Pathway of Hsp70 interactions at the ribosome

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.

Structural remodeling of ribosome associated Hsp40-Hsp70 chaperones during co-translational folding

“…Although the RNC was prepared with a highly engineered nascent chain, our analyses indicate that these structures could at least structurally reflect distinct functional stages of RAC on the ribosome. Through integration of recent data 15 , 17 , 27 – 29 , 32 , 34 , and in light of the proposed substrate-relay model 34 , our results reveal a structural framework for understanding the interplay between RAC and Ssb, involving sequential conformational changes of Zuo1, Ssz1 and Ssb1, during co-translational folding (Fig. 7 ).…”

“…Given the relative size of these two HSP70 proteins in the limited space of the PTE, upon the addition of Ssb1-ATP, Ssb would compete with Ssz1 for the general location of the PTE. Indeed, Ssb SBD was reported to be capable of interacting with the ribosome 27 , 28 , and its SBDβ could be crosslinked with uL24 in vivo 29 . Therefore, successive extensive structural remodeling of RAC ought to take place, first upon Ssb-ATP binding and then after ATP-hydrolysis of Ssb.…”

“…7f ). Ssb-ADP after these conformational changes loses its contact with Zuo1 and establishes its direct interaction with the ribosome through its Ssb SBD 27 – 29 . With a further growing of nascent chain, substrate-engaged Ssb-ADP may completely detach from the ribosome, and this is likely the reason that we did not observe solid density for Ssb1 in the structures from the RNC-RAC-Ssb1 dataset.…”

“…The ATP-hydrolysis on Ssb enables the conformational cycling of Ssb to a high-affinity state (ADP-bound) for stable nascent peptide binding 1 , 2 until productive folding occurs. Ssb is a typical HSP70, and attaches to the peptide tunnel exit (PTE) region of the ribosome through its SBD 27 – 29 . The function of Zuo1 in the ATPase stimulation of Ssb requires the RAC as a whole 25 .…”

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex

“…The position of Ssb at the ribosome has remained quite puzzling despite several cross-link studies 3,5,[19][20][21][22][23][24] . Recent data place Ssb next to the tunnel exit with different binding modes (with bound ATP or ADP) 24 .…”