Multivalent contacts of the Hsp70 Ssb contribute to its architecture on ribosomes and nascent chain interaction

Hsp70 chaperones assist de novo folding of newly synthesized proteins in all cells. In yeast, the specialized Hsp70 Ssb directly binds to ribosomes. The structural basis and functional mode of recruitment of Ssb to ribosomes is not understood. Here, we present the molecular details underlying ribosome binding of Ssb in Saccharomyces cerevisiae. This interaction is multifaceted, involving the co-chaperone RAC and two specific regions within Ssb characterized by positive charges. The C-terminus of Ssb mediates the key contact and a second attachment point is provided by a KRR-motif in the substrate binding domain. Strikingly, ribosome binding of Ssb is not essential. Autonomous ribosome attachment becomes necessary if RAC is absent, suggesting a dual mode of Ssb recruitment to nascent chains. We propose, that the multilayered ribosomal interaction allows positioning of Ssb in an optimal orientation to the tunnel exit guaranteeing an efficient nascent polypeptide interaction.

The manuscript significantly advances and clarifies the knowledge about the mechanism by which Ssb interacts with the ribosome. The background is presented well, the data is nicely presented and rigorously tested and supports the authors' conclusions. The authors also include a useful discussion of the parallels with the analogous system in mammalian cells.
Suggested improvements 1. The authors show that binding of the APPY peptide to Ssb was reduced in the presence of ATP versus ADP ( Figure S5B). How does this information fit with what is known about the role of RAC in stimulating the ATPase activity of Ssb and the interaction of Ssb with nascent polypeptides?
2. Have the authors examined whether RAC is able to stimulate the ATPase activity of  3. For clarification purposes, it would be helpful to note whether RAC is present in the experiments where purified Ssb is added to salt-stripped and puromycin treated ribosomes ( Figure S2). 4. Figure 6E showing levels of ribosomal proteins in the Ssb-RAC delta strain complemented with WT Ssb or the Ssb mutant would be more convincing if quantification of the protein levels was provided.
5. Figure 7C shows that, in the absence of RAC, the Ssb1∆601-13 mutant does not crosslink to nascent chains. The authors claim this points to a critical role for RAC in directing Ssb to the nascent chains. It would be helpful to also show that Ssb1∆601-13 is able to bind nascent chains in otherwise WT cells. This would provide additional evidence that the primary defect of Ssb1∆601-13 is ribosome interaction rather than nascent chain interaction.

Reviewer #2 (Remarks to the Author):
Many fungi harbor a special form of the molecular chaperone Hsp70, Ssb, which is associated with the ribosome. Together with the ribosome-associated J-protein complex RAC, Ssb supports de novo protein folding at the ribosome. Furthermore Ssb is involved in ribosome biogenesis. The present manuscript investigates the molecular basis for the interaction of Ssb with ribosomes.
The authors identify two regions enriched in basic residues that are conserved in Ssb sequences but not in general cytosolic Hsp70s. These regions are located in the substrate binding domain  and close to the C-terminus (K603, R604, K608 and R613). Mutational analysis showed that the latter is essential for constitutive interaction with the ribosome, while the former improves ribosome binding. However, both the 601-613 deletion and KRKR-AAAA mutants appear to be functional as long as RAC is present, suggesting that RAC transiently recruits cytosolic Hsp70 isoforms Ssb and Ssa to the ribosomal exit tunnel. In absence of RAC, wt Ssb interacts with nascent chain whereas the mutant does not. This suggests that Ssb can also cooperate with other J-domain proteins. This aspect could be investigated further.
Somewhat unsurprisingly, interaction studies with a model substrate peptide showed no substantial differences of the Ssb mutant proteins compared to wildtype.
A limitation of the manuscript is that the binding site of Ssb on the ribosome was not addressed. Nevertheless, this is an interesting, well-written story that provides additional insight into cotranslational folding.

Reviewer #1
This reviewer stated that "the manuscript significantly advances and clarifies the knowledge about the mechanism by which Ssb interacts with the ribosome", but suggested five points for improvement: 1. The authors show that binding of the APPY peptide to Ssb was reduced in the presence of ATP versus ADP (Fig. S5B)

. How does this information fit with what is known about the role of RAC in stimulating the ATPase activity of Ssb and the interaction of Ssb with nascent polypeptides?
Authors: In general Hsp70s have a lower affinity to substrates in the ATP-bound state than in the ADP-bound one (for review see e.g. Mayer, 2013). This lower affinity is due to the fact that ATP binding increases both association and dissociation rates. In their allosteric cycle Hsp70s bind substrates in the ATP-induced open conformation. Substrate binding in synergism with a J-domain protein, which is RAC in the case of Ssb, trigger ATP hydrolysis to ADP, which leads to the closed conformation of Ssb and consequently tight peptide binding (see De Los Rios & Barducci, 2014). We analyzed the specificity of the Ssb-APPY interaction by adding apyrase or ATP, which both influence the accessibility of the substrate-binding pocket. Pretreatment with apyrase led to the expected reduction of peptide binding to Ssb, as the Hsp70 changed into its ADP-closed conformation with very low substrate association rates. In contrast, addition of ATP to Ssb led to the open conformation allowing efficient peptide interaction. ATP hydrolysis then induced lid closure and tight substrate binding. During the gel filtration ATP was absent allowing us to retain more peptide bound to Ssb when ATP was present than when apyrase was added previous to incubation with the peptide. We did not add RAC in this experiment as in vitro Hsp70 peptide-binding occurs without any co-factor albeit less efficient. However, published data show that RAC stimulates the ATPase of Ssb (Huang et al., 2005, see also next point) as well as binding of nascent chains (Gautschi et al., 2002;Willmund et al., 2013) and in general ATP-hydrolysis is stimulated most efficiently by a combination of both, substrate and Hsp40 co-chaperone (Mayer & Kityk, 2015). (Please note: Due to changes in the current manuscript the figure mentioned above is now labeled as Supplementary Fig. 6b).

Have the authors examined whether RAC is able to stimulate the ATPase activity of Ssb∆601-13?
Authors: We tested RAC stimulation for wt Ssb1 and Ssb1∆601-13 (Ssb1∆C) in vitro using purified proteins and found that RAC stimulates the ATPase activity of both wild type and mutant Ssb protein.
Much more effort has to be put into carefully elucidating the ATPase cycle of Ssb on ribosomes driven by substrates and co-chaperones. These analyses will be complex and time consuming and are thus beyond the scope of this study. As we consider the data as too premature in its current state, we would prefer not to add it to this manuscript. [Redacted] 3. For clarification purpose, it would be helpful to note whether RAC is present in the experiments where purified Ssb is added to salt-stripped and puromycin treated ribosomes (Fig. S2).

Authors:
The purified ribosomes that we used for the in vitro binding analyses of wt and mutant Ssb protein had been salt-stripped and puromycin treated to generate a homogeneous population of ribosomes. Thus, these ribosomes almost completely lack associated factors like NAC, RAC or Ssb. We did not include additional RAC to these binding analyses as Ssb interacts with ribosomes independently of RAC (Rakwalska et al., 2004). We now include data in the manuscript showing the Western blot analysis of NAC and RAC in our purified ribosome fraction (see Supplementary Fig. 2b) and changed the corresponding passage of the text: "To test ribosome binding in vitro, recombinantly purified wt Ssb1 and Ssb1∆601-13 protein was incubated with puromycin stripped and high salt washed yeast ribosomes ( Supplementary Fig. 2a) that were almost completely devoid of exit site associated factors like NAC or RAC ( Supplementary Fig. 2b)." Furthermore the figure legend was adapted as follows: "b) Salt-stripped and puromycin-treated yeast ribosomes were tested for the presence of ribosome associated factors by either SDS-PAGE and Coomassie staining (top) or by immunological detection of different proteins (bottom)." 4. Figure 6E showing levels of ribosomal proteins in the Ssb-RAC delta strain complemented with WT Ssb or the Ssb mutant would be more convincing if quantification of the protein levels was provided.
Authors: As suggested by the reviewer, we quantified the experimental data and show the statistics in Fig. 6f. This analysis clearly shows that in five independently performed biological replicates of Ssb-RAC∆ cells transformed with Ssb1∆601-13 the level of Rpl17A is strongly reduced in comparison to wt cells and also lower than in Ssb-RAC∆ cells transformed with wt Ssb1. For the sake of completeness we included this type of Rpl17A quantification also into the corresponding analysis in Fig. 3 (see Fig. 3f). This quantification clearly shows that in the presence of RAC the level of ribosomal proteins in ssb1,2∆ cells transformed with either Ssb1 or Ssb1∆601-13 is completely restored to wt levels in both cases. We adapted the figure legends of both figures as follows: "f) Quantification of Rpl17A protein levels as shown exemplarily in e). Rpl17A signals were normalized to Pgk1 loading control and wt cells transformed with empty vector were set as 100 %. Error bars represent SEM." 5. Figure 7C shows that, in the absence of RAC, the Ssb∆601-13 mutant does not crosslink to nascent chains. The authors claim this points to a critical role for RAC in directing Ssb to the nascent chains. It would be helpful to also show that Ssb∆601-13 is able to bind nascent chains in otherwise WT cells. This would provide additional evidence that the primary defect of Ssb∆601-13 is ribosome interaction rather than nascent chain interaction.