Human NFU has been implicated in the formation of inorganic sulfide required for cellular iron-sulfur cluster biosyntheses. The protein contains a well-structured N-terminal domain and a C-terminal domain with molten globule characteristics that also contains a thioredoxin-like pair of redox active Cys that promote persulfide reductase activity. Recent reports have highlighted the existence of structural flexibility in the ISU/IscU-type scaffold proteins that mediate Fe-S cluster assembly, which is also likely to serve an important role in the pathway to iron-sulfur cluster maturation. We have previously reported similar structural mobility for the C-terminal domain of human NFU, a protein that has been implicated in the production of sulfide for cluster synthesis, while homologous proteins have also been suggested to serve as Fe-S cluster carriers. Herein we quantitatively characterize the structural stability of the two domains of human NFU, and in particular the functional C-terminal domain. The results of differential scanning calorimetry (DSC) and variable temperature circular dichroism (VTCD) studies that have been used to analyze the temperature-dependent structural melting profiles of the N- and C-terminal domains, relative to both full-length NFU and an equimolar ratio of the N- and C-terminal domains, and correlated with structural information derived from NMR. Calorimetry results indicate that the C-terminal NFU domain undergoes a significant structural stabilization following interaction with the N-terminal domain, which resulted in a novel and distinctive transition melting profile (Tmsec = 58.1 ± 0.4 °C, ΔHvsec = 60.4 ± 5.3 kcal/mol, Tmter = 49.3 ± 0.3 °C, ΔHvter = 71.8 ± 5.8 kcal/mol). VTCD experiments also revealed a secondary structure transition at 59.2 °C in agreement with calorimetry results. The degree of stabilization was found to be more significant in the full-length NFU, as the C-terminal domain transitions were recorded at higher temperatures (Tmsec = 63.3 ± 3.4 °C, ΔHvsec = 41.8 ± 8.2 kcal/mol). The interactions between the two domains demonstrated the hallmarks of hydrophobic character, as increased ionic strength decreased the degree of stabilization of the C-terminal domain. An increase of 2% in α -helix content further supports interaction between the two domains leading to greater secondary structure stabilization. Heteronuclear single quantum coherence (HSQC) experiments indicate the C-terminal domain to adopt an alternate tertiary conformation following binding to the N-terminal domain. The structural rigidity of the N-terminal domain leads to an alternative conformation for the C-terminal domain, suggesting that such an interaction, although weaker compared to the covalently-attached native NFU, is important for the structural chemistry of the native full-length protein. The results also emphasize the likely general importance of such structural flexibility in select proteins mediating metal cofactor biosynthesis.
