Human Mitochondrial Chaperone (mtHSP70) and Cysteine Desulfurase (NFS1) Bind Preferentially to the Disordered Conformation, Whereas Co-chaperone (HSC20) Binds to the Structured Conformation of the Iron-Sulfur Cluster Scaffold Protein (ISCU)
Human Mitochondrial Chaperone (mtHSP70) and Cysteine Desulfurase (NFS1) Bind Preferentially to the Disordered Conformation, Whereas Co-chaperone (HSC20) Binds to the Structured Conformation of the Iron-Sulfur Cluster Scaffold Protein (ISCU)
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DOI：
10.1074/jbc.m113.482042
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发表时间：
2013-10-04
影响因子：
4.8
通讯作者：
Markley, John L.
中科院分区：
文献类型：
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作者：
Cai, Kai;Frederick, Ronnie O.;Markley, John L.
人ISCU是线粒体铁-硫（Fe-S）簇生物合成和转运的支架蛋白。NMR光谱显示ISCU占据两种构象状态;即，更结构化的状态（S）和部分无序的状态（D）。我们鉴定了两个稳定S-状态的单氨基酸取代（D39 V和N90 A）和两个稳定D-状态的单氨基酸取代（D39 A和H105 A）。我们分离的两个组成蛋白的人半胱氨酸脱硫酶复合物（NFS 1和ISD 11）分别和使用NMR光谱研究它们与ISCU的相互作用。我们发现ISD 11不直接与ISCU相互作用。相比之下，NFS 1优先与ISCU的D-状态结合，NFS 1-ISD 11复合物也是如此。体外Fe-S簇组装试验表明，当由NFS 1单独催化时，[2Fe-2S]和[4Fe-4S]簇组装在ISCU上，并且当由NFS 1-ISD 11复合物催化时，以更高的速率组装。DnaK型伴侣蛋白（mtHSP 70）和DnaJ型共伴侣蛋白（HSC 20）参与了与ISCU结合的簇在ATP依赖性反应中向受体蛋白的转移。结果表明，HSC 20对mtHSP 70的ATP酶活性有促进作用，HSC 20 + ISCU对mtHSP 70的ATP酶活性有进一步的促进作用。NMR研究表明，mtHSP 70优先结合ISCU的D-态，HSC 20优先结合ISCU的S-态。
Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state. We isolated the two constituent proteins of the human cysteine desulfurase complex (NFS1 and ISD11) separately and used NMR spectroscopy to investigate their interaction with ISCU. We found that ISD11 does not interact directly with ISCU. By contrast, NFS1 binds preferentially to the D-state of ISCU as does the NFS1-ISD11 complex. An in vitro Fe-S cluster assembly assay showed that [2Fe-2S] and [4Fe-4S] clusters are assembled on ISCU when catalyzed by NFS1 alone and at a higher rate when catalyzed by the NFS1-ISD11 complex. The DnaK-type chaperone (mtHSP70) and DnaJ-type co-chaperone (HSC20) are involved in the transfer of clusters bound to ISCU to acceptor proteins in an ATP-dependent reaction. We found that the ATPase activity of mtHSP70 is accelerated by HSC20 and further accelerated by HSC20 plus ISCU. NMR studies have shown that mtHSP70 binds preferentially to the D-state of ISCU and that HSC20 binds preferentially to the S-state of ISCU.