Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 2 Caused by CYS59TYR BOLA3 Mutation.

Multiple mitochondrial dysfunctions syndrome (MMDS) is a rare neurodegenerative disorder associated with mutations in genes with a vital role in the biogenesis of mitochondrial [4Fe-4S] proteins. Mutations in one of these genes encoding for BOLA3 protein lead to MMDS type 2 (MMDS2). Recently, a novel phenotype for MMDS2 with complete clinical recovery was observed in a patient containing a novel variant (c.176G > A, p.Cys59Tyr) in compound heterozygosity. In this work, we aimed to rationalize this unique phenotype observed in MMDS2. To do so, we first investigated the structural impact of the Cys59Tyr mutation on BOLA3 by NMR, and then we analyzed how the mutation affects both the formation of a hetero-complex between BOLA3 and its protein partner GLRX5 and the iron-sulfur cluster-binding properties of the hetero-complex by various spectroscopic techniques and by experimentally driven molecular docking. We show that (1) the mutation structurally perturbed the iron-sulfur cluster-binding region of BOLA3, but without abolishing [2Fe-2S]2+ cluster-binding on the hetero-complex; (2) tyrosine 59 did not replace cysteine 59 as iron-sulfur cluster ligand; and (3) the mutation promoted the formation of an aberrant apo C59Y BOLA3-GLRX5 complex. All these aspects allowed us to rationalize the unique phenotype observed in MMDS2 caused by Cys59Tyr mutation.

IBA57 Recruits ISCA2 to Form a [2Fe-2S] Cluster-Mediated Complex.

The maturation of mitochondrial iron-sulfur proteins requires a complex protein machinery. Human IBA57 protein was proposed to act in a late phase of this machinery, along with GLRX5, ISCA1, and ISCA2. However, a molecular picture on how these proteins cooperate is not defined yet. We show here that IBA57 forms a heterodimeric complex with ISCA2 by bridging a [2Fe-2S] cluster, that [2Fe-2S] cluster binding is absolutely required to promote the complex formation, and that the cysteine of the conserved motif characterizing IBA57 protein family and the three conserved cysteines of the ISCA protein family act as cluster ligands. The [2Fe-2S] heterodimeric complex is the final product when IBA57 is either exposed to [2Fe-2S] ISCA2 or in the presence of [2Fe-2S] GLRX5 and apo ISCA2. We also find that the [2Fe-2S] ISCA2-IBA57 complex is resistant to highly oxidative environments and is capable of reactivating apo aconitase in vitro. Collectively, our data delinate a [2Fe-2S] cluster transfer pathway involving three partner proteins of the mitochondrial ISC machinery, that is, GLRX5, ISCA2 and IBA57, which leads to the formation of a [2Fe-2S] ISCA2-IBA57 complex.

Structural insights into the molecular function of human [2Fe-2S] BOLA1-GRX5 and [2Fe-2S] BOLA3-GRX5 complexes.

Members of the monothiol glutaredoxin family and members of the BolA-like protein family have recently emerged as specific interacting partners involved in iron-sulfur protein maturation and redox regulation pathways. It is known that human mitochondrial BOLA1 and BOLA3 form [2Fe-2S] cluster-bridged dimeric heterocomplexes with the monothiol glutaredoxin GRX5. The structure and cluster coordination of the two [2Fe-2S] heterocomplexes as well as their molecular function are, however, not defined yet. Experimentally-driven structural models of the two [2Fe-2S] cluster-bridged dimeric heterocomplexes, the relative stability of the two complexes and the redox properties of the [2Fe-2S] cluster bound to these complexes are here presented on the basis of UV/vis, CD, EPR and NMR spectroscopies and computational protein-protein docking. While the BOLA1-GRX5 complex coordinates a reduced, Rieske-type [2Fe-2S]1+ cluster, an oxidized, ferredoxin-like [2Fe-2S]2+ cluster is present in the BOLA3-GRX5 complex. The [2Fe-2S] BOLA1-GRX5 complex is preferentially formed over the [2Fe-2S] BOLA3-GRX5 complex, as a result of a higher cluster binding affinity. All these observed differences provide the first indications discriminating the molecular function of the two [2Fe-2S] heterocomplexes.

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation.

Strategies to prevent the recurrence of poliovirus (PV) after eradication may utilise non-infectious, recombinant virus-like particle (VLP) vaccines. Despite clear advantages over inactivated or attenuated virus vaccines, instability of VLPs can compromise their immunogenicity. Glutathione (GSH), an important cellular reducing agent, is a crucial co-factor for the morphogenesis of enteroviruses, including PV. We report cryo-EM structures of GSH bound to PV serotype 3 VLPs showing that it can enhance particle stability. GSH binds the positively charged pocket at the interprotomer interface shown recently to bind GSH in enterovirus F3 and putative antiviral benzene sulphonamide compounds in other enteroviruses. We show, using high-resolution cryo-EM, the binding of a benzene sulphonamide compound with a PV serotype 2 VLP, consistent with antiviral activity through over-stabilizing the interprotomer pocket, preventing the capsid rearrangements necessary for viral infection. Collectively, these results suggest GSH or an analogous tight-binding antiviral offers the potential for stabilizing VLP vaccines.

ISCA1 Orchestrates ISCA2 and NFU1 in the Maturation of Human Mitochondrial [4Fe-4S] Proteins.

The late-acting steps of the pathway responsible for the maturation of mitochondrial [4Fe-4S] proteins are still elusive. Three proteins ISCA1, ISCA2 and NFU1 were shown to be implicated in the assembly of [4Fe-4S] clusters and their transfer into mitochondrial apo proteins. We present here a NMR-based study showing a detailed molecular model of the succession of events performed in a coordinated manner by ISCA1, ISCA2 and NFU1 to make [4Fe-4S] clusters available to mitochondrial apo proteins. We show that ISCA1 is the key player of the [4Fe-4S] protein maturation process because of its ability to interact with both NFU1 and ISCA2, which, instead do not interact each other. ISCA1 works as the promoter of the interaction between ISCA2 and NFU1 being able to determine the formation of a transient ISCA1-ISCA2-NFU1 ternary complex. We also show that ISCA1, thanks to its specific interaction with the C-terminal cluster-binding domain of NFU1, drives [4Fe-4S] cluster transfer from the site where the cluster is assembled on the ISCA1-ISCA2 complex to a cluster binding site formed by ISCA1 and NFU1 in the ternary ISCA1-ISCA2-NFU1 complex. Such mechanism guarantees that the [4Fe-4S] cluster can be safely moved from where it is assembled on the ISCA1-ISCA2 complex to NFU1, thereby resulting the [4Fe-4S] cluster available for the mitochondrial apo proteins specifically requiring NFU1 for their maturation.

A pathway for assembling [4Fe-4S]2+ clusters in mitochondrial iron-sulfur protein biogenesis.

During its late steps, the mitochondrial iron-sulfur cluster (ISC) assembly machinery leads to the formation of [4Fe-4S] clusters. In vivo studies revealed that several proteins are implicated in the biosynthesis and trafficking of [4Fe-4S] clusters in mitochondria. However, they do not provide a clear picture into how these proteins cooperate. Here, we showed that three late-acting components of the mitochondrial ISC assembly machinery (GLRX5, BOLA3, and NFU1) are part of a ISC assembly pathway leading to the synthesis of a [4Fe-4S]2+ cluster on NFU1. We showed that the [2Fe-2S]2+ GLRX5-BOLA3 complex transfers its cluster to monomeric apo NFU1 to form, in the presence of a reductant, a [4Fe-4S]2+ cluster bound to dimeric NFU1. The cluster formation on NFU1 does not occur with [2Fe-2S]2+ GLRX5, and thus, the [4Fe-4S] cluster assembly pathway is activated only in the presence of BOLA3. These results define NFU1 as an 'assembler' of [4Fe-4S] clusters, that is, a protein able of converting two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. Finally, we found that the [4Fe-4S]2+ cluster bound to NFU1 has a coordination site which is easily accessible to sulfur-containing ligands, as is typically observed in metallochaperones. This finding supports a role for NFU1 in promoting rapid and controlled cluster-exchange reaction.

In-house high-energy-remote SAD phasing using the magic triangle: how to tackle the P1 low symmetry using multiple orientations of the same crystal of human IBA57 to increase the multiplicity.

This article describes the approach used to solve the structure of human IBA57 in-house by 5-amino-2,4,6-triiodoisophthalic acid (I3C) high-energy-remote single-wavelength anomalous dispersion (SAD) phasing. Multiple orientations of the same triclinic crystal were exploited to acquire sufficient real data multiplicity for phasing. How the collection of an in-house native data set and its joint use with the I3C derivative through a SIRAS approach decreases the data multiplicity needed by almost 50% is described. Furthermore, it is illustrated that there is a clear data-multiplicity threshold value for success and failure in phasing, and how adding further data does not significantly affect substructure solution and model building. To our knowledge, this is the only structure present in the PDB that has been solved in-house by remote SAD phasing in space group P1 using only one crystal. All of the raw data used, derived from the different orientations, have been uploaded to Zenodo in order to enable software developers to improve methods for data processing and structure solution, and for educational purposes.

Structural properties of [2Fe-2S] ISCA2-IBA57: a complex of the mitochondrial iron-sulfur cluster assembly machinery.

In mitochondria, a complex protein machinery is devoted to the maturation of iron-sulfur cluster proteins. Structural information on the last steps of the machinery, which involve ISCA1, ISCA2 and IBA57 proteins, needs to be acquired in order to define how these proteins cooperate each other. We report here the use of an integrative approach, utilizing information from small-angle X-ray scattering (SAXS) and bioinformatics-driven docking prediction, to determine a low-resolution structural model of the human mitochondrial [2Fe-2S]2+ ISCA2-IBA57 complex. In the applied experimental conditions, all the data converge to a structural organization of dimer of dimers for the [2Fe-2S]2+ ISCA2-IBA57 complex with ISCA2 providing the homodimerization core interface. The [2Fe-2S] cluster is out of the ISCA2 core while being shared with IBA57 in the dimer. The specific interaction pattern identified from the dimeric [2Fe-2S]2+ ISCA2-IBA57 structural model allowed us to define the molecular grounds of the pathogenic Arg146Trp mutation of IBA57. This finding suggests that the dimeric [2Fe-2S] ISCA2-IBA57 hetero-complex is a physiologically relevant species playing a role in mitochondrial [4Fe-4S] protein biogenesis.

Protein networks in the maturation of human iron-sulfur proteins.

The biogenesis of iron-sulfur (Fe-S) proteins in humans is a multistage process occurring in different cellular compartments. The mitochondrial iron-sulfur cluster (ISC) assembly machinery composed of at least 17 proteins assembles mitochondrial Fe-S proteins. A cytosolic iron-sulfur assembly (CIA) machinery composed of at least 13 proteins has been more recently identified and shown to be responsible for the Fe-S cluster incorporation into cytosolic and nuclear Fe-S proteins. Cytosolic and nuclear Fe-S protein maturation requires not only the CIA machinery, but also the components of the mitochondrial ISC assembly machinery. An ISC export machinery, composed of a protein transporter located in the mitochondrial inner membrane, has been proposed to act in mediating the export process of a still unknown component that is required for the CIA machinery. Several functional and molecular aspects of the protein networks operative in the three machineries are still largely obscure. This Review focuses on the Fe-S protein maturation processes in humans with the specific aim of providing a molecular picture of the currently known protein-protein interaction networks. The human ISC and CIA machineries are presented, and the ISC export machinery is discussed with respect to possible molecules being the substrates of the mitochondrial protein transporter.

Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins.

Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3.