Thermodynamic and Structural Analysis of Human NFU Conformational Chemistry
Thermodynamic and Structural Analysis of Human NFU Conformational Chemistry
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DOI：
10.1021/bi400320s
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发表时间：
2013-07-23
期刊：
影响因子：
2.9
通讯作者：
Cowan, J. A.
中科院分区：
文献类型：
--
作者：
Li, Jingwei;Ding, Shu;Cowan, J. A.
人NFU与细胞铁硫簇生物合成所需的无机硫化物的形成有关。该蛋白质含有一个结构良好的N-末端结构域和一个具有熔融球特征的C-末端结构域，该结构域还含有一对硫氧还蛋白样氧化还原活性Cys残基，其促进过硫化物还原酶活性。最近的报道强调了在介导Fe-S簇组装的ISU/IscU型支架蛋白中存在结构灵活性，这也可能在Fe-S簇成熟的途径中发挥重要作用。我们以前曾报道过类似的结构迁移率的C-末端结构域的人NFU，一种蛋白质，已被牵连在生产硫化物的灰尘合成，而同源蛋白质也被建议作为Fe-S簇载体。在这里，我们定量表征的两个结构域的人NFU，特别是功能的C-末端结构域的结构稳定性。差示扫描量热法和变温圆二色性（VTCD）研究的结果已被用来分析的N-和C-末端域的温度依赖性的结构熔融配置文件，相对于全长NFU和等摩尔比的N-和C-末端域，并与来自NMR数据的结构信息。量热法结果表明，C-末端NFU结构域在与N-末端结构域相互作用后经历显著的结构稳定化，这导致新的和独特的转变熔化曲线（T-m（sec）= 58.1 +/-0.4 ° C，Δ H-v（sec）= 60.4 +/-5.3kcal/mol，T-m（ter）49.3 +/-0.34 ° C，Δ H-v（ter）= 71.8 +/-5.8 kcal/mol）。VTCD实验还揭示了二级结构转变在59.2摄氏度与量热法的结果一致。发现在全长NFU中稳定化程度更显著，因为在较高温度下记录到C-末端结构域转变（T-m（sec）= 63.3 +/-3.4 ℃，Δ H-v（sec）= 41.8 +/-8.2 kcal/mol）。两个域之间的相互作用表现出疏水性的特点，增加离子强度降低的C-末端域的稳定程度。α-螺旋含量增加2%进一步支持两个结构域之间的相互作用，导致更大的二级结构稳定性。异源单量子相干实验表明，C-末端结构域采用交替的三级构象结合到N-末端结构域。N-末端结构域的结构刚性导致C-末端结构域的替代构象，这表明这种相互作用虽然弱于共价连接的天然NFU，但对于天然全长蛋白质的结构化学是重要的。结果还强调了这种结构灵活性在选择介导金属辅因子生物合成的蛋白质中的可能的普遍重要性。
Human NFU has been implicated in the formation of inorganic sulfide required for cellular iron-sulfur cluster biosynthesis. 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 residues 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 Fe-S 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 duster 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 and variable temperature circular dichroism (VTCD) studies 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 data. 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 (T-m(sec) = 58.1 +/- 0.4 degrees C, Delta H-v(sec) = 60.4 +/- 5.3 kcal/mol, T-m(ter) 49.3 +/- 0.3 4 degrees C, Delta H-v(ter) = 71.8 +/- 5.8 kcal/mol). VTCD experiments also revealed a secondary structure transition at 59.2 degrees 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 degrees C, Delta H-v(sec) = 41.8 +/- 8.2 kcal/mol). The interactions between the two domains demonstrated the hallmarks of a hydrophobic character, as increased ionic strength decreased the degree of stabilization of the C-terminal domain. An increase of 2% in alpha-helix content further supports interaction between the two domains, leading to greater secondary structure stabilization. Heteronuclear single-quantum coherence experiments indicate that the C-terminal domain adopts an alternate tertiary conformation following binding to the N-terminal domain. The structural rigidity of the N-terminal domain leads to an alternative conformation of the C-terminal domain, suggesting that such an interaction, although weaker than that of 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.