Thermodynamic and Structural Analysis of Human NFU Conformational Chemistry

Jingwei Li, Shu Ding, J. A. Cowan
2013 Biochemistry  
Human NFU has been implicated in the formation of inorganic sulfide required for cellular ironsulfur cluster biosyntheses. The protein contains a well-structured N-terminal domain and a Cterminal 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
more » ... s 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 Nterminal domain, which resulted in a novel and distinctive transition melting profile (T m sec = 58.1 ± 0.4 °C, ΔH v sec = 60.4 ± 5.3 kcal/mol, T m ter = 49.3 ± 0.3 °C, ΔH v ter = 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 (T m sec = 63.3 ± 3.4 °C, ΔH v sec = 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.
doi:10.1021/bi400320s pmid:23796308 pmcid:PMC3855582 fatcat:uexkbjayezegrehl3ml2pmujay