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. 2017 Mar 24;429(6):790-807.
doi: 10.1016/j.jmb.2017.01.021. Epub 2017 Feb 1.

Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)-Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway

Christine Wachnowsky  1 , Nathaniel A Wesley  2 , Insiya Fidai  3 , J A Cowan  4
Affiliations

Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)-Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway

Christine Wachnowsky et al. J Mol Biol. .

Abstract

Iron-sulfur (Fe/S)-cluster-containing proteins constitute one of the largest protein classes, with varied functions that include electron transport, regulation of gene expression, substrate binding and activation, and radical generation. Consequently, the biosynthetic machinery for Fe/S clusters is evolutionarily conserved, and mutations in a variety of putative intermediate Fe/S cluster scaffold proteins can cause disease states, including multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalomyopathy. Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (Gly208Cys) near the active site of NFU1, an Fe/S scaffold protein, via an in vitro investigation into the structural and functional consequences. Analysis of protein stability and oligomeric state demonstrates that the mutant increases the propensity to dimerize and perturbs the secondary structure composition. These changes appear to underlie the severely decreased ability of mutant NFU1 to accept an Fe/S cluster from physiologically relevant sources. Therefore, the point mutation on NFU1 impairs downstream cluster trafficking and results in the disease phenotype, because there does not appear to be an alternative in vivo reconstitution path, most likely due to greater protein oligomerization from a minor structural change.

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

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Figures

Figure 1
Figure 1
(A) A representation of the two-domain composition of the native and mutant NFU1 proteins, with the N-terminal domain in blue and the C-terminal domain in red. NFU1 features the functional CXXC Fe/S cluster binding motif in its C-terminal domain. This pattern is altered in the mutant protein with the mutation of a nearby glycine residue at position 208 to cysteine, which gives a CXCXXC motif. (B) Solution NMR structure of human C-terminal domain of the NFU1 protein (PDB ID: 2M5O) with the cluster binding cysteines shown in yellow [5]. The glycine at position 208 in the full length protein (58 above), which is mutated to a cysteine in MMDS1, is colored red. Numbering is consistent with the C-terminal construct used in the structure determination.
Figure 2
Figure 2
VTCD traces for the melting of 10 μM native human NFU1 (A) and G208C NFU1 (B) in 40 mM phosphate, pH 7.4. Data were fit to equation 1 to obtain Tm and ΔHV, which are shown in Table 3. 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 [4].
Figure 3
Figure 3
Differential scanning calorimetry profiles for (A) 0.2 mM native human NFU1, (B) 0.3 mM G208C human NFU1. Both of the proteins were in 50 mM HEPES, 100 mM NaCl, and pH 7.4. The data were fit using Origin 7.0 to obtain Tm, ΔHcal, and ΔHV, all of which are listed in Tables 1
Figure 4
Figure 4
Analytical ultracentrifugation profiles for G208C NFU1. (A) Apo G208C was sedimented in the absence of TCEP (black) and in the presence of 1 mM TCEP (red). Sedimentation was monitored at 280 nm. The first peak of the black trace at 22.6 kDa accounts for 29% of the sample, the second peak at 41.6 kDa accounts for 58%, and the third peak at 74.0 kDa accounts for 6%. The first peak of the red trace at 22.9 kDa accounts for 30% of the sample, the second peak at 41.9 kDa accounts for 60%, and the third peak at 83.5 kDa accounts for 1.5%. The AUC results were fit to the Lamm equation [6, 7] using a continuous distribution model to obtain the peaks and molecular weights shown above.
Figure 5
Figure 5
UV (A) and CD (B) spectra following reconstitution of native and G208C NFU1. In both spectra, the black trace corresponds to apo G208C NFU1, and the red trace to native reconstituted holo NFU1. In (A), the blue trace is holo G208C NFU1 reconstituted with Tm NifS and L-cysteine, while the green trace is holo G208C NFU1 reconstituted with ferric chloride and sodium sulfide. In (B), the blue trace is holo reconstituted NFU1, since both reconstitution methods resulted in the same spectra.
Figure 6
Figure 6
GSH extraction of the [2Fe-2S] cluster from 10 μM reconstituted holo G208C human NFU1 to form the [2Fe-2S](GS)4 complex. The change in absorbance at 420 nm was monitored over the course of an hour and data were fit to an exponential to obtain the kobs. (A) Shows a representative trace of extraction by 1 mM GSH (red trace). The black trace shows a control of reconstituted holo G208C human NFU1 in the absence of GSH to demonstrate the rate of general cluster instability or breakdown. The concentration of GSH was varied, while keeping the concentration of G208C NFU1 constant to obtain a second-order rate constant. (B) The kobs data were plotted against the concentration of GSH and fit to a linear equation to determine an overall second-order rate constant of 140 ± 20 M−1min−1.
Figure 7
Figure 7
Kinetics of [2Fe-2S] cluster transfer from holo reconstituted human G208C NFU1 to apo human ferredoxins. (A) Time course for cluster transfer to ferredoxin 1(Fdx1) monitored by CD in 50 mM HEPES, 100 mM NaCl, pH 7.5. Spectra were recorded every 2 min after the addition of holo NFU1, and converted to percent cluster transfer (B) to yield an apparent second-order rate constant from DynaFit of 2600 ± 300 M−1min−1 based on the concentration of the [2Fe-2S] cluster [3]. (C) Time course for cluster transfer from holo human NFU1 to apo human ferredoxin 2 (Fdx2) recorded by CD under the same conditions as for Fdx1. (D) The CD signal was again converted to the percentage of cluster transferred with time to yield an apparent second-order rate constant of 1200 ± 200 M−1min−1.
Figure 8
Figure 8
Kinetics of [2Fe-2S] cluster transfer from holo reconstituted human G208C NFU1 to apo glutaredoxins. (A) Time course for cluster transfer to apo human Grx2 monitored by CD in 50 mM HEPES, 100 mM NaCl, and pH 7.5 with 3 mM GSH. Spectra were recorded every 2 min after the addition of holo NFU1. However transfer was too rapid to monitor, so cluster transfer was monitored from 435–445 nm every 10 sec and converted to percent cluster transfer (B) to determine an apparent second-order rate constant using DynaFit of 22400 ± 5000 M−1min−1 based on the concentration of the [2Fe-2S] cluster [3]. (C) Time course for cluster transfer to apo S. cerevisiae Grx3 monitored by CD under identical conditions. Again, cluster transfer was too rapid to monitor, and so transfer was monitored from 450–460 nm every 10 sec and converted to percent cluster transfer (D) to determine an apparent second-order rate constant of 14500 ± 3500 M−1min−1.
Figure 9
Figure 9
Alignment of the human C-terminal domain of the NFU1 protein (PDB ID: 2M5O) (green trace) and corresponding cysteines in red. The Phyre2 homology modeled structure generated for G208C NFU1 is shown in blue with the cysteines colored yellow.
Figure 10
Figure 10
(A) A model for [2Fe-2S] cluster uptake by monomeric NFU1, represented by the N- and C-terminal domains, from the [2Fe-2S](GS)4 cluster complex to form an intermediate [2Fe-2S] species with two exogenous GSH ligands. A second monomeric NFU1 displaces the GSH molecules to form [2Fe-2S] dimeric NFU1. Adapted from [1]. (B) A model for [2Fe-2S] cluster uptake by monomeric NFU1, from a [2Fe-2S] cluster scaffold, such as IscU. Holo IscU interacts with monomeric NFU1 to form a transient heterodimeric complex. A second NFU1 monomeric displaces the now apo IscU to form the holo NFU1 dimer. In a case where NFU1 is a pre-formed dimer (C), such as in the G208C mutant, the dimeric NFU1 is unable to form the transient complex with holo IscU and therefore unable to receive the [2Fe-2S] cluster.
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