Structural and mutational analysis of the ribosome-arresting human XBP1u.

Vivekanandan Shanmuganathan,Roland Beckmann,Kenji Kohno,Katharina Braunger,Florian Cymer,Nina Schiller,Gunnar Von Heijne,Otto Berninghausen,Birgitta Beatrix,Anastasia Magoulopoulou,Jingdong Cheng

doi:10.7554/elife.46267

Vivekanandan Shanmuganathan, Roland Beckmann + Show 9 more

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https://doi.org/10.7554/elife.46267

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Abstract

XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.

Highlights

Polypeptide stretches, which can induce ribosomal stalling to regulate gene expression, are called ribosomal arrest peptides (AP)
The construct used for the RNC preparation encompassed only the hydrophobic region 2 (HR2) domain and the XBP1u pausing sequence denoted as AP, with N- and C-terminal tags for affinity purification and detection purposes, Figure 2A
A major portion of the XBP1u peptide in the exit tunnel was resolved to between 3.0–3.5 Afor both classes (Figure 2—figure supplement 3A,B), whereas the resolution in the part distal to the PTC near the exit was worse than 4 A, apparently due to flexibility of the nascent chain

Summary

Introduction

Polypeptide stretches, which can induce ribosomal stalling to regulate gene expression, are called ribosomal arrest peptides (AP). Depending on the context APs can inhibit translation during elongation (SecM, VemP) (Ishii et al, 2015; Su et al, 2017; Tsai et al, 2014), termination (TnaC, CMV uORF2 (cytomegalovirus upstream open reading frame 2) and SAM-DC uORF (S-adenosyl-methionine decarboxylase) (Gong et al, 2001; Janzen et al, 2002; Raney et al, 2002) or both in some cases (ErmCL, MifM and AAP) (Chiba and Ito, 2012; Fang et al, 2000). Properties of these APs vary in a way that some have a defined stall position (Ishii et al, 2015), while others have multiple stalling sites (Chiba and Ito, 2012; Tsai et al, 2014)

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