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. 2023 Jul 21;24(14):11734.
doi: 10.3390/ijms241411734.

Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3

Beatrice Bargagna  1   2 , Lucia Banci  1   2   3 , Francesca Camponeschi  1   2   3
Affiliations

Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3

Beatrice Bargagna et al. Int J Mol Sci. .

Abstract

Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation.

Keywords: BOLA3; GLRX5; ISC machinery; MMDS2; iron-sulfur cluster biogenesis; iron-sulfur clusters; mitochondria; multiple mitochondrial dysfunctions syndrome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
His96Arg mutation involves a highly conserved residue of BOLA3. (A) Superimposition of the solution structure of WT BOLA3 (green, PDB: 2NCL, [19]) and of the putative structure of H96R BOLA3 (violet, see Section 4). The histidine at position 96 in the wild-type protein, which is mutated to arginine in MMDS2, is colored blue on the ribbon structure of WT BOLA3. The pathogenic Arg96 mutation is shown in red on the ribbon structure of H96R BOLA3. (B) Multiple sequence alignment of BOLA3 with BolA homologues in several model organisms using Clustal Omega [26]. h = Homo sapiens; mm = Mus musculus; dr = Danio rerio; sc = Saccharomyces cerevisiae. Identical residues are colored in red, and similar residues are shown in green. Red and yellow asterisks in (A,B) indicate the location of conserved Cys59 and His96 [2Fe-2S] cluster ligands in yeast human BOLA3, respectively.
Figure 2
Figure 2
The His96Arg mutation does not affect the overall fold of BOLA3 protein. (A) Superimposition of the 1H-15N HSQC spectra of 15N-labeled H96R BOLA3 (magenta) and 15N-labeled WT BOLA3 (black), acquired at 500 MHz at 298 K; (B) mapping of the chemical shift changes (shown in cyan) due to the His96Arg mutation on the model structure of H96R BOLA3. Far-UV CD spectra (C) and analytical SEC (D) of WT BOLA3 (black) and H96R BOLA3 (magenta). The model shown in panel 2B was rendered with Pymol 2.3.5.
Figure 3
Figure 3
The His96Arg mutation does not impact the formation of the apo H96R BOLA3-GLRX5 complex. (A) Superimposition of the 1H-15N HSQC spectra of 15N-labeled H96R BOLA3 in the absence (black) and the presence of 0.5 eq. (blue) and 1.0 eq. (red) of apo unlabeled GLRX5, acquired at 500 MHz and 298 K; (B) chemical shift perturbations of 15N-labeled H96R BOLA3 before and after the addition of 1.0 eq. of unlabeled GLRX5, calculated as (((ΔH)2 + (ΔN/5)2)/2)1/2. The indicated threshold value (obtained by averaging CSP values plus 1σ) were used to define meaningful chemical shift differences. (C) Mapping of the meaningful chemical shift changes due to the interaction with GLRX5 on the model structure of H96R BOLA3. Residues experiencing chemical shift variation are shown in orange, while residues experiencing line-broadening are shown in magenta in both (B,C) panels. The model shown in panel 3C was rendered with Pymol 2.3.5.
Figure 4
Figure 4
Identification of the interacting surface of apo GLRX5 with H96R BOLA3. (A) Superimposition of the 1H-15N HSQC spectra of apo 15N-labeled GLRX5 in the absence (black) and in the presence of 1.0 eq. of unlabeled H96R BOLA3 (cyan), acquired at 500 MHz and 298 K; (B) chemical shift perturbations of apo 15N-labeled GLRX5 before and after the addition of unlabeled H96R BOLA3, calculated as (((ΔH)2 + (ΔN/5)2)/2)1/2. The indicated threshold value was obtained by averaging CSP values plus 1σ and was used to define meaningful chemical shift differences. (C) Mapping of the meaningful chemical shift changes due to the interaction with H96R BOLA3 on the solution structure of apo GLRX5 (PDB: 2MMZ, [27]). Residues experiencing chemical shift variation are shown in orange, while residues experiencing line-broadening are shown in magenta in both (B,C) panels. (D) Analytical SEC of isolated apo GLRX5 (black line), isolated apo H96R BOLA3 (orange line) and a 1:1 mixture of apo GLRX5 and apo H96R BOLA3 (cyan line). The structure shown in panel 3C was rendered with Pymol 2.3.5.
Figure 5
Figure 5
H96R BOLA3 interacts with [2Fe-2S]-GLRX52. (A) Chemical shift perturbations of apo 15N-labeled H96R BOLA3 before and after the addition of 0.5 eq. of unlabeled [2Fe-2S]-GLRX52, calculated as (((ΔH)2 + (ΔN/5)2)/2)1/2. The indicated threshold value was obtained by averaging CSP values plus 1σ and was used to define meaningful chemical shift differences. (B) Mapping of the meaningful chemical shift changes on the model structure of apo H96R BOLA3. Residues experiencing chemical shift variation are shown in orange, while residues experiencing line-broadening are shown in magenta in both (B,C) panels. (C) Chemical shift perturbations of apo 15N-labeled [2Fe-2S]-GLRX52 before and after the addition of 2.0 eq. of unlabeled H96R BOLA3, calculated as (((ΔH)2 + (ΔN/5)2)/2)1/2. The indicated threshold value (obtained by averaging CSP values plus 1σ) was used to define meaningful chemical shift differences. (D) Mapping of the meaningful chemical shift changes on the solution structure of GSH-bound GLRX5 (PDB: 2MMZ, [27]). Residues experiencing chemical shift variation are shown in orange, while residues experiencing line-broadening are shown in magenta. (E) Analytical SEC of [2Fe-2S]-GLRX52 before (blue line) and after (red line) the addition of 2.0 eq. of apo H96R BOLA3. The analytical SEC of isolated apo GLRX5 (black line), isolated apo H96R BOLA3 (orange line) and of a 1:1 GLRX5:H96R BOLA3 mixture (cyan line) are reported for reference. The model shown in panel 3B and 3D was rendered with Pymol 2.3.5.
Figure 6
Figure 6
The [2Fe-2S]-GLRX52 homo-dimeric complex is converted into the H96R BOLA3-[2Fe-2S]-GLRX5 heterodimeric complex. UV-Vis (A), CD (B), 1D 1H paramagnetic NMR spectra (C) of [2Fe-2S]2+-GLRX52 before (blue) and after (red) the addition of 2.0 eq. of H96R BOLA3. (D) EPR spectra of dithionite reduced [2Fe-2S]+-GLRX52 before (blue) and after (red) the addition of 2.0 eq. of H96R BOLA3, recorded at 45 K and 0.5 mW.
Figure 7
Figure 7
His96Arg mutation abolish the ability of the BOLA3-[2Fe-2S]-GLRX5 complex to assemble a [4Fe-4S]2+ cluster on NFU1. [4Fe-4S]2+-NFU1 hyperfine-shifted signals regions of the 1D 1H NMR spectra of 2:1 mixtures of WT BOLA3-[2Fe-2S]2+-GLRX5 (a) or H96R BOLA3-[2Fe-2S]2+-GLRX5 hetero-complex (b) and apo NFU1.
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Supplementary concepts

Grants and funding

The authors acknowledge the support by the Italian Ministry for University and Research (FOE funding) to the CERM/CIRMMP Italian Centre of Instruct-ERIC, a ESFRI Landmark. FC and LB acknowledge the project “Potentiating the Italian Capacity for Structural Biology Services in Instruct Eric (ITACA.SB)” (Project n° IR0000009) within the call MUR 3264/2021 PNRR M4/C2/L3.1.1, funded by the European Union NextGenerationEU.