The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.
The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.
复制标题
DOI：
10.1021/bi5010552
复制
发表时间：
2014-11-25
期刊：
影响因子：
2.9
通讯作者：
Markley, John L.
中科院分区：
文献类型：
--
作者：
Alderson, T. Reid;Kim, Jin Hae;Cai, Kai;Frederick, Ronnie O.;Tonelli, Marco;Markley, John L.
来自大肠杆菌isc操纵子的蛋白质构成用于合成铁-硫（Fe-S）簇以递送至受体脱辅基蛋白的机器。[2Fe-2S]簇从全支架蛋白IscU的有效和快速转移取决于HscA的核苷酸结合结构域（NBD）中的ATP水解，HscA是一种具有低内在ATP酶活性（在25 °C时为0.02 min−1，此后以min − 1为单位报告）的专门的Hsp 70型分子伴侣。HscB是一种Hsp 40型辅伴侣，与HscA结合并刺激ATP水解以促进簇转移，但虽然HscA和HscB之间的相互作用已被研究，但HscA的域间连接体在调节ATP酶活性中的作用尚未被探索。为了解决这个问题，我们创建了HscA的40 kDa NBD的三种变体：单独的NBD（HscA 386）、具有部分接头的NBD（HscA 389）和具有完整接头的NBD（HscA 395）。我们发现HscA 395的ATP水解速率（0.45 min-1）比HscA 386的ATP水解速率（0.035 min-1）高近15倍，尽管它们对ATP的表观亲和力是相等的。HscA 395，其中包含完整的共价连接的接头肽，表现出内在的色氨酸荧光发射和基础的热稳定性高于HscA 386。此外，与HscA 386相比，HscA 395在其二维1H-15 N TROSY-HSQC光谱中显示更窄的1HN线宽，表明顺式构型的肽结合到NBD的结构并使其稳定。添加到HscA 386的合成肽的序列相同的结构域间连接器（L387 LLDVIPLS 395）刺激其ATP酶活性和诱导广泛的NMR化学位移扰动指示的结合相互作用的反式配置。
Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron–sulfur (Fe–S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min−1 at 25 °C, henceforth reported in units of min–1). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min–1) is nearly 15-fold higher than that of HscA386 (0.035 min–1), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower 1HN line widths in its two-dimensional 1H–15N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L387LLDVIPLS395) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration.
登录
查看更多内容
影响因子：
7.7
作者：
De Los Rios P;Barducci A
通讯作者：
Barducci A
影响因子：
4.8
作者：
Cai, Kai;Frederick, Ronnie O.;Markley, John L.
通讯作者：
Markley, John L.
影响因子：
5.6
作者：
Bhattacharya, Akash;Kurochkin, Alexander V.;Yip, Grover N. B.;Zhang, Yongbo;Bertelsen, Eric B.;Zuiderweg, Erik R. P.
通讯作者：
Zuiderweg, Erik R. P.
DOI：
10.1073/pnas.0903503106
发表时间：
2009-05-26
影响因子：
11.1
作者：
Bertelsen, Eric B.;Chang, Lyra;Zuiderweg, Erik R. P.
通讯作者：
Zuiderweg, Erik R. P.
影响因子：
5.6
作者：
Isaya G
通讯作者：
Isaya G