Iron-sulfur (Fe-S) clusters are crucial co-factors that participate in several key biological events such as mitochondrial respiration, photosynthesis and nitrogen fixation. In eukaryotes, Fe-S cluster biogenesis is conducted in mitochondria by the ISC (iron-sulfur cluster) machinery that composed of multiple proteins. Previous studies suggest that an Hsp70 chaperone might participate in Fe-S cluster biogenesis via interaction with Fe-S cluster assembly scaffold protein, ISU/IscU. Nevertheless, details regarding the underlying mechanism for this interaction and the role of chaperones in Fe-S cluster biogenesis are unclear.
Mortalin, a Hsp70 chaperone that has been implicated in mitochondrial import and multiple human diseases, is suggested to be the mitochondrial Hsp70 chaperone that participates in Fe-S cluster biogenesis in humans. However, there is no direct evidence to support this hypothesis. One major obstacle to settle this question is the lack of sufficient material to characterize human mortalin and its biochemistry with partner proteins. Herein, a protocol has been established to express and purify mortalin in sufficient amount for biochemical and biophysical studies. The characterization of mortalin through spectroscopic methods such as fluorescence and circular dichroism has been conducted and compared with another Hsp70 chaperone, namely Thermotoga maritima DnaK (Tm DnaK). In addition, interactions between chaperones proposed to participate in cluster assembling and cognate cluster scaffold proteins have been investigated through studies using mortalin and Tm DnaK for Gram(+) for human and bacteria / archaea model chaperones, respectively. Interactions of chaperones and peptides that mimic plausible binding motifs on scaffold proteins (human ISU and Tm SufU) were examined by use of fluorescence anisotropy; furthermore, interactions of chaperones and scaffold proteins were determined by use of isothermal titration calorimetry (ITC).
Intrinsic properties of mortalin were determined, and the comparison of human mortalin and Tm DnaK shows that they share similar secondary structures (about 30% α-helix and 15% β-sheet) and slow turnover rate in ATP hydrolysis (khyd = 0.00060 ± 0.00006 s-1 and 0.00036 ± 0.00004 s-1 for mortalin and Tm DnaK, respectively). Although tentative, the analysis of chaperone-peptide interactions suggests that, mortalin and Tm DnaK exhibit different behaviors in terms of peptide binding. Mortalin is more promiscuous toward peptide substrates and the presence of nucleotides does not appear to be required. Dissociation constants for all chaperone-peptide interactions fall into the sub-μM range, which is consistent with observations from other Hsp70 chaperones. ITC results support the hypothesis that mortalin interacts with the human scaffold protein, ISU, with a stoichiometric binding ratio of 1:1. This is the first direct evidence to corroborate the possible involvement of mortalin in Fe-S cluster biogenesis in humans. A cross-system comparison suggests that chaperone-scaffold protein pairs from those two distinct systems cannot generally communicate with each other.
This work provides a protocol that produces ample amount of mortalin sample. Also, results here offer insight toward the mechanistic understanding of the role of chaperones in Fe-S cluster biogenesis, as well as serve as a foundation for future studies in connecting the link between mortalin and human diseases.