Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase
Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase
复制标题
DOI：
10.1073/pnas.1114372109
复制
发表时间：
2012-01-10
影响因子：
11.1
通讯作者：
Markley, John L.
中科院分区：
文献类型：
--
作者：
Kim, Jin Hae;Tonelli, Marco;Markley, John L.
用于铁-硫簇组装的支架蛋白apo-IscU占据两种相互转换的构象状态，一种是无序的（D），一种是结构化的（S），如通过广泛的NMR分配所揭示的。在pH 8和25 ℃下，大约70%的蛋白质是S，状态的寿命为1.3 s（S）和0.50 s（D）。Zn（II）和Fe（II）各自结合并稳定结构化（类S）状态。发现保守残基处的单个氨基酸取代使平衡向S或D状态移动。簇组装发生在IscU和半胱氨酸脱硫酶IscS之间的复合物中，并且我们的NMR研究表明IscS优先结合D形式的apo-IscU。发现向IscU中加入10%IscS大大增加了IscU的保护酰胺处的H/D交换，增加了S -> D反应的速率，并降低了D -> S反应的速率。在饱和的IscU：IscS复合物中，IscU基本上是无序的。在体外集群组装反应提供了证据的功能的重要性的S可逆箭头D平衡。发现有利于S状态的IscU变体经历了一个滞后期，而野生型没有观察到，这延迟了簇组装;发现有利于D状态的变体以中间速率组装不太稳定的簇，而没有滞后。看来，ISCU已经演变为存在于一个无序的构象状态，这是脱硫酶的初始底物，并转换为结构化状态，稳定的集群，一旦它组装。
The scaffold protein for iron-sulfur cluster assembly, apo-IscU, populates two interconverting conformational states, one disordered (D) and one structured (S) as revealed by extensive NMR assignments. At pH 8 and 25 degrees C, approximately 70% of the protein is S, and the lifetimes of the states are 1.3 s (S) and 0.50 s (D). Zn(II) and Fe(II) each bind and stabilize structured (S-like) states. Single amino acid substitutions at conserved residues were found that shift the equilibrium toward either the S or the D state. Cluster assembly takes place in the complex between IscU and the cysteine desulfurase, IscS, and our NMR studies demonstrate that IscS binds preferentially the D form of apo-IscU. The addition of 10% IscS to IscU was found to greatly increase H/D exchange at protected amides of IscU, to increase the rate of the S -> D reaction, and to decrease the rate of the D -> S reaction. In the saturated IscU: IscS complex, IscU is largely disordered. In vitro cluster assembly reactions provided evidence for the functional importance of the S reversible arrow D equilibrium. IscU variants that favor the S state were found to undergo a lag phase, not observed with the wild type, that delayed cluster assembly; variants that favor the D state were found to assemble less stable clusters at an intermediate rate without the lag. It appears that IscU has evolved to exist in a disordered conformational state that is the initial substrate for the desulfurase and to convert to a structured state that stabilizes the cluster once it is assembled.