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. 2021 Apr 14;13(4):mfab010.
doi: 10.1093/mtomcs/mfab010.

Biochemical impact of a disease-causing Ile67Asn substitution on BOLA3 protein

Sambuddha Sen  1 , Zechariah Thompson  1 , Christine Wachnowsky  1   2 , Sean Cleary  3   4 , Sophie R Harvey  3   4 , J A Cowan  1   2
Affiliations

Biochemical impact of a disease-causing Ile67Asn substitution on BOLA3 protein

Sambuddha Sen et al. Metallomics. .

Abstract

Iron-sulfur (Fe-S) cluster biosynthesis involves the action of a variety of functionally distinct proteins, most of which are evolutionarily conserved. Mutations in these Fe-S scaffold and trafficking proteins can cause diseases such as multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalopathy. Herein, we investigate the effect of Ile67Asn substitution in the BOLA3 protein that results in the MMDS2 phenotype. Although the exact functional role of BOLA3 in Fe-S cluster biosynthesis is not known, the [2Fe-2S]-bridged complex of BOLA3 with GLRX5, another Fe-S protein, has been proposed as a viable intermediary cluster carrier to downstream targets. Our investigations reveal that the Ile67Asn substitution impairs the ability of BOLA3 to bind its physiological partner GLRX5, resulting in a failure to form the [2Fe-2S]-bridged complex. Although no drastic structural change in BOLA3 arises from the substitution, as evidenced by wild-type and mutant BOLA3 1H-15N HSQC and ion mobility native mass spectrometry experiments, this substitution appears to influence cluster reconstitution on downstream proteins leading to the disease phenotype. By contrast, substituted derivatives of the holo homodimeric form of BOLA3 are formed and remain active toward cluster exchange.

Keywords: BOLA; GLRX5; MMDS; glutaredoxin; iron-sulfur cluster.

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Figures

Graphical Abstract
Graphical Abstract
Ile67Asn substitution of human BOLA3 results in the disease phenotype of multiple mitochondrial dysfunctions syndrome-2. Substitution introduces no drastic structural change but impacts the ability of BOLA3 to interact with partner GLRX5 and impairs formation of a [2Fe-2S]-bridged complex of BOLA3 with GLRX5 and subsequent downstream trafficking. The homodimeric form of the BOLA3 derivative can be formed and is active in cluster exchange.
Fig. 1
Fig. 1
Solution structure of apo BOLA3 (PDB-2NCL) with Ile67 circled. His96 and Cys59 are cluster-coordinating residues.
Fig. 2
Fig. 2
Overlay of 15N HSQC spectra of native (red), Ile67Asn (black), Ile67Ala (magenta), Ile67Val (purple), and Ile67Arg (orange) derivatives of BOLA3.
Fig. 3
Fig. 3
ITC plots for (a) Apo Ile67Asn BOLA3 titrated to apo GLRX5 that show no meaningful binding and (b) Apo Ile67Val BOLA3 titrated to apo GLRX5 (N = 1.16 ± 0.03, K = 1.5 × 105 ± 2 × 104 M−1, ΔH = −3294 ± 121 J mol−1, ΔS = 13 J mol−1 K−1). The experiments were performed in the presence of 5 mM GSH in 50 mM HEPES, 100 mM NaCl, pH 7.5. One millimolar BOLA3 was titrated into 50 µM GLRX5 in 10 µl aliquots over a period of 24 s with a 5 min interval at 30°C.
Fig. 4
Fig. 4
Ion mobility chromatograms of the 5+ charge state of BOLA3 WT (a) and the 167N mutant (b). Chromatograms demonstrate that no structural change is seen between wild-type and mutant species.
Fig. 5
Fig. 5
Comparison of the CD spectra of reconstituted holo GLRX5-BOLA3 derivatives. The extinction coefficients are based on the cluster concentrations.
Fig. 6
Fig. 6
Time course measurements for cluster transfer from (a) holo Ile67Asn to apo FDX1 and (b) holo Ile67Val to apo FDX1 under anaerobic conditions. The CD signal intensity was monitored at room temperature for a 1 : 1 donor:acceptor cluster stoichiometry in the presence of 3 mM GSH. CD spectra in the 300–600 nm region were recorded every 2 min.
Fig. 7
Fig. 7
Time course measurements for the cluster transfer experiment from holo ISCU to (a) apo Ile67Asn BOLA3–GLRX5 and (b) apo Ile67Val BOLA3–GLRX5 under anaerobic conditions. The CD response was monitored at room temperature for a 1 : 1 donor:acceptor cluster stoichiometry in the presence of 3 mM GSH. CD spectra were collected in the 300–600 nm range every 2 min.
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