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. 2017 Nov;284(22):3838-3848.
doi: 10.1111/febs.14271. Epub 2017 Oct 12.

Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1

Nathaniel A Wesley  1 , Christine Wachnowsky  1   2 , Insiya Fidai  1   3 , J A Cowan  1   2   3
Affiliations

Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1

Nathaniel A Wesley et al. FEBS J. 2017 Nov.

Abstract

Iron-sulfur (Fe/S) cluster-containing proteins constitute one of the largest protein classes, with highly varied function. Consequently, the biosynthesis of Fe/S clusters is evolutionarily conserved and mutations in intermediate Fe/S cluster scaffold proteins can cause disease, including multiple mitochondrial dysfunctions syndrome (MMDS). Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (p.Gly189Arg) near the active site of NFU1, an Fe/S scaffold protein. In vitro investigation into the structure-function relationship of the Gly189Arg derivative, along with two other variants, reveals that substitution at position 189 triggers structural changes that increase flexibility, decrease stability, and alter the monomer-dimer equilibrium toward monomer, thereby impairing the ability of the Gly189X derivatives to receive an Fe/S cluster from physiologically relevant sources.

Keywords: NFU1; cluster exchange; iron-sulfur cluster; mitochondrial disease; protein stability.

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Figures

Figure 1
Figure 1
(A) Solution NMR structure of the C-terminal domain of the human NFU1 protein (PDB ID: 2M5O) with corresponding cluster ligating cysteines in yellow and the glycine at position 189 in red. Although only the C-terminal domain is shown, numbering corresponds to the positions in the full length protein. (B) Sequence chain view to illustrate the residues near the glycine at position 189. The coloring scheme matches part (A). The glycine at position 208 that is also implicated in MMDS1 is colored orange.
Figure 2
Figure 2
VTCD trace for the melting of (A) 10 μM Gly189Arg, (B) Gly189Ala and (C) Gly189Lys human NFU1 in 40 mM phosphate, pH 7.4. Data were fit to a two-phase model (Eq. 1) to obtain Tm and ΔHV, which are shown in Table 2. CD units of ellipticity (mdeg) were used directly without conversion to molar ellipticity, because the van’t Hoff enthalpies are independent of such a factor [25].
Figure 3
Figure 3
Differential scanning calorimetry profiles for (A) 0.2 mM Gly189Arg, (B) 0.2 mM Gly189Ala, and (C) 0.15 mM Gly189Lys human NFU1. The protein was in 50 mM HEPES, 100 mM NaCl, and pH 7.4. The data were fit using Origin 7.0 to obtain the Tm and ΔHV values listed in Tables 3 and 4.
Figure 4
Figure 4
AUC profile for (A) apo Gly189Arg, (B) Gly189Ala, and (C) Gly189Lys NFU1. Apo protein was sedimented in the absence of TCEP (black) and in the presence of 1 mM TCEP (red). Sedimentation was monitored at 280 nm. The AUC results were fit to the Lamm equation [2, 4] using a continuous distribution model to obtain the peaks and molecular weights shown above. The peak percentages are listed in Table 5.
Figure 5
Figure 5
GSH extraction of the [2Fe-2S] cluster from holo (A) 10 μM Gly189Arg NFU1, (B) 10 μM Gly189Ala NFU1, and (C) 10 μM Gly189Lys NFU1, to form the [2Fe-2S](GS)4 complex. The change in absorbance at 420 nm was monitored over the course of one hour. The change in absorbance at 420 nm was plotted against time for each of the different concentrations of GSH and fit to an exponential to obtain the kobs for each concentration. kobs data was plotted against GSH concentration and fit to equation 2 to determine an apparent dissociation constant K and a first-order rate constant k1 for formation of the [2Fe-2S](GS)4 complex (Table 6). k1 was divided by K to determine an overall second-order rate constant of 4400 ± 630 M−1min−1 for (A), 2000 ± 330 M−1min−1 for (B), and 2590 ± 420 M−1min−1 for (C) [31]. Error bars are defined as ± SD, with n=3.
Figure 6
Figure 6
Representative [2Fe-2S] kinetic cluster transfer reactions. (A) Time course of cluster transfer from holo reconstituted human Gly189Arg NFU1 to apo human ferredoxin 1 (Fdx1). Time course for cluster transfer to Fdx1 monitored by CD in 50 mM HEPES and 100 mM NaCl (pH 7.5). Spectra were recorded every 2 min after the addition of holo Gly189Arg NFU1 and were converted to percent cluster transfer (B) to yield an apparent second-order rate constant from DynaFit of 12,500 ± 1000 M−1 min−1 based on the concentration of the [2Fe-2S] cluster [27]. (C) Time course of [2Fe-2S] cluster transfer from holo reconstituted human Gly189Arg NFU1 to apo S. cerevisiae glutaredoxin 3 (Grx3) monitored by CD in 50 mM HEPES and 100 mM NaCl (pH 7.5). Spectra were recorded every 2 min after the addition of holo Gly189Arg NFU1. However transfer was too rapid to monitor, so cluster transfer was monitored from 465-455 nm every 10 sec and converted to percent cluster transfer (D) to determine an apparent second-order rate constant using DynaFit of 39,500 ± 8000 M−1 min−1 based on the concentration of the [2Fe-2S] cluster [27].
Figure 7
Figure 7
Representative cluster transfer reaction for delivery of a [2Fe-2S] cluster into Nfu from a physiologically relevant donor. Kinetics of [2Fe-2S] cluster transfer from holo reconstituted human IscU to human Gly189Arg NFU1. (A) Time course for cluster transfer to human Gly189Arg NFU1 monitored by CD in 50 mM HEPES and 100 mM NaCl (pH 7.5). Spectra were recorded every 2 min following addition of holo IscU and were converted to (B) percent cluster transfer to yield an apparent second-order rate constant from DynaFit of 5700 ± 1400 M−1 min−1 based on the concentration of the [2Fe-2S] cluster [27].
Figure 8
Figure 8
A schematic illustration, highlighting the distinct impact of Gly189Arg and Gly208Cys substitutions on the NFU1 monomer-dimer equilibrium and uptake of [2Fe-2S] cluster to form holo dimer. The Gly208Cys derivative is incapable of receiving cluster from any source [1], while Gly189Arg accepts cluster from IscU.
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