Pathway of Hsp70 interactions at the ribosome

Kanghyun Lee,Wojciech Delewski,Thomas Ziegelhoffer,Grzegorz Sabat,Elizabeth A Craig,Scott E Berger

doi:10.1038/s41467-021-25930-8

Abstract

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb’s ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

Highlights

In eukaryotes, an Hsp[70] molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel
The cycle is completed by the action of a nucleotide-exchange factor (NEF), facilitating release of ADP, that is followed by binding of ATP and substrate release[10,26]
Starting with Ssz[1], we incorporated Bpa at 15 positions distributed across the surface of its nucleotide-binding domain (NBD)

Summary

Introduction

An Hsp[70] molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In a manner consistent with the ADP conformation, crosslinks to ribosomal proteins across the tunnel exit from Zuo[1]. These two modes of Hsp[70] Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz[1], allowing optimal J-domain and nascent chain engagement; after ATP hydrolysis, Ssb(ADP) directly with the ribosome. In that a high proportion is found associated with ribosomes, Ssbs are canonical Hsp70s—they have the typical Hsp70-domain architecture and undergo efficient, adenine nucleotide-dependent cycles of interaction with substrate polypeptides[24,25]. The cycle is completed by the action of a nucleotide-exchange factor (NEF), facilitating release of ADP, that is followed by binding of ATP and substrate release[10,26]

Methods

Results

Conclusion