Journal of Biological Chemistry
Volume 285, Issue 5, 29 January 2010, Pages 3227-3234
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Protein Structure and Folding
Structural Analysis of the Ribosome-associated Complex (RAC) Reveals an Unusual Hsp70/Hsp40 Interaction*

https://doi.org/10.1074/jbc.M109.075804Get rights and content
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Yeast Zuotin and Ssz are members of the conserved Hsp40 and Hsp70 chaperone families, respectively, but compared with canonical homologs, they atypically form a stable heterodimer termed ribosome-associated complex (RAC). RAC acts as co-chaperone for another Hsp70 to assist de novo protein folding. In this study, we identified the molecular basis for the unusual Hsp70/Hsp40 pairing using amide hydrogen exchange (HX) coupled with mass spectrometry and mutational analysis. Association of Ssz with Zuotin strongly decreased the conformational dynamics mainly in the C-terminal domain of Ssz, whereas Zuotin acquired strong conformational stabilization in its N-terminal segment. Deletion of the highly flexible N terminus of Zuotin abolished stable association with Ssz in vitro and caused a phenotype resembling the loss of Ssz function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuotin, and their mutual stabilization are the major structural determinants for RAC assembly. We furthermore found dynamic changes in the J-domain of Zuotin upon complex formation that might be crucial for RAC co-chaperone function. Taken together, we present a novel mechanism for converting Zuotin and Ssz chaperones into a functionally active dimer.

Chaperones
Chaperones/Protein Folding
Chaperones/Heat Shock
Protein/Folding
Protein/Protein-Protein Interactions
Hsp40
Hsp70
Ribosome

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*

This work was supported in part by the Konstanz Graduate School Chemical Biology and the Interdisciplinary Research Center Proteostasis, a fellowship of the Zukunftskolleg (to S. P.), an HFSP fellowship (to J. F.), and DFG Grants DE-783/3-1 (to E. D.) and SFB638 (to B. B.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental materials and Figs. S1–S5.

1

Present address: Novartis Pharma AG, 4057 Basel, Switzerland.

2

Both authors contributed equally to this work and are listed alphabetically.