期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：50
页码：E6872-E6881
DOI：10.1073/pnas.1512783112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceClpB/Hsp100 chaperones protect cells from the devastating effects of protein inactivation and aggregation arising from extreme stress. This function is accomplished first by binding to the aggregates and then forcibly unraveling individual proteins by passing them through the central channel in the hexameric chaperones. Here, we investigate the role of the ClpB/Hsp100 N-terminal domain (NTD) in protein disaggregation. Our results demonstrate that ClpB recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins via a substrate-binding groove in its NTD. We further show that the NTD has regulatory roles that include blocking the translocation channel in the absence of substrate and destabilizing client proteins upon binding, thus priming them for subsequent unfolding and disaggregation. ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.
