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ClpB N-terminal domain plays a regulatory role in protein disaggregation.

ABSTRACT: 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.

SUBMITTER: Rosenzweig R

PROVIDER: S-EPMC4687599 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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ClpB N-terminal domain plays a regulatory role in protein disaggregation.

Rosenzweig Rina R Farber Patrick P Velyvis Algirdas A Rennella Enrico E Latham Michael P MP Kay Lewis E LE

Proceedings of the National Academy of Sciences of the United States of America 20151130 50

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 ...[more]

PMID: 26621746

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Project description:Temperature is considered one of the critical factors directly influencing grapevine during the three primary growth and development stages: sprout, flowering, and fruit-coloring, which is strongly correlated to the yield and quality of the grape. The grapevine is frequently exposed to high-temperature conditions that are detrimental to growth. However, the mechanisms of the heat stress response and adaptation in grapevine are not adequately studied. The Arabidopsis copine gene AtBON1 encodes a highly conserved protein containing two C2 domains at the amino terminus, participation in cell death regulation and defense responses. Previously, we showed that a BON1 association protein from the grapevine, VvBAP1, plays a positive role in cold tolerance. Similarly, the involvement of VvBAP1 in the resistance to heat stress was also found in the present study. The results indicated VvBAP1 was significantly induced by high temperature, and the elevated expression of VvBAP1 was significantly higher in the resistant cultivars than the sensitive cultivars under heat stress. Seed germination and phenotypic analysis results indicated that overexpression of VvBAP1 improved Arabidopsis thermoresistance. Compared with the wild type, the chlorophyll content and net photosynthetic rate in VvBAP1 overexpressing Arabidopsis plants were markedly increased under heat stress. At high temperatures, overexpression of VvBAP1 also enhanced antioxidant enzyme activity as well as their corresponding gene transcription levels, to reduce the accumulation of reactive oxygen species and lipid peroxidation. Besides, the transcriptional activities of HSP70, HSP101, HSFA2, and HSFB1 in VvBAP1 overexpressing Arabidopsis plants were significantly up-regulated compare to the wild type. In summary, we propose that VvBAP1 may play a potential important role in enhanced grapevine thermoresistance, primarily through the enhancement of antioxidant enzyme activity and promoted heat stress response genes expression.

Dataset Information

ClpB N-terminal domain plays a regulatory role in protein disaggregation.

Publications

ClpB N-terminal domain plays a regulatory role in protein disaggregation.

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