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