The Specialized Hsp70 (HscA) Interdomain Linker Binds to Its Nucleotide-Binding Domain and Stimulates ATP Hydrolysis in Both cis and trans Configurations

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• 作者：T. Reid Alderson ; Jin Hae Kim ; Kai Cai ; Ronnie O. Frederick ; Marco Tonelli ; John L. Markley
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：November 25, 2014
• 年：2014
• 卷：53
• 期：46
• 页码：7148-7159
• 全文大小：584K
• ISSN：1520-4995

文摘

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron鈥搒ulfur (Fe鈥揝) 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^鈭? at 25 掳C, henceforth reported in units of min^鈥?). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer; however, while the interactions between HscA and HscB have been investigated, the role of HscA鈥檚 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^鈥?) is nearly 15-fold higher than that of HscA₃₈₆ (0.035 min^鈥?), although their apparent affinities for ATP are equivalent. HscA₃₉₅, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence and basal thermostability that were higher than those of HscA₃₈₆. Furthermore, HscA₃₉₅ displayed narrower ¹H^N line widths in its two-dimensional ¹H鈥?sup>15N TROSY-HSQC spectrum in comparison to HscA₃₈₆, indicating that the peptide in the cis configuration binds to and stabilizes 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.