Investigating the roles of the conserved Cu2+-binding residues on Brucella FtrA in producing conformational stability and functionality.
Investigating the roles of the conserved Cu2+-binding residues on Brucella FtrA in producing conformational stability and functionality.
复制标题
研究布鲁氏菌 FtrA 上保守的 Cu2 结合残基在产生构象稳定性和功能中的作用。
DOI：
10.1016/j.jinorgbio.2020.111162
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发表时间：
2020
影响因子：
3.9
通讯作者：
Dasgupta,Saumya
中科院分区：
文献类型：
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作者：
Banerjee,Sambuddha;Garrigues,RyanJ;Chanakira,MinaN;Negron-Olivo,JacobJ;Odeh,YasmeneH;Spuches,AnneM;MartinRoop2nd,R;Pitzer,JoshuaEdison;Martin,DanielW;Dasgupta,Saumya
布鲁氏菌是一种人畜共患病原体，需要铁的生存，并获得这种金属通过几个高亲和力摄取系统的表达。其中，新发现的亚铁转运蛋白FtrABCD被认为参与亚铁的摄取。序列同源性分析表明，FtrA是一种P19型蛋白（C.显示Cu2+依赖性铁亲和性的jejuni）。先前对其他P19系统的结构和生化研究已经建立了Cu2+依赖性Mn2+亲和力以及这些系统的同源二聚体的形成。来自这些蛋白质的Cu 2+配位氨基酸在布鲁氏菌FtrA中是保守的，暗示了相似的性质。然而，迄今为止，还没有实验证据证明布鲁氏菌FtrA与金属的亲和力和形成二聚体的可能性。使用野生型FtrA和Cu2+结合突变体（H65 A、E67 A、H118 A和H151 A），我们采用同源性建模、分析凝胶过滤、量热和光谱方法研究了这种P19型蛋白的金属亲和力、折叠稳定性、二聚体形成能力以及Cu2+依赖性的分子基础。本文报告的数据证实了野生型FtrA的Cu2+依赖性、低μM Mn2+（Fe2+模拟物）亲和力。此外，我们的数据清楚地显示了Mn 2+亲和力的丧失，以及由于突变这些保守的Cu 2+结合残基而形成的不太稳定的蛋白质构象，表明这些残基在产生布鲁氏菌FtrA的天然和功能性折叠中发挥重要作用。
Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein fromC. jejuniwhich shows Cu2+dependent iron affinity). Previous structural and biochemical studies on other P19 systems have established a Cu2+dependent Mn2+affinity as well as formation of homodimers for these systems. The Cu2+coordinating amino acids from these proteins are conserved inBrucellaFtrA, hinting towards similar properties. However, there has been no experimental evidence, till date, establishing metal affinities and the possibility of dimer formation byBrucellaFtrA. Using wild-type FtrA and Cu2+-binding mutants (H65A, E67A, H118A, and H151A) we investigated the metal affinities, folding stabilities, dimer forming abilities, and the molecular basis of the Cu2+dependence for this P19-type protein employing homology modeling, analytical gel filtration, calorimetric, and spectroscopic methods. The data reported here confirm a Cu2+-dependent, low-μM Mn2+(Fe2+mimic) affinity for the wild-type FtrA. In addition, our data clearly show the loss of Mn2+affinity, and the formation of less stable protein conformations as a result of mutating these conserved Cu2+-binding residues, indicating the important roles these residues play in producing a native and functional fold ofBrucellaFtrA.