Ag(+) Ion Binding to Human Metallothionein-2A Is Cooperative and Domain Specific.
Ag(+) Ion Binding to Human Metallothionein-2A Is Cooperative and Domain Specific.
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DOI：
10.1021/acs.analchem.0c00829
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发表时间：
2020-07-07
影响因子：
7.4
通讯作者：
Russell DH
中科院分区：
文献类型：
--
作者：
Dong S;Shirzadeh M;Fan L;Laganowsky A;Russell DH
金属硫蛋白（MT）是一类富含半胱氨酸的蛋白质，对多种金属离子起着关键的生物学作用，但与许多其他金属蛋白不同的是，脱辅基和部分金属化的MT的结构尚不清楚。在这里，我们结合联合收割机纳米电喷雾电离质谱（ESI-MS）和纳米ESI-离子迁移率（IM）-MS与碰撞诱导解折叠（CIU），使用N-乙基马来酰亚胺（NEM）的化学标记，以及自下而上和自上而下的蛋白质组学，以更好地了解部分金属化形式的人MT-2A的金属结合位点，即，Ag4-MT。然后将Ag 4-MT的结果与Cd 4-MT的类似结果进行比较。结果表明，Ag 4-MT是一个协同产物，自上而下和自下而上的蛋白质组学质谱分析结合NEM标记的数据显示，Ag 4-MT的4个Ag+离子均结合在β结构域。结合位点被鉴定为Cys 13、Cys 15、Cys 19、Cys 21、Cys 24和Cys 26。而Ag+和Cd ~（2+）均与MT反应生成协同产物，Ag 4-MT和Cd 4-MT，这些产物非常不同; Ag 4-MT的Ag+离子位于β-结构域，而Cd 4-MT的Cd 2+离子位于α-结构域。据报道，Ag 6-MT在β结构域中完全金属化，但我们的数据表明，另外两个Ag+离子的结合比其他四个更弱。高阶Agi-MT复合物（i = 7-17）在含有过量Ag+离子的溶液中形成，并且这些被认为与α结构域结合或在两个结构域之间共享。有趣的是，在向该溶液中加入NEM后，过量的Ag+离子被置换，主要产生Ag 4 NEM 14-MT。CIU的结果表明，Agi-MT复合物在结构上更加有序，并且展开这些复合物所需的能量随着配位Ag+数量的增加而增加。
Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biological roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chemical labeling using N-ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag4-MT. The results for Ag4-MT are then compared to similar results obtained for Cd4-MT. The results show that Ag4-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry analysis combined with NEM labeling revealed that all four Ag+ ions of Ag4-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag+ and Cd2+ react with MT to yield cooperative products, i.e., Ag4-MT and Cd4-MT, these products are very different; Ag+ ions of Ag4-MT are located in the β-domain, whereas Cd2+ ions of Cd4-MT are located in the α-domain. Ag6-MT has been reported to be fully metalated in the β-domain, but our data suggest the two additional Ag+ ions are more weakly bound than are the other four. Higher order Agi-MT complexes (i = 7–17) are formed in solutions that contain excess Ag+ ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag+ ions are displaced upon addition of NEM to this solution to yield predominantly Ag4NEM14-MT. Results from CIU suggest that Agi-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag+ increases.
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影响因子：
4.1
作者：
Hogstrand, C;Galvez, F;Wood, CM
通讯作者：
Wood, CM
影响因子：
2.9
作者：
Chen, Shu-Hua;Russell, David H.
通讯作者：
Russell, David H.
影响因子：
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作者：
Grabenauer, Megan;Wyttenbach, Thomas;Bowers, Michael T.
通讯作者：
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影响因子：
5.6
作者：
ARSENIEV, A;SCHULTZE, P;WUTHRICH, K
通讯作者：
WUTHRICH, K
影响因子：
15
作者：
Chen, Shu-Hua;Chen, Liuxi;Russell, David H.
通讯作者：
Russell, David H.