Revealing the interaction mode of the highly flexible Sorghum bicolor Hsp70/Hsp90 organizing protein (Hop): A conserved carboxylate clamp confers high affinity binding to Hsp90
Graphical abstract
Introduction
A polypeptide usually requires folding to reach its folded and functional state and the opposite, partial unfolding or misfolding may eventually result in loss of function [1,2]. Non-folded proteins may be degraded by the proteasome, an important part of the cellular Protein Quality Control (PQC) system, which has evolved to maintain proteostasis [3]. Besides the proteasome, chaperones also are part of the PQC and have a central role in proteostasis since they are involved in aiding folding and avoiding, as well as rescuing, misfolded proteins [[4], [5], [6]]. Since stress is one of the main conditions that perturb proteostasis most chaperones are heat shock proteins (Hsps).
The machinery formed by the Hsp70/Hsp90 system is one of the most important subsystems of the PQC system and is involved in the maintenance of several physiological functions [[7], [8], [9]]. Hsp70 is dedicated to the folding of nascent proteins and is also part of a disaggregation system in metazoans (for review see [10,11]) while Hsp90 interacts with about 10% of the proteome [12] and has been investigated as a target for cancer therapy (for reviews see [13,14]). Besides Hsp70 and Hsp90, the subsystem is formed by several co-chaperones that facilitate the interactions between the two chaperones and their substrates. Among these co-chaperones is Hsp70/Hsp90 organizing protein (Hop), or stress-inducible protein 1 (Sti1) as it is known in yeast, which binds the Hsp70 and Hsp90 chaperones and enables complex formation with both at the same time [15,16]. Hop has been implicated in several physiological functions linked to disease states, for instance cancer and transmissible spongiform encephalopathies [[17], [18], [19]].
Hop/Sti1 is built from an N-terminal tetratricopeptide repeat (TPR) domain (TPR1) associated with an aspartate-proline (DP) rich domain (DP1), which together with the subsequent long linker create a flexible module that is connected to a rigid C-terminal module made from the core TPR2A-TPR2B domain and a second DP domain (DP2) [15,16]. Tetratricopeptide repeats are all α-helical structural motifs, whose basic repeat is a sequence of 34 amino acids [[20], [21], [22]]. Usually, 3–16 of these repeats pack together in tandem to form a spiral of antiparallel α-helices that creates an amphipathic groove, thereby providing an interaction site for the target protein. The TPR domains of Hop/Sti1 have been extensively studied and it is known that both the N-terminal TPR1 domain and the TPR2B domain interact with the Hsp70 C-terminal hexapeptide, while the TPR2A domain has been shown to bind the C-terminal pentapeptide of Hsp90 [23,24]. Both chaperones contain an EEVD motif in the C-terminus, which provides affinity for the binding to Hop, while specificity is mediated by amino acids immediately N-terminal to this motif [25]. Furthermore, also other parts of the chaperones are in contact with Hop, thereby strengthening the interaction. On the other hand, the specific function of the DP domains has not yet been fully established, but studies of the Sti1 DP2 domain indicate that it is important for client activation [26].
Most of the studies concerning Hop/Sti1 have been done with the human and yeast proteins and specific information about homologs in plants is still mainly related to functional studies in a small number of species [27,28]. The study of plant chaperones, especially from sorghum and sugar cane species [29,30], has both agronomic and economic benefits, as well as importance for bioethanol production. Sorghum bicolor is a C4 grass that is among the five most important cereals for human agriculture. More recently, Sorghum has been considered as a biomass feedstock. Biomass is the largest renewable resource for producing biofuels to address the diminishing fossil energy reserves and to mitigate the environmental issues from the petroleum-based fuels. Also, among energy crops, Sorghum is a unique plant feedstock in which its aboveground growth components (stem sap sugar, biomass or grain) can be converted into biofuel. Thus, understanding the functional and structural mechanism of Sorghum chaperones, which are directly involved in stress responses, has the potential to bring benefits to both agriculture and biofuel studies.
Here we present the production, purification and structural characterization of Sorghum bicolor Hop (SbHop), which show that the protein folds into a stable monomeric fold typically seen for Hop/Sti1 proteins. Furthermore, the results indicate a conserved interaction pattern between SbHop and the Hsp90 chaperone. This study not only contributes to the still limited information on Hop/Sti1 homologs from plants, but can also help to develop tools for enhancing the environmental stress tolerance of S. bicolor, which is an important crop plant.
Section snippets
Protein and peptide preparation
Sorghum bicolor hop gene was cloned into a pET28a vector and transformed into BL21(DE3)pRare by heat shock. The cells were grown overnight at 37 °C in LB medium with kanamycin until the absorbance (A600nm) reached 0.6–0.8. Protein expression was induced with 0.4 mM isopropyl β-d-thiogalactoside at 20 °C under constant shaking for 4 h. The cells were centrifuged and 15 mL of 25 mM Tris-HCl, 150 mM NaCl, pH 7.4 buffer mixed with 5 μL DNase (1000 U, Promega, WI, USA), 9 μL Lysozyme (50 mg/mL) and
SbHop was purified as a folded monomer
Recombinant SbHop was obtained in the soluble fraction after cell lysis (lane 3 in Fig. S1) and was >84% pure after Ni2+-affinity chromatography (lane 4 in Fig. S1) and >95% pure after size exclusion chromatography (lane 5 in Fig. S1 and Table 1). Circular dichroism (CD) experiments showed spectra with minima of molar ellipticity at 208 and 222 nm (Fig. S2), which is typical for mainly α-helical proteins. Based on the signal at 222 nm, the helical content was predicted to be of about 60% (Table
Conclusions
Hop/Sti1 is one of the most important co-chaperones of the Hsp70/Hsp90 system because of its function as a scaffold for the interaction of these two chaperones, which both are involved in a plethora of important functions related to cell proteostasis. As a matter of fact, several recent findings establish Hop in the center of many diseases. Currently, there is much more information on human and yeast homologs than for plants, although plants are exposed to continuous environmental stress. Since
Acknowledgements
This study was funded by Fundação de Amparo do Estado de São Paulo FAPESP (2012/50161-8, 2014/17264-3 and 2015/15822-1), CNPq (305018/2015-9 and 306943/2015-8) and CAPES (88887.125517/2016-00). We thank the National Laboratory of Synchrotron Light (Campinas, SP, Brazil) and its staff for the use of SAXS beam line facilities. We acknowledge the Protein Analysis Facility (Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Switzerland) for cross-linking MS
Author contributions
Cloning and purification: LMZ; Initial characterization: RA, LMZ; Calorimetry: RA; Mass spectroscopy: DS, TBL; HDX: TBL, FCG; SAXS: GMSP, LB; Modeling: KMD; Conception and design of the work: CHIR; Wrote the paper: all authors.
Conflict of interest
The authors declare that they have no conflict of interest.
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- 1
The authors contributed equally to this work.
- 2
Present address: R.A.: Center for Natural and Human Science (CCNH), Federal University of ABC (UFABC), Santo André, São Paulo, Brazil; L.M.Z.: Brazilian Bioethanol Science and Technology Laboratory (CTBE), National Center for Research in Energy and Materials, Campinas, São Paulo, Brazil.