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J Am Chem Soc. 2014 Aug 20;136(33):11586-9. doi: 10.1021/ja5055252. Epub 2014 Aug 6.

Nucleotide-dependent interactions within a specialized Hsp70/Hsp40 complex involved in Fe-S cluster biogenesis.

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Mitochondrial Protein Partnership, Center for Eukaryotic Structural Genomics, and ‡Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.


The 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.

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