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Structurally restricted Bi(III) metallation of apo-βMT1a: metal-induced tangling.
Metallomics ( IF 2.9 ) Pub Date : 2021-04-26 , DOI: 10.1093/mtomcs/mfab023
Natalie C Korkola 1 , Elyse Hudson 1 , Martin J Stillman 1
Affiliation  

Non-toxic bismuth salts are used in anti-ulcer medications and to protect against nephrotoxicity from anti-cancer drugs. Bismuth salts also induce metallothionein (MT), a metal-binding protein that lacks a formal secondary structure. We report the impact on the metallation properties of Bi(III) to the 9-cysteine β fragment of MT as a function of cysteine accessibility using electrospray ionization mass spectrometry. At pH 7.4, Bi2βMT formed cooperatively. Cysteine modification shows that each Bi(III) was terminally bound to 3 cysteinyl thiolates. Non-cooperative Bi(III) binding was observed at pH 2.3, where cysteine accessibility is increased. However, competition from H4EDTA inhibited Bi(III) binding. When GdmCl, a well-known denaturing agent, was used to increase cysteine accessibility of the apoβMT at pH 7.4, a greater fraction of Bi3βMT formed using all 9 cysteines. The change in binding profile and equilibrium of Bi2βMT was determined as a function of acidification, which changed as a result of competition with H4EDTA. There was no Bi(III) transfer between Bi2βMT, Cd3βMT, and Zn3βMT. This lack of metal exchange and the resistance towards binding the third Bi(III) suggests a rigidity in the Bi2βMT binding sites that inhibits Bi(III) mobility. These experiments emphasize the conformational control of metallation that results in substantially different metallated products: at pH 7.4 (many cysteines buried) Bi2βMT, whereas at pH 7.4 (all cysteines accessible) enhanced formation of Bi3βMT. These data suggest that the addition of the first 2 Bi(III) cross-link the protein, blocking access to the remaining 3 cysteines for the third Bi(III), as a result of tangle formation.

中文翻译:

apo-βMT1a在结构上受限制的Bi(III)金属化:金属诱导的缠结。

无毒的铋盐可用于抗溃疡药物,并保护其免受抗癌药物的肾毒性。铋盐还诱导金属硫蛋白(MT),一种缺乏正式二级结构的金属结合蛋白。我们报告了使用电喷雾电离质谱法对半胱氨酸可及性的影响,对MT的9-半胱氨酸β片段Bi(III)的金属化性质的影响。在pH 7.4时,Bi2βMT协同形成。半胱氨酸修饰表明,每个Bi(III)末端都与3个半胱氨酸硫醇盐结合。在pH 2.3处观察到非合作Bi(III)结合,其中半胱氨酸的可及性增加。但是,来自H4EDTA的竞争抑制了Bi(III)的结合。当使用著名的变性剂GdmCl来增加pH 7.4时apoβMT的半胱氨酸可及性时,使用所有9个半胱氨酸形成的Bi3βMT的比例更高。Bi2βMT的结合曲线和平衡的变化被确定为酸化的函数,这是与H4EDTA竞争的结果。Bi2βMT,Cd3βMT和Zn3βMT之间没有Bi(III)转移。这种金属交换的缺乏和对结合第三个Bi(III)的抵抗力表明,Bi2βMT结合位点具有刚性,从而抑制了Bi(III)的迁移。这些实验强调了金属化的构象控制,其导致了实质上不同的金属化产物:在pH 7.4(许多半胱氨酸被掩埋)下的Bi2βMT,而在pH 7.4(所有半胱氨酸可接近)下增强了Bi3βMT的形成。这些数据表明,添加前两个Bi(III)会交联蛋白质,从而阻止第三个Bi(III)进入其余3个半胱氨酸,
更新日期:2021-04-26
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