Structure and evolution of the 4-helix bundle domain of Zuotin, a J-domain protein co-chaperone of Hsp70

Abstract

The J-domain protein Zuotin is a multi-domain eukaryotic Hsp70 co-chaperone. Though it is primarily ribosome-associated, positioned at the exit of the 60S subunit tunnel where it promotes folding of nascent polypeptide chains, Zuotin also has off-ribosome functions. Domains of Zuotin needed for 60S association and interaction with Hsp70 are conserved in eukaryotes. However, whether the 4-helix bundle (4HB) domain is conserved remains an open question. We undertook evolutionary and structural approaches to clarify this issue. We found that the 4HB segment of human Zuotin also forms a bundle of 4 helices. The positive charge of Helix I, which in Saccharomyces cerevisiae is responsible for interaction with the 40S subunit, is particularly conserved. However, the C-termini of fungal and human 4HBs are not similar. In fungi the C-terminal segment forms a plug that folds back into the bundle; in S . cerevisiae it plays an important role in bundle stability and, off the ribosome, in transcriptional activation. In human, C-terminal helix IV of the 4HB is extended, protruding from the bundle. This extension serves as a linker to the regulatory SANT domains, which are present in animals, plants and protists, but not fungi. Further analysis of Zuotin sequences revealed that the plug likely arose as a result of genomic rearrangement upon SANT domain loss early in the fungal lineage. In the lineage leading to S . cerevisiae , the 4HB was subjected to positive selection with the plug becoming increasingly hydrophobic. Eventually, these hydrophobic plug residues were coopted for a novel regulatory function—activation of a recently emerged transcription factor, Pdr1. Our data suggests that Zuotin evolved off-ribosome functions twice—once involving SANT domains, then later in fungi, after SANT domain loss, by coopting the hydrophobic plug. Zuotin serves as an example of complex intertwining of molecular chaperone function and cell regulation.

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex

“…Different studies already investigated the 4HB interaction with ES12 22,40 , however, so far only with perturbed or truncated systems. While it was previously envisaged that a direct coupling between RAC binding and the ribosome active center occurs via the central rRNA helix H44 including ES12 at its tip 23 , our structures suggest the RAC influence on fidelity to depend on its constant binding probably by modulating the speed of ratcheting. Further functional and especially high-resolution structural studies of all components of stalled on-pathway complexes are needed to finally unveil the complete movie of this unique co-translational chaperone triad in protein biosynthesis.…”

“…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 .…”

“…Further functional and especially high-resolution structural studies of all components of stalled on-pathway complexes are needed to finally unveil the complete movie of this unique co-translational chaperone triad in protein biosynthesis. The absence of a Ssz1 homolog in humans and the presence of additional domains in hs Zuo1 together with off-ribosomal transcriptional functions of Zuo1 and Ssz1 23,41 promise further surprises from this puzzling Hsp70 chaperone system.…”

“…RAC binding to the ribosome has been thoroughly studied by cross-linking experiments and low-resolution cryo-EM structures showing flexible conformations on idle 80S ribosomes and interactions with both the 40S and 60S subunits 5,[19][20][21][22][23][24] .…”

Zuo1 supports G4 structure formation and directs repair toward nucleotide excision repair

“…Although the function of ZRF1 is not clear, yet, it is conceivable to expect similarities with Zuo1 in supporting G4 structures and NER recruitment. Indeed, it has been shown that zuo1Δ growth defect can be rescued by expressing the human orthologue ZRF1 69 . Interestingly, the NER complex component Mms1 (DDB1 in human) can bind to G4 structures 58 , further underlining the importance of G4 formation for NER function.…”