Deciphering molecular details of the RAC\u2013ribosome interaction by EPR spectroscopy

Sandra J Fries,Christoph Globisch,Theresa S Braun,Christine Peter,Malte Drescher,Elke Deuerling

doi:10.1038/s41598-021-87847-y

Sandra J Fries, Christoph Globisch + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-87847-y

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Journal: Scientific Reports	Publication Date: Apr 21, 2021
Citations: 5	License type: open-access

Affiliation: University of Konstanz

Abstract

The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC–ribosome contact affecting translational accuracy. We identified a local contact point of RAC to the ribosome. The data provide the first experimental evidence for the existence of a four-helix bundle as well as a long α-helix in full-length RAC, in solution as well as on the ribosome. Additionally, we complemented the structural picture of the region mediating this functionally important contact on the 40S ribosomal subunit. In sum, this study constitutes the first application of SDSL-EPR spectroscopy to elucidate the molecular details of the interaction between the 3.3 MDa translation machinery and a chaperone complex.

Highlights

The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding
For a strategic cysteine placement, we built a working model (Fig. 1b) combining available structural fragments of Zuo[1]: the crystal structure of the Zuo[1] homology domain (ZHD)[6] and the NMR structure of the 4HB8 connected by a modeled α-helix as middle domain (Supplementary Fig. S1)
Earlier cryo-EM analysis[5] revealed that yeast RAC contacts both subunits of the 80S ribosome using three binding sites in Zuo[1], with C1 and C2 binding to the 60S subunit close to the polypeptide exit tunnel and C3 in the C-terminus of Zuotin contacting a helical RNA element (ES12) of the 40S subunit that elongates into the decoding center of the ribosome (Fig. 1B)

Summary

Introduction

The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. A modeled α-helix as middle domain combined with the NMR structure of the 4 HB8 were recently fitted into the cryo-EM density of the RAC–ribosome complex[5], providing a structural model for Zuo[1] C-terminal region. Whether this model reflects the situation of full-length RAC on the ribosome is unknown. In this study we applied EPR spectroscopic methods combined with site-directed spin labeling (SDSL)[9] to analyze the functionally important Zuo1-40S contact on a molecular level. We examined the structure of Zuo1’s C-terminus as well as potential conformational changes upon ribosome binding by double electron–electron resonance (DEER) distance measurements, which are suitable to reveal movements within DNA and proteins in the nanometer r ange[12,13,14]

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Results

Conclusion