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

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 iron-sulfur 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 iron-sulfur 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.

Analysis of NFU‐1 metallocofactor binding‐site substitutions—impacts on iron–sulfur cluster coordination and protein structure and function

“…; Table ). No higher order species were observed for G208S, although a peak at 102 kDa could reflect a small tendency of G208A toward tetramer formation, similar to prior observations for G208C . Even in the presence of TCEP, these values remain essentially unchanged, supporting a crucial role for residue 208 and changes to the secondary structure composition in dictating oligomerization trends independent of disulfide bonding or cofactor binding.…”

“…This GSH complex has been shown to be taken up by a number of apo Isc proteins and is a viable substrate for the mitochondrial ABCB7 transporter , suggesting a possible role for the [2Fe–2S](GS) 4 complex as a component of the labile iron pool. Native human NFU1 can take up a [2Fe–2S] cluster from the complex with a second‐order rate constant of 1930 m −1 ·min −1 , as observed by CD, but the G208C construct could not . Similar to the native protein, the G208A construct was also able to take up a [2Fe–2S] cluster moderately faster with a larger second‐order rate constant of 3500 m −1 ·min −1 (Fig.…”

“…Therefore, we have examined the ability of the single‐substitution proteins to be reconstituted by two donor proteins Schizosaccharomyces pombe Isc assembly protein (Isa1) and human IscU by use of CD spectroscopy. Like the native, the G208S construct was able to receive cluster from the two donor proteins examined, in contrast to both the G208C and the G208A constructs that could not be reconstituted by the donors . Holo reconstituted human IscU was added to apo G208S (Fig.…”

“…A). Based on iron quantitation, reconstitution yields varied from 0.62 cluster/dimer for G208C/C210A, 0.86 for G208C/C213A, 0.65 for G08C/C210S, and 0.57 for G208C/C213S, which are slightly lower than those typically achieved for the native construct . Protein oligomerization around the cluster was confirmed by use of AUC, while monitoring at 420 nm to assess the cluster‐bound form of the protein.…”

Privileged Electrophile Sensors: A Resource for Covalent Drug Development

“…However, we state upfront that to our knowledge, none of these cysteines have been shown to be privileged RES-sensors and a therapy based around RES would need to be administered likely for life. In multiple mitochondrial dysfunctions syndrome 1 (MMDS1—a disease that often causes death in perinatal stages), a recessive cysteine mutation in Nfu1 (G208C) promotes dimerization and perturbs secondary structure, likely impairing iron-sulfur-cluster biogenesis (Wachnowsky, et al, 2017). Because this mutation is recessive (i.e., the gene is haplosufficient), it is possibly an ideal candidate for a molecular intervention approach.…”