57) for [4Fe-4S] cluster biosynthesis, which additionally requires the ferredoxin FDX2 electron transfer chain. Subsequently, the newly formed [4Fe-4S] cluster is delivered to recipient apoproteins by dedicated Fe-S-targeting proteins (e.g., NFU1, IND1 directly binding the [4Fe-4S] cluster) via parallel pathways. The major role of BOLA3 protein is in lipoyl synthase (LIAS) maturation. BOLA3 might be involved in such function via an alternative pathway, which consists on the [2Fe-2S] cluster donation from [2Fe-2S] GLRX5-BOLA3 complex to apo NFU1 to assemble a [4Fe-4S] cluster on NFU1 thanks to the delivery of two electrons, whose physiological source needs, however, to be identified.
Figure 2
Figure 2
Pathogenic missense mutations mapped on the crystallographic structure of human GLRX5. (A) The backbone of the residues involved in pathogenic missense mutations of GLRX5 is shown in red on the ribbon structure of homodimeric [2Fe-2S] GLRX5 (PDB ID 2WUL). The sidechain of the conserved cluster coordinating Cys67 residue is shown in yellow. The iron and sulfur atoms of the [2Fe-2S] cluster are in red and yellow spheres, respectively; and glutathione (GS) ligand is shown in blue. (B) The electrostatic interaction established between the sidechain of Lys101 (in magenta) and the carboxylate group of the glutathione cluster ligand, shown in ball and stick mode, is shown as a dotted line. (C) The sidechains of the hydrophobic residues (in blue) interacting with the sidechain of Met128 (in magenta) are shown.
Figure 3
Figure 3
Pathogenic missense mutations mapped on the solution structures of the isolated N- and C-terminal domains of human NFU1 and on a structural docking model describing their interaction. (A–C) The backbone of the residues involved in pathogenic missense mutations in the C-terminal (A) and N-terminal (C) domains of NFU1 is shown in red on the ribbon structures of these domains (PDB ID 2LTM and 2M5O, respectively). The sidechains of the two conserved cluster coordinating Cys residues on the C-terminal domain of NFU1 are shown in yellow. (B) The electrostatic interactions established by sidechain of Arg182 (magenta) and the sidechains of the surrounding Asp residues (in orange) are shown as dotted black lines. (D) Docking structural model of the interaction between the N-terminal and C-terminal domains of NFU1 is shown. Ala100 of the N-terminal domain (in red) is close to Arg101 and Arg 105 (both in blue), which are in turn involved in electrostatic interactions with Glu and Asp residues of the C-terminal domain (all in orange). Interacting hydrophobic patches between the two domains (Van der Waals surfaces are in blue and red for the C- and N-domains, respectively), which involve both aromatic and aliphatic residues (sidechains are in green) and are close in space to Ala100 (sidechain is in red), are also shown.
Figure 4
Figure 4
Pathogenic missense mutations mapped on the structures of human BOLA3 and [2Fe-2S] GLRX5-BOLA3 complex. (A) The backbone of the residues involved in pathogenic missense mutations of BOLA3 is colored in red on the solution structure of apo BOLA3 (PDB ID 2NCL), and the sidechains of the conserved cluster coordinating Cys59 and His96 residues are shown in yellow and cyan, respectively. The sidechains of Arg99 and Ile67 residues involved in pathogenic missense mutations are shown in magenta. The sidechains of the hydrophobic residues interacting with Ile67 are shown in blue. (B) The ribbon diagram of the structural model of [2Fe-2S] GLRX5-BOLA3 available from a data-driven docking approach [45] is shown. GLRX5 is in grey and BOLA3 is in green. The sidechains of the two Cys ligands and of the His ligand are shown as yellow and cyan stick, respectively; the iron and sulfur atoms of the [2Fe-2S] cluster are in red and yellow spheres, respectively; and GS cluster ligand is shown in blue. The sidechains of the residues involved in pathogenic missense mutations at the interface between BOLA3 and GLRX5 are shown in magenta. Electrostatic interactions involving these residues are shown as dotted lines.
Figure 5
Figure 5
Pathogenic missense mutations mapped on the crystallographic structure of human IBA57. (A) The backbones of the buried and solvent-accessible residues involved in pathogenic missense mutations of IBA57 are colored in red and blue, respectively, on the ribbon structure of IBA57 (PDB ID 6QE4). The sidechain of the conserved cluster coordinating Cys259 is shown in yellow. (B) The sidechains of the residues involved in pathogenic missense mutations are shown in magenta. Hydrogen bond interaction is shown as a dotted black line. The positively charged, negatively charged and hydrophobic residues interacting with the residues shown in magenta are shown in cyan, red and blue, respectively.
Figure 6
Figure 6
Effects of the pathogenic missense mutations on the heterodimeric [2Fe-2S] ISCA2-IBA57 complex. Ribbon diagrams of the two structural models of [2Fe-2S] ISCA2-IBA57 available from a data-driven docking approach [29] are shown in panels (A,B). IBA57 is in cyan and ISCA2 is in green. The sidechains of the four Cys ligands are shown as yellow stick, the iron and sulfur atoms of the [2Fe-2S] cluster are in red and yellow spheres, respectively. The side chains (involved in intermolecular electrostatic interactions) of Asp111 (red, ISCA2) and Arg146 (blue, IBA57) and of Glu126 (red, ISCA2) and Arg268 (blue, IBA57) in (A), and of Glu75 (red, ISCA2) and Arg268 (blue, IBA57) and Glu126 (red, ISCA2) and Arg146 (blue, IBA57) in (B) are shown as sticks. The backbone of Gly77 (magenta, ISCA2) and the side chain of Ile261 (green IBA57) are also shown as sticks on both models.
Figure 7
Figure 7
Pathogenic missense mutations mapped on the structural model of human ISCA2 obtained by AlphaFold2. (A) The backbone of the residues involved in pathogenic missense mutations of ISCA2 is colored in red on the ribbon structure of monomeric apo ISCA2, and the sidechains of the three conserved cluster coordinating Cys residues are shown in yellow. (B) The electrostatic interaction established between the negatively charged carboxylate of the sidechain of Glu100 (in red) and the positively charged guanidinium group of the sidechain of Arg105 (in magenta) is shown.
Figure 8
Figure 8
Pathogenic missense mutations mapped on the structural models of human ISCA1 and ISCA1-ISCA2 complex. (A) The backbone of the residues involved in pathogenic missense mutations of ISCA1 is colored in red on the structural model of monomeric apo ISCA1 obtained by AlphaFold2, and the sidechains of the three conserved cluster coordinating Cys residues are shown in yellow. (B) The hydrogen bond formed between the side-chains of Glu87 (in magenta) and Lys49 (in blue) is shown as a white dotted line. The sidechains of the positively charged Lys88 and Lys89 residues spatially close to Glu87 are also shown. (C) The sidechain of Tyr101 is shown in red on the hetero-dimeric apo ISCA1 (green)-ISCA2 (cyan) complex. The interacting hydrophobic patches formed by the sidechains of Tyr101 (in red), Phe110, Leu38 and Ala31 (all in blue) on ISCA1 protomer and by the sidechains of Leu127 and Ile138 (both in blue) on ISCA2 protomer are shown as Van der Waals surfaces in blue and cyan, respectively.
Figure 9
Figure 9
Pathogenic missense mutations mapped on the structural model of human IND1 obtained by AlphaFold2. (A) The backbone of the residues involved in pathogenic missense mutations of IND1 are colored in red on the ribbon structure of IND1, and the sidechains of the two conserved cluster coordinating Cys residues are shown in yellow. (B) The sidechains of Asp105, Leu104 and Val182 are shown in red. The Asp and Lys residues involved in the electrostatic interactions with Asp105 are shown in orange and cyan, respectively. The sidechains of the hydrophobic residues close in space to Leu104 and Val182 are shown in blue.
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This research was funded by Instruct-ERIC, a Landmark ESFRI project, and specifically the CERM/CIRMMP Italian Instruct Centre, and by Ministero dell’Istruzione.

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