AtTPR10 Containing Multiple ANK and TPR Domains Exhibits Chaperone Activity and Heat-Shock Dependent Structural Switching

Abstract

Among the several tetratricopeptide (TPR) repeat-containing proteins encoded by the Arabidopsis thaliana genome, AtTPR10 exhibits an atypical structure with three TPR domain repeats at the C-terminus in addition to seven ankyrin (ANK) domain repeats at the N-terminus. However, the function of AtTPR10 remains elusive. Here, we investigated the biochemical function of AtTPR10. Bioinformatic analysis revealed that AtTPR10 expression is highly enhanced by heat shock compared with the other abiotic stresses, suggesting that AtTPR10 functions as a molecular chaperone to protect intracellular proteins from thermal stresses. Under the heat shock treatment, the chaperone activity of AtTPR10 increased significantly; this was accompanied by a structural switch from the low molecular weight (LMW) protein to a high molecular weight (HMW) complex. Analysis of two truncated fragments of AtTPR10 containing the TPR and ANK repeats showed that each domain exhibits a similar range of chaperone activity (approximately one-third of that of the native protein), suggesting that each domain cooperatively regulates the chaperone function of AtTPR10. Additionally, both truncated fragments of AtTPR10 underwent structural reconfiguration to form heat shock-dependent HMW complexes. Our results clearly demonstrate that AtTPR10 functions as a molecular chaperone in plants to protect intracellular targets from heat shock stress.

1. Introduction

Terrestrial plants face diverse abiotic and biotic stresses during their life cycles. To cope with these environmental stresses, plants employ various important defense-associated proteins, such as pathogenesis-related (PR) proteins (chitinase, glucanase, thaumatin, defensin, and thionin), stress-resistant peptides, and highly efficient defense signaling systems. Among the defense-related proteins, tetratricopeptide (TPR) repeat proteins, which mediate protein–protein interactions, assemble as multiprotein complexes to defend against external stresses [1,2,3]. The Arabidopsis thaliana genome encodes more than 16 TPR-containing proteins. Previously, TPR motifs were detected in the FKBP52 protein, which facilitates the folding of target proteins with the help of heat shock protein 90 (HSP90) [4], and in cyclophilin 40 (CyP40), a cyclosporine A-binding immunophilin that supports protein stability and refolding of protein aggregates through the action of its hydrophobic regions [5]. In addition, CHIP, a ubiquitin E3-ligase protein containing three TPR repeats and a U-box domain, protects membrane channel proteins and degrades damaged proteins under extreme temperature conditions [6,7]. In Arabidopsis, TPR motif-containing proteins have been reported to function in the regulation of plant growth and development and hormone signaling [8]. Thus, the TPR-repeat thioredoxin (Trx)-like 1 (TTL1) protein affects abscisic acid (ABA) sensitivity and osmotic stress responses in plants [9]. Ectopic expression of the spindly (SPY) gene showed that SPY functions as a negative regulator of gibberellin (GA) response, affecting hypocotyl elongation and flowering time in plants [10]. In rice (Oryza sativa L.), the Trx-like protein OsTDX, which contains three N-terminal TPR motifs and a C-terminal Trx domain, exhibits defense activity against various fungal pathogens [11]. Moreover, OsTPR1 participates in the immune response by disturbing the interaction between chitin and MoChia1, a suppressor of chitin-triggered plant immunity [12].

In contrast to other types of Arabidopsis TPRs, AtTPR10 contains seven N-terminal repeats of the ankyrin (ANK) domain and three C-terminal repeats of the TPR domain. Similar to the TPR motifs, the ANK repeat domains mediate protein–protein interaction in diverse organisms including eukaryotes, prokaryotes, and some viruses [13,14]. The domains also contain several conserved hydrophobic residues necessary for maintaining their secondary and tertiary structures [15,16,17]. Genomic analyses revealed a total of 509 ANK repeats in more than 100 proteins in Arabidopsis with structurally similar characteristics. The ANK repeat-containing plant proteins regulate the cell cycle, mitochondrial enzyme activity, ion transport, and signal transduction [13].

These data suggest that the two domains play diverse physiological roles in plants by associating with different partner proteins. Although the AtTPR10 protein consists of two highly important domains (TPR and ANK) in a single polypeptide structure, its molecular function remains unknown. Here, we performed bioinformatics analysis of AtTPR10 gene expression and investigated the biochemical characteristics of the AtTPR10 protein. Our results show that AtTPR10 functions as a molecular chaperone to protect plants from diverse abiotic stresses that can be used for the development of stress-tolerant crops.

2. Materials and Methods

2.1. Plasmid Construction and Recombinant Protein Purification

Sequences of the full-length AtTPR10 gene, ankyrin domain (ANK-D), and TPR domain (TPR-D) were amplified from the Arabidopsis cDNA library by PCR using the primer pairs AtTPR10 forward (F)/AtTPR10 reverse (R) (5′-GGATCCATGGCTCCTGATGC-TTCTACT-3′/5′-GTCGACTTATGA-TTTGGCCTTAACCTC-3′), ANK-D_F/ANK-D_R (5′-GGATCCATGAACAAACGTGGAGCGCTT-CACT-3′/5′-GTCGACTTAAGTTTCTGGTTTTGTTGTCAATGGG-3′), and TPR-D_F/TPR-D_R (5′-GGATCCATGGCCGCAGAAGCAAAAGCCA-3′/5′-GTCGACTTAGCTTTCAGGGCTAAGCAAT-ACTCC-3′), respectively. The PCR products were cloned into the pGEM-T^easy vector (Promega Co., Madison, USA), and the resulting pGEM-T^easy-AtTPR10, -ANK-D, and -TPR-D constructs were sequenced. Then, these plasmids were digested with BamHI and SalI, and cloned into the pGEX-2T expression vector (New England Biolab, Ipswich, MA, USA) to generate fusions with the glutathione S-transferase (GST) gene, and the resulting DNA constructs were used for protein expression in Escherichia coli BL21 (DE3) cells. The transformed bacteria were grown in Luria broth (LB) containing 50 μg/μL ampicillin at 37 °C until the optical density at 600 nm absorbance (O.D.₆₀₀) approached 0.7. Then, expression of the recombinant GST fusion proteins was induced by the addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the cells were cultured for another 5 h. The cells were then harvested by centrifugation at 6000× g for 5 min, and the pellet was resuspended in 1X phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄ [pH 7.6]). The GST-AtTPR10, GST-ANK-D, and GSR-TPR-D fusion proteins were purified from the cells using GST agarose resin, and the eluted GST-tagged proteins were cleavaged by thrombin. The mature proteins were then analyzed by dialysis using 50 mM HEPES-KOH (pH 8.0). Each protein was further purified using a TSK G4000SW_XL high performance liquid chromatography (HPLC) column (30 cm × 7.8 mm), as described previously [18,19].

2.2. Determination of Holdase Chaperone Activity

Holdase chaperone activity was measured using malate dehydrogenase (MDH; Sigma-Aldrich) and citrate synthase (CS) as substrates [20,21]. MDH and CS were incubated with AtTPR10, ANK-D, and TPR-D in 50 mM HEPES-KOH (pH 8.0) at 43 °C for 15 min, and the heat-induced thermal aggregation of both substrates was monitored using a DU800 spectrophotometer (Beckman, Brea, CA, USA) equipped with a thermostatic cell holder preheated to 43 °C, as described previously [22].

2.3. Size Exclusion Chromatography (SEC)

HPLC (Dionex, Sunnyvale, CA, USA) was carried out at 23 °C on a Superdex 200 HR 10/30 column equilibrated with 50 mM HEPES-KOH (pH 8.0) containing 100 mM NaCl. Proteins were eluted using the same buffer at a flow rate of 0.5 mL/min, as described previously [23]. The fractionated protein peaks (A₂₈₀) were isolated and concentrated at 4 °C using a Centricon YM-10 (Millipore, Billerica, MA, USA).

2.4. Measurement of Bis-ANS Fluorescence

A mixture of AtTPR10, ANK-D, and TPR-D (20 µg/mL each) in 50 mM HEPES (pH 8.0) was added to 10 µM 4,4′-bis(phenylamino)-[1,1′-binaphthalene]-5,5′-disulfonic acid dipotassium salt (bis-ANS), and the resulting mixture was incubated at 23 °C for 30 min. Excess bis-ANS was removed by dialysis using the sample buffer, and its fluorescence intensity was measured using a SFM25 spectrofluorometer (Kontron, Instruments SpA, Milan, Italy), as described previously [22].

2.5. Abiotic Stress Treatments and AtTPR10 Expression Analysis

Two-week-old seedlings of Arabidopsis thaliana ecotype Columbia (Col-0; wild-type [WT]) were grown in Murashige and Skoog (MS) medium containing 3% sucrose. To perform the heat shock treatment, temperature was increased from 23 °C to 37 °C or 45 °C and then held constant for 3 h [24]. To conduct the cold treatment, the temperature of soil-grown plants was decreased from 23 °C to 4 °C and then held constant for 48 h [25]. To induce salt stress, soil-grown plants were incubated in a solution containing 250 mM NaCl for 48 h [26]. To induce drought stress, plants grown in MS medium for seven days were transferred to soil and grown for one week with sufficient watering; then, watering was withheld for two days [27,28].

To determine AtTPR10 expression under various abiotic stresses, samples were collected at the indicated time points, immediately frozen in liquid nitrogen, and used for RNA extraction and subsequent analysis. Real-time polymerase chain reaction (RT-PCR) was performed to amplify AtTPR10 using gene-specific primers (AtTPR10 RT_F, 5′-CTGTGGAAGTCTTGTTAGAAC-ACAATGCC-3′; AtTPR10 RT_R, 5′-GGTATGATCCGTTGGGTCAAAATCAATT-3′). Tubulin, which was used as the loading control, was amplified using the following primers: Tubulin F (5′-CTCAAGAGGTTCTCAGCAGTA-3′) and Tubulin R (5′-TCACCTTCTTCATCCGCAGTT-3′).

2.6. Heat Stability Analysis of AtTPR10

To determine the heat stability of AtTPR10, 1 µg each of the recombinant MDH and AtTPR10 proteins was incubated at 23 °C, 50 °C, and 60 °C for 15 min. Proteins were then centrifuged at 13,000× g for 20 min. The supernatant and pellet fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% gel, followed by Coomassie brilliant blue staining. The solubility ratio of each protein was calculated by dividing the band density of the supernatant fraction with that of the pellet fraction by densitometer.

2.7. Validation of AtTPR10 Expression in Response to Abiotic Stresses

Expression patterns of AtTPR10 under various abiotic stresses were analyzed using the Arabidopsis eFP Browser v2.0 (http://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBro-wser2.html). This browser contained expression data from Arabidopsis shoots subjected to different abiotic stresses for 12 h.

2.8. Native Gel Electrophoresis

Native PAGE gel was used for separation of proteins in their native conformation. Heat shock treated protein samples were isolated from insoluble proteins by centrifugation at 13,000× g for 15 min; the supernatants were obtained to new e-tubes, and the supernatant fraction was measured by Bradford’s method. Protein samples were mixed with nonreducing–nondenaturing sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, and 0.1% Bromophenol blue). Equal amounts of protein samples were subjected to 10% native PAGE gel [23,29]. After finishing the native gel electrophoresis, their structural properties were analyzed by silver staining method.

3. Results

3.1. Bioinformatic Analysis of AtTPR10 Function

The Arabidopsis genome encodes more than 21 AtTPR proteins. These proteins are classified on the basis of their molecular structure and the presence or absence of the TPR domain and/or other motifs (Figure 1). Among the proteins, AtTPR10 (At3g04710) showed a peculiar structure with both TPR and ANK domains in a single polypeptide that is possibly involved in protein–protein interactions, protein assembly, and signal transduction [1,30,31,32,33]. The AtTPR10 protein is a 456 amino acid (aa) protein with seven N-terminal ANK domains (54–280 aa) and three C-terminal TPR domains (328–429 aa) (Figure 2).

To determine the function of AtTPR10 in plants, we investigated its mRNA expression under diverse abiotic stresses using microarray data (Arabidopsis eFP Browser v2.0; http://bar.u-toronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html). Results showed that the transcript level of AtTPR10 was specifically upregulated by heat stress (Figure 3a) but not by the other abiotic stresses, such as cold, salt, and drought. This prediction was confirmed by real-time polymerase chain reaction (RT-PCR), which revealed the upregulation of AtTPR10 transcripts by heat stress treatment at 37 °C and 45 °C (Figure 3b). Additionally, AtTPR10 expression was gradually induced by heat stress (37 °C) within 1 h after treatment (Figure 3c), suggesting that the functional role of the protein might be involved in heat resistance in plants. By contrast, AtTPR10 was non-responsive to other abiotic stresses, including cold, salt, and drought (Figure 3d).

In addition to AtTPR10 expression analysis under heat shock treatment, we also examined the heat stability of the AtTRP10 protein. Recombinant AtTPR10 purified from E. coli was treated with high temperatures (50 °C, and 60 °C) for 15 min and then centrifuged at 13,000× g for 20 min. The supernatant and pellet fractions were analyzed by SDS-PAGE, followed by Coomassie brilliant blue staining. Most of the AtTPR10 protein showed high stability at all temperatures tested, unlike the malate dehydrogenase MDH used as a control (Figure 4). At 60 °C, more than 95% of the AtTPR10 protein was present in the supernatant fraction; by contrast, less than 2% of the MDH remained in the supernatant, while most of the MDH was precipitated by heat-induced aggregation (Figure 4a,b). These results strongly suggest that AtTPR10 is highly thermo-stable and its function possibly includes heat shock resistance in plant cells.

3.2. Heat Shock-Dependent Structural Switching of AtTPR10

Next, we examined the structure of AtTPR10 under heat stress by native gel electrophoresis and size exclusion chromatography (SEC) using HPLC. Heat-shock treated recombinant AtTPR10 protein samples produced a single band on SDS-PAGE gel (Figure 5a, bottom panel). While the AtTPR10 showed a single protein band in SDS-PAGE gel, it showed multiple bands in the native gel at 23 °C, indicating that the AtTPR10 protein exists as different structural isoforms including monomers, oligomers, and high molecular weight (HMW) complexes (Figure 5a, upper panel, lane #1). However, heat treatment induced significant structural shifts from low MW (LMW) protein species to HMW complexes. Thus, upon incubation of the protein at 60 °C for 30 min, most of the LMW species formed HMW complexes (Figure 5a, upper panel, lane #3). This structural shift was confirmed by SEC. Under normal conditions (23 °C), AtTPR10 displayed several discrete peaks representing monomers, oligomers and HMW complexes; however, under heat stress, most of the peaks representing LMW species in the SEC chromatogram disappeared, and the peak representing HMW complexes increased in size, indicating that the LMW species formed HMW complexes at high temperatures (Figure 5b). The heat shock-dependent formation of HMW complexes suggests that the AtTPR10 protein interacts with itself and forms higher MW complexes mainly by hydrophobic interaction. Therefore, we examined changes in the surface hydrophobicity of AtTPR10 in response to high temperature by incubating the protein with bis-ANS (probe) (Figure 5c). Bis-ANS binds to hydrophobic patches of proteins and emits fluorescence at 470 nm [22]. The fluorescence intensity of the bis-ANS peak (observed at 480 nm) was significantly higher at 60 °C than at 23 °C, indicating that several hydrophobic patches of AtTPR10 are exposed at high temperatures, enabling the formation of supra-structures. This confirms the propensity of AtTPR10 to form HMW complexes at high temperatures.

3.3. AtTPR10 Acts as a Molecular Chaperone

Since heat stability and temperature-dependent structural shifts are representative characteristics of molecular chaperones [5,6,7,34,35], we investigated whether AtTPR10 acts as a molecular chaperone. The chaperone activity of AtTPR10 was measured by its ability to protect target substrate proteins, such as MDH and CS, from denaturation under heat stress (Figure 6). During the heat treatment, formation of insoluble aggregates of denatured CS and MDH was measured by monitoring the degree of light scattering using a temperature-controlled spectrophotometer at 340 nm. In this experiment, 2-Cys Prx (a strong molecular chaperone) and GST were used as positive and negative controls, respectively [23,36].

Both AtTPR10 and 2-Cys Prx effectively prevented the thermal denaturation of the target substrate (MDH) at 43 °C; however, GST showed no protective function with either substrate (MDH or CS) under the same conditions (Figure 6a,b). When the molar ratio of AtTPR10 to MDH was increased, the protective function of AtTPR10 increased significantly; at the AtTPR10:MDH molar ratio of 5:1, nearly perfect protection was achieved within 15 min of the heat treatment. This result clearly demonstrates that AtTPR10 functions as a molecular chaperone under heat stress. Additionally, the chaperone activity of AtTPR10 was critically enhanced by a 15 min heat shock treatment at 60 °C (Figure 6c). These results strongly suggest that AtTPR10 performs the biochemical function of a molecular chaperone to protect intracellular molecules from heat stress.

3.4. ANK and TPR Domains Work Together as a Molecular Chaperone

Because AtTPR10 showed molecular chaperone activity (Figure 6) and contains two domains, ANK and TPR (Figure 2), which are reported to be involved in protein–protein interactions and intracellular protein complex assembly [4,5,7,21,34], we analyzed the contribution of each domain to the chaperone function of AtTPR10. To do the experiment, two truncated versions of the AtTPR10 protein, ANK-D (1–300 aa) and TPR-D (300–456 aa), were constructed (Figure 7a). These truncated recombinant proteins were expressed in E. coli, homogeneously purified, and used for biochemical analysis (Figure 7b). Chaperone activity of the truncated proteins was measured using MDH and CS as substrates at a truncated protein:substrate molar ratio of 1:5. Both domains exhibited a similar level of chaperone activity (Figure 7c), but each truncated protein showed approximately 30%–40% lower chaperone activity than the native AtTPR10. This suggests that each domain contributes to the chaperone function of AtTPR10 in a synergistic manner. To investigate the structural changes in the truncated proteins, we analyzed the hydrophobicity of these proteins by incubation with bis-ANS. Consistent with the result of chaperone activity, the hydrophobicity of AtTPR10 was considerably higher than that of each truncated protein (Figure 7d).

Furthermore, because the chaperone activity of AtTPR10 increased upon heat shock treatment (Figure 6c), we investigated changes in the chaperone activity and hydrophobicity of the truncated mutants under different temperatures (Figure 8). Similar to AtTPR10 (Figure 6c), chaperone activity of ANK-D and TPR-D increased significantly upon increasing the temperature from 23 °C to 60 °C (Figure 8a). Additionally, exposure of the hydrophobic surfaces of the truncated proteins at different temperatures was verified by incubation with bis-ANS (Figure 8b,c).

In contrast to the hydrophobicity of proteins under normal temperature (23 °C), the proportion of hydrophobic patches on the surface was greatly increased to allow the binding of denatured substrates under heat stress. In addition, chaperone activities of both truncated proteins were greatly enhanced by the heat shock treatment, suggesting that the structure of these proteins was transformed by thermal exposure. Because AtTPR10 polymerized and formed HMW complexes in response to high temperature (Figure 5a,b), we examined the heat stress-induced structural shift in the two truncated proteins by native gel electrophoresis and SEC. The results of native gel electrophoresis showed that the MW of both ANK-D and TPR-D increased significantly under high temperatures (Figure 9a,c), similar to ATPR10. These structural shifts were confirmed by the HPLC–SEC experiment (Figure 9b,d). Upon exposure to 60 °C for 30 min, both ANK-D and TPR-D were predominantly present as HMW complexes, indicating that the monomeric and oligomeric forms of each truncated protein interacted to form HMW complexes.

Overall, our results demonstrate that AtTPR10 acts as a molecular chaperone to protect plants from heat stress, and the ANK and TPR domains of AtTPR10 work together to enhance its chaperone function. Given that Arabidopsis contains a number of AtTPR isotypes (Figure 1), it is possible that most of these proteins perform the same basic function as AtTPR10, in addition to other specific roles in the regulation of diverse plant processes.

4. Discussion

Because plants are sessile organisms, protection from temperature fluctuations is critical for their survival. Thus, plants have developed a number of tools or systems to combat environmental stresses. Among these, molecular chaperone proteins that support the structural assembly or disassembly of intracellular target substrates and maintain their conformational folding contribute to the protection of plants from diverse external stresses. Most of the HSPs, including HSP60, HSP70, HSP90, and small HSPs, are distributed in all living organisms, ranging from bacteria to humans, and function as chaperones. The expression of genes encoding these HSPs is induced by diverse environmental factors, including cold, chemical treatment, wounding, and ultraviolet (UV) light. HSPs not only stabilize newly synthesized proteins to ensure correct folding but also repair misfolded proteins damaged by internal or external stresses. As the name implies, HSPs primarily function under heat shock by stabilizing heat stress-denatured molten-globular target substrates and renaturing these proteins to form native structures after the stresses are removed [28,29]. To mediate interaction between the partially denatured and unstructured proteins/peptides and to prevent the self-aggregation of substrates, chaperone proteins contain hydrophobic surfaces. The TPR and ANK domains are highly optimized to perform this function since they have an irregular structure with a large proportion of hydrophobic residues. Based on these structural characteristics, the TPR domain is reported to function as a co-chaperone in association with HSP90/70 [37,38,39]. Other Arabidopsis proteins such as protein phosphatase 5 (PP5) [34] and the Trx domain-containing protein AtTDX, which functions as a disulfide reductase [21], also contain TPR domains. In these proteins, TPR domains are involved in protein–protein interactions, protein assembly, and signal transduction. Overexpression of genes encoding TPR domain-containing proteins has been shown to enhance heat shock tolerance in plants [40,41].

Similarly, the ANK domain first discovered in the yeast Cdc10 protein contains 4–6 repeats of a 33 aa motif, forming two α-helices and a separate type 1 β-bulge loop [14,42]. The lateral surface structure of these repeats is hydrophobic with changeable conformation, which is mainly responsible for protein–protein interactions. The ANK domain forms a linear solenoid structure and performs multiple functions, including cell cycle control, ion transport, and signal transduction via protein–protein interactions. The ANK domain is present in several important proteins including the non-expressor of PR1 (NPR1), which functions as a key regulator of systemic acquired resistance (SAR) [43,44]; accelerated cell death 6 protein (ACD6), which contains one ANK motif and regulates plant defense via salicylic acid signaling [45]; and the nuclear-located ANK protein in tobacco, which enhances the auxin-mediated bZIP transcription factors, BZI-1 and BZI-2, through its regulation of protein complex formation [46]. These proteins also participate in pathogen resistance and hormone signaling [44,45,46]. In addition, the ANK repeat-containing protein 2A (AKR2A) protects its targets from stress-induced aggregation. In Arabidopsis, APX3 and the outer envelope protein 7 (OEP7) work together in chloroplasts to maintain plant growth and development [47]. In humans, mutation of ANK genes has a critical effect on the association of the encoded protein with the cell cycle inhibitor p16 or Notch protein, resulting in diverse diseases such as cancer, neurological disorders including alcoholism, nicotine dependency, obesity, and juvenile delinquency [14,48,49]. Thus, investigation of ANKs and TPRs is highly important to understand the regulation of their protein–protein interaction activity, identify the crucial residues needed for signal transduction, and control their chaperone function in plants. Understanding the regulation of AtTPR10 expression and chaperone function in future studies will enable the development of stress-tolerant crops.