Ccr4 Caf1
II. The eukaryotic ribosome-associated protein quality control system
4. Results and discussion
4.5. Structural analysis of the ribosome-associated complex (RAC) reveals an unusual Hsp70/Hsp40 interaction
Fiaux J., Horst J., Scior A., Preissler S., Koplin A., Bukau B., Deuerling E.
J Biol Chem. 2010 Jan 29;285(5):3227-34.
Contributions
1. Design of the cloning strategy for Zuotin fusion constructs 2. Contribution to pulldown experiments
3. Discussion of experiments and proofreading the manuscript
Abbreviations: Zuo, Zuotin; RAC, ribosome-associated complex; ATP, adenosine triphosphate; HDX, hydrogen-deuterium exchange; MS, mass spectrometry; aa, amino acid
4.5.1. Objective
In Saccharomyces cerevisiae, Ssz and Zuotin (Zuo) belong to the Hsp70 and Hsp40 chaperone families, respectively (see introduction). Whereas Hsp70/Hsp40 interactions are usually transient, Ssz and Zuo form a stable heterodimer termed ribosome-associated complex (RAC). Another special feature is that RAC localizes to ribosomes presumably through a positively charged region in the C-terminus of Zuo (Gautschi et al, 2001; Peisker et al, 2008; Yan et al, 1998). Zuo acts as J-protein partner for the ribosome-associated Hsp70 Ssb by stimulating its ATPase activity (Gautschi et al, 2002; Huang et al, 2005). However, this function critically depends on complex formation with Ssz (Huang et al, 2005), suggesting that RAC and Ssb form a functional chaperone triad (Ssb-RAC) on ribosomes (Figure 8). The observation that yeast strains lacking Ssb have almost the same phenotype as ssz1Δ and zuo1Δ cells, and that combinations of these deletions have no additive effects, supports this hypothesis (Gautschi et al, 2001; Jones et al, 2003; Nelson et al, 1992; Yan et al, 1998). The role of Ssz in this tripartite chaperone system is particularly difficult to interpret. Its in vivo function does neither depend on ATP binding and hydrolysis (Conz et al, 2007; Huang et al, 2005), nor on substrate interactions via its putative peptide-binding domain (Hundley et al, 2002). It was therefore proposed that Ssz might be required to facilitate the ability of Zuo to act as a cofactor for Ssb (Huang et al, 2005). Interestingly, a mammalian heterodimeric ribosome-associated complex, composed of Hsp70L1 and the J-protein Mpp11, was recently discovered (Hundley et al, 2005; Otto et al, 2005), indicating
In order to understand how unusual Hsp70/Hsp40 interactions evolved to fulfill specialized tasks, it is necessary to study their molecular design. However, no structural data for yeast RAC or its orthologs could be obtained so far. We thus investigated the architecture as well as the conformational dynamics of yeast RAC by combining biophysical and biochemical methods.
4.5.2. Summary of the experimental data
A central objective of the project was to identify the binding interface of Zuo and Ssz in the RAC complex. Therefore, amide hydrogen-deuterium exchange (HDX) in combination with mass spectrometry (MS) was used to detect conformational changes in the individual subunits of RAC upon complex formation (Rist et al, 2006; Rist et al, 2003; Wales & Engen, 2006). HDX experiments are based on the exchange of amide protons of a protein with solvent deuterons. Thereby, the exchange rate depends on the accessibility of the protons.
Whereas exposed protons exchange fast for a deuteron, internal protons or the ones, which participate in hydrogen bonding, remain protected. Conformational transitions or the interactions with binding partners thus result in shifts of the molecular mass in the affected protein areas due to changes in the accessibility of protons.
The HDX experiments were performed with recombinant proteins expressed in E. coli cells.
Ssz and Zuo were purified individually to study conformational changes caused by complex formation. Additionally, Ssz and Zuo were coexpressed to isolate preformed RAC complexes. The proteins were diluted into a D2O containing buffer solution to start the HDX reaction. Samples were then taken at different time points and the exchange process was quenched. Deuteron incorporation was subsequently analyzed by MS.
Determination of the total number of incorporated deuterons in each individual protein revealed that Zuo is structurally highly dynamic. Ssz showed even faster exchange kinetics, suggesting a rather loose conformation. However, addition of ATP resulted in decreased exchange rates, indicating a structural stabilization of Ssz in the ATP-bound state. In the next step, HDX experiments were performed with Zuo and Ssz bound to each other. Whereas Zuo showed a small but significant decrease in deuteron incorporation when assembled into the RAC complex, a pronounced HDX protection was observed for Ssz. This suggests that Zuo and Ssz are both stabilized by RAC formation.
To map the regions within Zuo and Ssz, which are affected by complex formation and thus may contribute to the interaction surface, the HDX samples were digested into peptides prior MS analysis. The data showed that Ssz interacts mainly via the C-terminal domain with Zuo.
Vice versa, the N-terminal segment of Zuo, which appeared to be highly dynamic in the
Results and discussion
absence of Ssz, was significantly stabilized in RAC. Thus, the mutual stabilization of the C-terminal domain of Ssz and the N-terminus of Zuo are crucial for RAC assembly.
The interaction between Zuo and Ssz resulted also in HDX deprotection in the J-domain of Zuo, indicating a conformational opening of this region upon RAC formation. It is therefore tempting to speculate, that the increased dynamics of the J-domain might be important for the function of RAC as a co-chaperone for Ssb on the ribosome.
Figure 25: The formation of the ribosome-associated complex (RAC) is based on an unusual Hsp70/Hsp40 interaction. A) Ssz associates with chimeric fusion proteins carrying the terminal region of Zuo in vivo. Two N-terminal fragments of Zuo (aa 1-62 and 1-102, upper panel) were fused to the J-domain of Ydj1 and a C-N-terminal His6-tag, respectively. The constructs were expressed individually in zuo1Δ cells from a plasmid. Afterwards, the fusion constructs were affinity purified via the His6-tag using a Ni-NTA affinity matrix. The eluates were analyzed for copurification of Ssz by Western blotting. The Zuo fusion constructs were visualized by immunoblotting against the His6-tag. As a control, the zuo1Δ cells were transformed with an empty plasmid (vector). The totals show that the levels of Ssz were similar in the lysates used for the pulldown experiment. Ssz signals were obtained specifically in the eluate fractions of samples in which the fusion constructs were expressed. B) Model for RAC complex formation and its architecture. Binding of ATP (yellow) stabilizes the ATPase domain of Ssz (blue).
However, complex formation with Zuo (pink) is most likely independent of the ATP status of Ssz. The formation of a kinetically stable RAC complex involves the N-terminus of Zuo and the C-terminal domain of Ssz, and leads to significant stabilization of the participating regions in both proteins. The regions in Ssz and Zuo, which show changes in the conformational dynamics upon RAC formation, are indicated in dark blue and purple, respectively.
Binding of Zuo to Ssz results in increased dynamics around the HPD motif within the J-domain of Zuo. The increased dynamics of the J-domain may enable RAC to act as a co-chaperone. Binding of ATP to RAC further stabilizes the ATPase domain of Ssz but has no effect on Zuo.
The observation that the N-terminus of Zuo is stabilized in the RAC complex was confirmed B
Totals
Eluates
α-Ssz α-Ssz
α-His6 vector Zuo-(1-62)Zuo-(1-102)
Zuo-(1-102)
Zuo-(1-62)
A
J-domain (Ydj1) His6 Zuo N-term.
1-62
1-102 J-domain (Ydj1) His6
terminal 62 aa (Zuo-ΔN), was unable to interact with Ssz in vitro. This suggests that the N-terminus of Zuo (aa 1-62) indeed is the major contact region for Ssz. Accordingly, zuo1Δ cells expressing Zuo-ΔN showed a phenotype similar to the one of ssz1Δ cells. Next, we asked whether the N-terminus of Zuo is sufficient to establish a stable interaction with Ssz.
Therefore, a reverse experiment was performed where the N-terminus of Zuo (either aa 1-62 or aa 1-102) was fused to the J-domain of another yeast Hsp40, which does not interact with Ssz or Ssb on its own, as well as to a C-terminal His6-tag (Figure 25A). These constructs, designated Zuo-(1-62) and Zuo-(1-102), were expressed individually in zuo1Δ yeast cells.
The association of Ssz with these constructs was tested by pull-down assays using the His6 -tag of the fusion proteins. As shown in Figure 25A, Ssz copurified specifically with both fusion variants carrying N-terminal Zuo fragments, whereas no Ssz was detected in the control sample. Together, the experiments show that the N-terminus of Zuo is required and sufficient to mediate complex formation with Ssz in vivo. Based on these results we propose a model according to which the N-terminal extension of Zuo and the C-terminal domain of Ssz, as well as their mutual stabilization, provide the structural basis for RAC formation (Figure 25B).
Results and discussion