Characterizing the nucleotide-binding domain of Escherichia coli HscA, a specialized Hsp70 chaperone in Fe-S cluster biogenesis

Abstract

Proteins from the ISC operon of E. coli constitute the machinery used for the synthesis of iron-sulfur clusters and their delivery to recipient apo-proteins. [2Fe-2S] cluster transfer from the holo-scaffold protein lscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70 chaperone. HscB, an Hsp40-type co-chaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer. However, the interactions between HscA and HscB have remained uncharacterized, and, moreover, the role of HscA's interdomain linker in modulating ATPase activity has not been explored. I have created three variants of the isolated HscA NBD truncated at different residues within the linker, and have shown that the linker binds to the NBD and autoactivates ATP hydrolysis. Using solution-state NMR spectroscopy and chemical shift perturbations, I illustrated that a synthetic linker peptide is capable of binding to the NBD and stimulating ATP hydrolysis. Moreover, the interdomain linker altered the intrinsic tryptophan fluorescence emission of Trp291 in HscA's NBD and enhanced stability of the NBD, as measured with differential scanning fluorimetry. With these data in hand, further experiments are well-poised to identify the NBD residues involved in binding to the interdomain linker and elucidate the mechanism by which ATPase activity is enhanced