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

• Published in: Biochemistry (Easton) Vol. 53; no. 46; pp. 7148 - 7159
• Main Authors: Alderson, T. Reid, Kim, Jin Hae, Cai, Kai, Frederick, Ronnie O, Tonelli, Marco, Markley, John L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 25.11.2014
• Subjects: Accelerated Publication; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Binding Sites; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; HSP70 Heat-Shock Proteins - chemistry; HSP70 Heat-Shock Proteins - metabolism; Hydrolysis; Models, Molecular; Protein Stability; Protein Structure, Tertiary
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi5010552

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; however, 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 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 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.