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Nucleotide-Dependent Interactions within a Specialized Hsp70/Hsp40 Complex Involved in Fe–S Cluster Biogenesis
journal contribution
posted on 2015-12-17, 03:53 authored by Jin Hae Kim, T. Reid Alderson, Ronnie
O. Frederick, John L. MarkleyThe
structural mechanism by which Hsp70-type chaperones interact
with Hsp40-type co-chaperones has been of great interest, yet still
remains a matter of debate. Here, we used solution NMR spectroscopy
to investigate the ATP-/ADP-dependent interactions between Escherichia coli HscA and HscB, the specialized Hsp70/Hsp40
molecular chaperones that mediate iron–sulfur cluster transfer.
We observed that NMR signals assigned to amino acid residues in the
J-domain and its “HPD” motif of HscB broadened severely
upon the addition of ATP-bound HscA, but these signals were not similarly
broadened by ADP-bound HscA or the isolated nucleotide binding domain
of HscA complexed with either ATP or ADP. An HscB variant with an
altered HPD motif, HscB(H32A,P33A,D34A), failed to manifest WT-like
NMR signal perturbations and also abolished WT-like stimulation of
ATP hydrolysis by HscA. In addition, residues 153–171 in the
C-terminal region of HscB exhibited NMR signal perturbations upon
interaction with HscA, alone or complexed with ADP or ATP. These results
demonstrate that the HPD motif in the J-domain of HscB directly interacts
with ATP-bound HscA and suggest that a second, less nucleotide-dependent
binding site for HscA resides in the C-terminal region of HscB.