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Contribution of the C-Terminal Region of a Group II Chaperonin to its Interaction with Prefoldin and Substrate Transfer
Contribution of the C-terminal region of a group II chaperonin to its interaction with prefoldin and substrate transfer Tamotsu Zako, Muhamad Sahlan, Sayaka Fujii, Yohei Y. Yamamoto, Phan The Tai, Kotaro Sakai, Mizuo Maeda, Masafumi Yohda PII: DOI: Reference:
S0022-2836(16)30050-X doi: 10.1016/j.jmb.2016.04.006 YJMBI 65057
To appear in:
Journal of Molecular Biology
Received date: Revised date: Accepted date:
19 February 2016 23 March 2016 4 April 2016
Please cite this article as: Zako, T., Sahlan, M., Fujii, S., Yamamoto, Y.Y., Tai, P.T., Sakai, K., Maeda, M. & Yohda, M., Contribution of the C-terminal region of a group II chaperonin to its interaction with prefoldin and substrate transfer, Journal of Molecular Biology (2016), doi: 10.1016/j.jmb.2016.04.006
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Contribution of the C-terminal region of a group II chaperonin to its
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interaction with prefoldin and substrate transfer
Tamotsu Zako1, $, &, Muhamad Sahlan2,3, $, Sayaka Fujii2, $, Yohei Y. Yamamoto2$,
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Phan The Tai2, Kotaro Sakai1,2, Mizuo Maeda1 and Masafumi Yohda2*
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Bioengineering Laboratory, RIKEN Institute, Saitama, Japan.
Department of Biotechnology and Life Science, Tokyo University of Agriculture and
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Department of Chemical Engineering, University of Indonesia, Depok, Indonesia. Equal contribution
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$
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Technology, Tokyo, Japan.
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Current address: Department of Chemistry and Biology, Graduate School of Science and
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Engineering, Ehime University
Correspondence: Masafumi Yohda, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-4-16 Naka-cho, Koganei-shi, Tokyo, Japan 1848588; Tel/fax: +81-420-388-7479; email: [email protected]
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Abbreviations CPN, chaperonin; T.KS-1, hyperthermophilic archaeon Thermococcus sp. strain KS-1; PFD,
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prefoldin; PFD11, prefoldin of Thermococcus sp. strain KS-1 composed of 1 and 1
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subunits; PFD22, prefoldin of Thermococcus sp. strain KS-1 composed of 2 and 2
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subunits; CS, citrate synthase from porcine heart; GFP, green fluorescent protein; SPR, surface plasmon resonance; PAGE, polyacrylamide gel electrophoresis; CPN, T.KS-1
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chaperonin homo-oligomer; CPNTc1, T.KS-1 chaperonin mutant with the truncation of one amino acid from the C-terminus; CPNTc2, T.KS-1 chaperonin mutant with the
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truncation of two amino acids from the C-terminus; CPNTc6, T.KS-1 chaperonin truncation mutant with the truncation of six amino acids from the C-terminus; IPMDH, 3-
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Isopropylmalate dehydrogenase from Thermus thermophilus HB8; dIPMDH, denatured IPMDH; IPMA, isopropylmalate acid; Cy3-dIPMDH, Cy3-labeled dIPMDH; 488-PFDα1β1,
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AlexaFluor 488-labeled PFDα1β1; Cy5-CPNβWT, Cy5-labeled CPNβW; Cy5-CPNβTc6,
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Cy5-labeled CPNTc6; Cy5-CPNβs, Cy5-labeled CPNβs; PFD1tc1, prefoldin of Thermococcus sp. strain KS-1 composed of two 1 subunits with truncation from the Cterminus and four 1 subunits; RU, Resonance Unit; TKM buffer, 50 mM Tris-HCl pH 7.5, 100 mM KCl and 25 mM MgCl2.
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Abstract Prefoldin is a molecular chaperone that captures an unfolded protein substrate and transfers it
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to a group II chaperonin. Previous studies have shown that the interaction sites for prefoldin are located in the helical protrusions of group II chaperonins. However, it does not exclude
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the possibility of the existence of other interaction sites. In this study, we constructed Cterminal truncation mutants of a group II chaperonin and examined the effects of these
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mutations on the chaperone’s function and interaction with prefoldin. Whereas the mutants with up to six amino acids truncation from the C-terminus retained more than 90% chaperone
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activities for protecting citrate synthase from thermal aggregation and refolding of green fluorescent protein and isopropylmalate dehydrogenase, the truncation mutants showed
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decreased affinities for prefoldin. Consequently, the truncation mutants showed reduced transfer efficiency of the denatured substrate protein from prefoldin and subsequent
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chaperonin-dependent refolding. The results clearly show that the C-terminal region of group
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II chaperonins contributes to their interactions with prefoldin, the transfer of the substrate protein from prefoldin and its refolding.
Keywords
Chaperone; Chaperonin; Prefoldin; Protein interaction; Protein folding
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Introduction Chaperonins (CPNs) are ubiquitous, double ring-shaped molecular chaperones that
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capture unfolded proteins in their cavities and assist in protein folding in an ATP-dependent manner [1, 2]. CPNs are subdivided into group I and group II [2, 3]. Group I CPNs are
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present in bacteria and in the organelles of eukaryotes. Group II CPNs exist in the cytosol of archaea and eukaryotes. Studies of the reaction cycle of a group I CPN using Escherichia coli
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GroEL have advanced our knowledge [1, 4, 5]. The heptameric ring-shaped complex of a cochaperonin, GroES, acts as a lid for the central cavity. Group II CPNs exist in archaea and the
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eukaryotic cytosol [3, 6, 7]. The group II CPNs do not require a GroES-like co-chaperonin but have a built-in lid that is composed of a helical protrusion in the apical domain [8-11].
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We have been studying the molecular mechanisms of group II CPNs using the chaperonin from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1 (T. KS-1).
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There are two CPN subunits (α and β) in T. KS-1 [12]. Their amino acid sequences are highly
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similar throughout their entire length, except for approximately 20 amino acid residues at the C-terminus. The T. KS1 CPN α subunit contains the GGM repeat, which is also observed in the C-terminal region in the group II CPNs. However, the GGM repeat does not exist in the C-terminal region of the β subunit. Both subunits can assemble into functional double ring complexes. The stoichiometry of the natural complex varies with temperature [13]. The β subunit content increases with temperature. We have shown that the C-terminal segment correlates with the thermal stability of the complex [14]. The C-terminal segments of the CPNs protrude from the bottom into the cavity but are not resolved in the crystal structure [8]. Tang et al. postulated that the C-terminal GGM repeats of GroEL facilitate the rearrangement of certain folding intermediates by providing a mildly hydrophobic interactive surface [15]. We have determined the crystal structure of the T. KS1 CPN α subunit homo-oligomer [11]. Similar to those of other CPNs, the structure of the 19 C-terminal amino acid residues was 4
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disordered. The high mobility of this region was confirmed by NMR analysis [16]. The fact that the T. KS-1 CPN with a His-tag sequence at the C-terminus can be purified by a nickel
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chelating column also supports the idea that the C-terminal residues protrude into the cavity. Prefoldin (PFD) is a jellyfish-like hexameric protein that is exclusively found in
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archaea and eukaryotes [17-19]. Biochemical studies have shown that PFDs bind to and stabilize unfolded target polypeptides and subsequently deliver them to group II CPNs to
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complete the folding process [20-22]. The transfer of a substrate from PFD to CPN involves a direct interaction [21, 23]. The three-dimensional reconstructed image of a complex of a
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eukaryotic CPN and PFD showed that PFD interacts with the apical domain of CPN [23]. We have investigated the protein-folding mechanism of PFD and CPN using the
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PFD-CPN systems of the hyperthermophilic archaea, T. KS-1 and Pyrococcus horikoshii [16, 20-22, 24-26]. We have shown that both the N- and C-terminal regions of the β subunit of
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PFD are important for its interaction with CPN [16, 26]. The rate of transfer of a substrate
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protein from PFD to CPN correlated with the strength of the PFD-CPN interaction [22]. We have also determined the location of the PFD interaction site that lies on the helical protrusion of CPN. However, based on our study using NMR, we speculated that the C-terminus CPN might also contributes to the interaction with PFD [16]. There are two pairs of PFD subunits (α1, α2 and β1, β2) in T. KS1 [24]. PFDα1β1 and PFDα2β2 are thought to be the physiological complexes. T. KS-1 PFDα1β1 binds to the T.KS-1 CPN β homo-oligomer (CPNβ) at high affinity [24]. In this study, we constructed Cterminal truncation mutants of CPNβ and examined the effects of the truncations on their chaperone functions and interactions with PFDα1β1. The results indicate that the C-terminal region of CPN also influences the transfer efficiency and CPN-mediated refolding of the substrate proteins. These results also imply a novel role for the CPN C-terminal region, which is buried in the cavity, in the interaction with PFD binding and substrate transfer from PFD. 5
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Results Construction and functional characterization of the CPN C-terminal truncation mutants
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In this study, we used CPNβ and PFDα1β1 from T. KS1, because these isoforms interact with each other at high affinity [24]. We expressed and purified three CPNβ mutants,
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with the truncation of one, two and six amino acids from the C-terminus, named CPNβTc1, CPNβTc2, and CPNβTc6, respectively. CPNβTc2 and CPNβTc6 were designed to delete two
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hydrophobic amino acid residues from the CPNβ C-terminus (Gly-Ser-Glu-Asp-/Phe-Gly-SerAsp-/Leu-Asp, (/, Tc6 and Tc2 truncation positions)). The mutants formed hexadecameric
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homo-oligomers, similar to the wild type CPNβ, CPNβWT (data not shown). First, we investigated the effects of the truncations on the ability of CPNβ to protect
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porcine citrate synthase (CS) from thermal aggregation (Fig. 1A). The thermal aggregation of CS was completely suppressed by the presence of an equimolar amount of the CPNβ variants.
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The truncation mutants exhibited the same protection abilities as CPNβWT. The protein
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folding activities of the CPNβ variants were examined using acid-denatured GFP as a substrate. The acid-denatured GFP refolds in a neutral pH environment to emit fluorescence. The amount of spontaneous refolding reached a maximum of approximately 28% at 400 sec after dilution and gradually decreased to approximately 24% at 1200 sec. When CPNβWT is present in the folding mixture, the spontaneous refolding of GFP is inhibited. The addition of ATP at 300 sec induced productive refolding of GFP, and the refolding yield reached approximately 35% at 900 sec after the addition of ATP (Fig. 1B). The refolding yields of the truncated CPNβ variants were more than 90% of as CPNβWT (102% for CPNβTc2 and 91% for CPNβTc6, respectively) (Fig. 1B). The ATP-dependent conformational changes of the truncation mutants were also investigated using a protease sensitivity assay (Fig. 1C). In the open state, the built-in lid is sensitive to protease digestion, while in the closed state, it is not [27]. The CPNβ variants 6
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were subjected to thermolysin digestion in the presence or absence of ATP at 60ºC. In the presence of ATP, all CPNβ variants were relatively resistant to thermolysin. In contrast, the
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CPNβ variants were nearly completely degraded in the absence of ATP. Therefore, like the wild-type protein, the confirmation of the truncated mutants can change to the closed state in
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an ATP-dependent manner.
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Interaction affinities of the CPNβ variants for PFDα1β1
To assess the interaction between PFDα1β1 and the CPNβ variants, we monitored
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their association and dissociation by surface plasmon resonance using Biacore. PFDα1β1 was immobilized on the sensor chip and the CPNβ variants were applied as analytes. The
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sensorgrams of these CPNβ variants injected at various concentrations are shown in Fig. 2. The dissociation constants (KD) for the interaction between the mutants and PFDα1β1 were
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calculated by steady-state affinity determination method (Fig. S1, Supplementary
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information). The KD values for the CPNβ variants are represented in Table 2. Interestingly, the KD values increased in a stepwise manner with the removal of 1, 2, and 6 residues from the C-terminus of CPNβ. These data demonstrated that the affinities of the CPNβ truncation mutants for PFDα1β1 decreased relative to CPNβWT and that the mutant of CPNβ lacking the 6 terminal amino acids showed the weakest affinity for PFDα1β1.
The C-terminal region contributes to the functional cooperation between PFDα1β1 and CPNβ In an aim to clarify the effect of the affinity between PFDα1β1 and the CPNβ variants (CPNβWT and CPNβTc6) on cooperative substrate refolding, substrate transfer and the subsequent refolding were examined. Substrate transfer was examined with FRET using fluorescently labeled PFDα1β1, the CPNβ variants and denatured 3-Isopropylmalate 7
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dehydrogenase (IPMDH) from Thermus thermophilus HB8 (dIPMDH). Substrate release from PFDα1β1 was estimated by dissolution of FRET between Cy3-labeled dIPMDH (Cy3-
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dIPMDH) captured by AlexaFluor 488-labeled PFDα1β1 (488-PFDα1β1) by the addition of non-labeled CPNβ variants. First, FRET between 488-PFDα1β1 and Cy3-dIPMDH was
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confirmed by a decrease in donor fluorescence (AlexaFluor 488) (Fig. 3A). The donor fluorescence increased with the addition of CPNβWT, indicating that the FRET between 488-
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PFDα1β1 and Cy3-dIPMDH was canceled by the substrate release from PFDα1β1. Although the FRET dissolution was also observed by the addition of CPNβTc6, the efficiency decreased
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to 49% of that with CPNβWT. FRET dissolution efficiency by CPNβWT was higher than that by CPNβTc6 at various CPN concentrations (Fig. S2, Supplementary information). This result
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clearly indicates that affinity between PFD and CPNβ correlates with the substrate transfer efficiency.
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To further confirm substrate transfer from PFD to CPNβ, the capture of the released
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substrate by CPN was examined by FRET between Cy3-labeled dIPMDH and Cy5-labeled CPNβs (Cy5-CPNβs). Cy5-CPNβs (Cy5-CPNβWT and Cy5-CPNβTc6) were added to the solution containing a complex of Cy3-dIPMDH and PFDα1β1. As shown in Fig. 3B, the Cy3 fluorescence decreased with the addition of Cy5-CPNβs, indicating the capture of the released substrate by the CPNβs. The FRET efficiency of CPNβTc6 was 64% of that of CPNβWT, supporting a correlation between affinity and substrate transfer. Finally, we examined the effect of the C-terminal truncations on the cooperative refolding of dIPMDH. First, the ATP-dependent refolding of IPMDH by the CPNβ variants was confirmed (Fig. 4A). Both CPNβWT and CPNβTc6 were able to refold dIPMDH in an ATP-dependent manner with the same efficiency, which is consistent with the results for GFP (Fig. 1B). Then, the refolding of dIPMDH captured by PFDα1β1 was applied to CPNβdependent folding. The dIPMDH-PFD complex was added to CPNβWT or CPNβTc6. After 8
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incubation for 300 sec, ATP was added to induce refolding of IPMDH by CPNs (Fig. 4B). The results clearly illustrate the role of PFD in transferring denatured proteins to CPN for
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productive folding. The refolding yields were significantly increased in this experiment compared with those without PFD. The refolding yield by CPNβTc6 was approximately 80%
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of that by CPNβWT. The difference in the refolding yield should be due to the difference in the affinities with PFD between CPNβWT and CPNβTc6. In both cases, initial refolding rate
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is significantly high, which is probably due to the accumulation of CPN-IPMDH complex by substrate transfer from PFD to CPN during the incubation before ATP addition. To observe the
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effect of the difference in the affinities between PFDα1β1 and CPNβ variants more clearly, the dIPMDH-PFD complex was prepared in the buffer containing ATP and was mixed with
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CPNβWT or CPNβTc6 in the presence of ATP (Fig. 4C). Addition of CPNs initiates transfer of dIPMDH from PFDα1β1 to CPNs and refolding of IPMDH by CPNs. In this condition, the
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transferred dIPMDH is immediately subjected to the refolding process by CPN. Thus, the
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yield of IPMDH refolding directly correlates with the interaction efficiency. The refolding yield of CPNβTc6 was approximately 55% of that of CPNβWT. Then, we compared the refolding yields of IPMDH by CPNβWT or CPNβTc6 at various CPN concentrations (Fig. 4D). As the increase of CPN concentration, the difference of the refolding yield increased and reached saturation. At high CPN concentrations, the difference should be determined by the difference of affinity between CPN and PFD. Taken together, the C-terminal region of CPNβ, which is located inside the cavity, affects the interaction affinity with PFD, substrate transfer efficiency and subsequent refolding of substrate proteins by CPNβ.
The PFD α subunit is not responsible for the interaction with the C-terminus of CPN Regarding the possible binding site in PFD for the C-terminal region in the cavity of CPN, we hypothesized that the PFD α subunit, which is relatively long, could contribute to 9
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the interaction. Although the tip of the PFD α subunit appears disordered in the crystal structure, the model of the complex structure indicates that the tip of the αsubunit may be
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deeply inserted into the cavity [23]. In particular, the C-terminal segment of the T. KS1-PFD α1 subunit is long and possesses three Lys residues, and it was likely that this region could be
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important for the interaction with the CPN C-terminal region, which has three Asp residues in the last 7 residues. To confirm this hypothesis, we constructed a PFD α1 subunit C-terminal
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truncation mutant (PFDα1tcβ1), where all three Lys residues were deleted (10 amino acid residues in total), and examined the interaction with CPNβWT. However, the result
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contradicted the hypothesis. The C-terminal truncation of the PFD α subunit did not decrease the interaction with CPNβWT (Fig. 5A), indicating that the C-terminal region of the PFD α
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subunit is not important for the interaction with the CPN C-terminal region. The effect of PFD α1 subunit C-terminal truncation on the cooperative folding of
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IPMDH with CPNβWT was also examined. As shown in Figure 5B, there was no significant
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difference between PFDα1delβ1 and the wild type PFDα1β1 on cooperative IPMDH refolding, supporting the result above. These results indicate that the C-terminus of the PFD α subunit is not important for the interaction with CPN and substrate transfer.
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Discussion Previous studies have shown that the C-terminus of the PFD β subunit interacts with
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the helical protrusions of CPNs via electrostatic interactions [22]. However, the thermodynamic parameters calculated from the association constants indicated that the
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interaction between PFD and CPN is entropy driven [28]. There are two pairs of prefoldin subunits (α1, α2 and β1, β2) in T. KS-1, and PFDα1β1 and PFDα2β2 are thought to be the
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physiological complexes. We could construct four different PFD complexes (PFDα1β1, PFDα1β2, PFDα2β1 and PFDα2β2) from them, and CPNβ exhibited higher affinities to all of
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them compared to CPNα [24]. Among all of the PFD complexes, PFD complexes containing the β1 subunit (PFDα1β1 and PFDα2β1) exhibited relatively higher affinity to CPNβ than
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those with the β2 subunit (PFDα1β2 and PFDα2β2), possibly because the PFD β2 subunit has shorter N- and C-termini compared to the β1 subunit and lacks one Lys residue that is thought
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to interact with the Glu residue on the CPN helical protrusion [22, 28].
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However an unexplained result remained. PFD complexes containing the β1 subunit have higher affinities to CPNα, which contains basic residues in the helical protrusion, than those with the β2 subunit [24]. This result suggests the existence of another interaction site. Our previous NMR-based study also demonstrated that the C-terminal region of the T.KS-1 CPN subunits is highly flexible in the large PFD-CPN complex, suggesting that it plays a role in the interaction between T.KS-1 CPN and PFD [16]. Thus, we postulated that the CPN Cterminal region would be another important site for the interaction with PFD. In this study, we constructed CPNβ with C-terminal truncations and examined the effects of these truncations on the interaction with PFD. The mutants exhibited the similar chaperone activity and ability to undergo a conformational change as the wild-type protein. We then examined the interaction between PFD and the CPNβ variants by surface plasmon resonance using the Biacore system. The comparison of the sensorgrams derived from the 11
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wild-type protein and the mutants showed that the affinities of all of the C-terminal truncation mutants for PFD were significantly decreased compared to the wild-type protein. The mutant
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with a 6 amino acid truncation from C-terminus had the lowest affinity of any mutant examined. In addition, the dissociation constants for the C-terminal truncation mutants with 2
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and 6 amino acid truncations were not significantly different (Fig. 2, Table 2). These results clearly demonstrate that the C-terminal segment of CPNβ contributes to the interaction of
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CPNβ with PFD. Of the 6 C-terminal amino acids (Phe-Gly-Ser-Asp-Leu-Asp), the last two, Asp and Leu, appear to be the most important for binding to PFD.
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Importantly, the C-terminal truncation of CPNβ also affected substrate transfer from PFD. The efficiency of substrate transfer, as estimated by FRET, and CPN-dependent
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refolding were decreased for the C-terminal truncation mutants. This result indicates that affinity between PFD and CPN correlates with substrate transfer efficiency and supports the
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importance of the CPN C-terminal region for cooperative refolding by PFD and CPNβ.
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Both the N- and C-termini of CPNβ are located at the bottom of the CPNβ cavity in the crystal structure of the T. KS-1 CPN αhomo-oligomer [11]. However, the N-terminal 8 and the C-terminal 19 residues are disordered and were therefore excluded from the final model. Combined with our previous NMR data, this observation suggests that the C-terminal segment might not be located at the bottom of the cavity and therefore may contribute to protein folding and interaction with PFD in the cavity. Tang et al. have reported that the Cterminal segment of GroEL contributes to the rapid folding of certain proteins encapsulated in the central cavity, supporting the flexible movement of the C-terminal segment [15]. We hypothesized that the C-terminal region of the PFD α subunit might be responsible for the interaction, because it is long and possesses three Lys residues. However, this was clearly not the case in the experiment using PFD containing a truncation mutant of the α1 subunit. 12
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Taking all of the results into account, it is plausible that the PFD β subunit could interact with the CPN C-terminal region; i.e., the PFD β subunit simultaneously interacts with
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the apical domain [23] and C-terminal region of CPNβ. This idea could be supported by our previous observation showing that T. KS1 PFD variants, including the β1 subunit, showed
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stronger affinity to CPNβWT than those including the β2 subunit [24], possibly because the longer β1 subunit (117 a.a.) could interact more effectively with the flexible C-terminal
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segment in the cavity than the β2 subunit (99 a.a.). Our other previous studies showing that truncation of PhPFD β subunit reduced its affinity to T. KS1 CPNs [16, 26] also support this
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idea. In addition, this is consistent with our previous finding showing that the interaction between PFD and T. KS1 CPN is also entropy-driven [28]. It is plausible that the disordered
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regions of the T. KS1 CPN C-terminus and PFD interact with each other. We have postulated that the difference in the interaction of CPN and the PFD complexes containing the β1 and β2
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subunits is caused by the difference in the interaction with helical protrusion, as β1 and lacks
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one Lys residue that is thought to interact with Glu residue. However, there are no basic residues in the extended region of the β1 subunit, and the β2 subunit retained some basic residues. Thus, it is reasonable to think that the difference between the β1 and β2 subunits should be caused by the differences in interaction with the C-terminal region and not with the helical protrusion. Studies on the PFD β subunit disruptants have shown that PFDα1β1 (PfdA/PfdB complex) plays a crucial role at all growth temperatures and PFDα2β2 (PfdC/PfdD complex) contributes to survival in a high-temperature [29]. The C-terminal region seems to be responsible for discrimination between PFDα1β1 and PFDα2β2. Disruption of CPNβ gene significantly affected the growth at the elevated temperatures [30]. We have thought that the effect is due to relatively low thermal stability of the remaining CPN, CPNα. However, it might be partly due to the lack of cooperation between CPNα and PFDα2β2 [24]. 13
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In conclusion, we demonstrated that in addition to the group II CPN apical domain, the C-terminal region in the cavity of the group II CPN is important for the interaction with
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PFD and their cooperative folding. Our results also suggested that the PFD β subunit would interact with both the CPN apical domain and C-terminal region simultaneously. Further
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studies, such as detailed cryo-EM observations, would be necessary to confirm this idea.
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Materials and Methods Proteins
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The genes for the truncation mutant CPNβ subunits were constructed by polymerase
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chain reaction (PCR) using the primers shown in Table 1. The expression vector pET9a (Merck Millipore, Billerica, MA, USA) was used to express the CPNβs. The CPNβ
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complexes with the wild-type and C-terminal truncation mutants were expressed and purified as described previously [31]. The gene for the truncation mutant (10 amino acid residues from
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the C-terminus) of the T. KS-1 PFD α1 subunit (PFDα1tc) was constructed with the
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QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) by replacing the corresponding sequence with a stop codon. The primers used are shown in Table 1. PFDα1β1
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and PFDα1tcβ1 were expressed and purified as described [24]. IPMDH was expressed in E. coli and purified as described [25].
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For site-specific fluorescent dye labeling of CPN, the intrinsic cysteine residue of CPNβ was replaced with serine (CPNβC366S), and cysteine was introduced in the C-terminal
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end of CPNβC366S (CPNβ_Cys) and C-terminal 6 amino acid truncation mutant of CPNβC366S (CPNβTc6_Cys) using the QuikChange Site-directed Mutagenesis Kit. The Cterminal end of the α subunit of PFDα1β1 was also replaced with cysteine (PFD_Cys). PFDα1β1 has no intrinsic cysteine residues. The primers used are shown in Table 1.
Characterization of T. KS1 chaperonins Thermal aggregation of citrate synthase from porcine heart (CS) was monitored using light scattering at 500 nm in a fluorophotometer (FP-6500, JASCO, Tokyo, Japan) at 50ºC. CS (120 nM, as monomer) was incubated in TKM buffer (50 mM Tris-HCl pH 7.5, 100 mM KCl and 25 mM MgCl2) with or without each CPN variant (120 nM). The assay buffer was pre-incubated at 50ºC and continuously stirred throughout the measurements.
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For the protease sensitivity assay, the CPNs were incubated with or without ATP (1 mM) at 60ºC in TKM buffer under continuous mixing. Digestion with thermolysin (1 ng/L)
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was then performed for 5 min at 60ºC. The proteins in the reaction mixture were precipitated by the addition of trichloroacetic acid and then analyzed by SDS polyacrylamide gel
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electrophoresis.
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Refolding assay
The CPN-dependent green fluorescence protein (GFP) refolding reactions were
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performed at 60ºC, as previously described [22, 31]. In brief, the acid-denatured GFP was diluted into TKM buffer with 5 mM DTT in the presence or absence of the CPNs. The
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fluorescence of GFP at 510 nm with excitation at 396 nm was monitored using a spectrofluorometer (FP-6500, JASCO). As a control, the native GFP protein was diluted into
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the folding buffer. The fluorescence intensity of native GFP was set to 100%.
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IMPDH refolding was performed as described, with slight modifications [25]. IPMDH (4 M) was unfolded in 6 M GdnHCl and 50 mM TrisHCl, pH 7.5, overnight at room temperature. The denatured IPMDH (dIPMDH) was diluted to 25 nM in an assay buffer (TKM buffer including 0.4 mM isopropylmalate acid (IPMA) and 1 mM NAD) in the presence or absence of 100 nM CPN. dIPMDH refolding was started with the addition of 1 mM ATP. IMPDH refolding was measured as the rate of increase in absorbance at 340 nm of NADH, which was converted from NAD by the refolded IMPDH, using a spectrophotometer (V-570 or V-650 JASCO). The increase in the rate of the native IPMDH was set to 100%. For ATP-dependent refolding experiment of IPMDH by CPN after the transfer from PFD, 25 nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer without ATP for 300 sec at 60ºC, and 100 nM CPN were then added. After a 300 sec incubation, 1 mM ATP was added to initiate refolding IPMDH by CPN. For the substrate transfer experiment from PFD to CPN, 16
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25 nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer with 1 mM ATP for 300 sec at 60ºC, and 100 nM CPN was then added for transfer and CPN-dependent refolding
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of dIPMDH. IPMDH refolding was monitored as described above. Spontaneous refolding
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without chaperones was performed as a negative control experiment.
Analysis of the PFD-CPN interaction by surface plasmon resonance
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The surface plasmon resonance (SPR) experiments were performed using the Biacore J or Biacore T-100 systems (GE Healthcare, Buckinghamshire, UK) at a sensor temperature
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of 25ºC, as previously described [26, 28]. The dissociation constants (KD) were calculated by steady-state affinity determination method. In brief, each CPN mutant was injected on
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immobilized PFD at various concentrations (at least 5 concentrations). Then KD was obtained by fitting to the SPR signal data with a recursive least-squares algorithm by KaleidaGraph
Req = Rmax ·C/(C + KD)
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(Synergy Software, Reading, PA, USA) using the following equation:
where Req represents the resonance units at equilibrium, Rmax is the resonance signal at saturation, and C is the injected CPN concentration. Req values were obtained by fitting to the sensorgrams using Langmuir binding model with BIAevaluation software (GE Healthcare). Relative KD values were calculated by dividing the KD values of CPN mutants by the KD value of the wild-type CPN.
Estimation of substrate transfer from PFD to CPN with fluorescent resonance energy transfer (FRET) Substrate transfer from PFD to CPN was also examined with FRET using fluorescently labeled dIPMDH, PFD and CPNβ. IPMDH was incubated with the Cy3 mono-reactive dye (GE Healthcare) (Cy3-IPMDH) overnight at 4°C. The labeled substrate proteins were 17
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separated from the unreacted dyes using a NAP5 gel filtration column (GE Healthcare) equilibrated with TKM buffer. For site-specific labeling of PFD and CPN, the C-terminal
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residues of the α subunit of PFDα1β1 and cysteine-free CPN mutant were replaced with cysteine residues, as described above (PFD_Cys and CPNβ_Cys). PFD_Cys and CPNβ_Cys
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were labeled with AlexaFluor 488-C5-maleimide (AlexaFluor 488, Invitrogen, OR, 488-PFD) and Cy5-maleimide (GE Healthcare, Cy5-CPN), as described above. The molar ratios of
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AlexaFluor 488 to PFDα1β1, Cy5 to CPNβWT/CPNβTc6 and Cy3 to IPMDH were 0.6 (per PFD complex), 3.3/2.8 (per CPN oligomer) and 2.5, respectively. The labeled PFD and CPN
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proteins showed similar chaperone activity as the unlabeled proteins (data not shown). Substrate transfer was estimated with two different FRET phenomena, as follows:
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1) denatured Cy3-IPMDH captured by 488-PFD was transferred to the non-labeled CPNs and 2) denatured Cy3-IPMDH captured by non-labeled PFD was transferred to Cy5-CPNs
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(CPNβWT and CPNβTc6). In the former case, FRET between AlexaFluor 488 and Cy3 was
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canceled by substrate release from PFD. Cy3-IPMDH was denatured as described above (Cy3-dIPMDH). 488-PFD (100 nM) was incubated with denatured Cy3-IPMDH (100 nM) in TKM buffer for 5 min at 60°C, and the non-labeled CPNs (100 nM) were then added and incubated for 5 min. The emission spectra were recorded upon excitation at 460 nm with a spectrofluorometer (FP-6500, JASCO). The emission spectra of the same concentrations of 488-PFD only, Cy3-dIPMDH only and 488-PFD/Cy3-dIPMDH without CPN were also obtained. In the latter case, FRET between the Cy3 and Cy5 dyes was observed by substrate capture by CPN. Denatured Cy3-dIPMDH (100 nM) was incubated with non-labeled PFD for 5 min at 60°C, and Cy5-CPNs (100 nM) were then added and incubated for 5 min. The emission spectra were recorded upon excitation at 540 nm with a spectrofluorometer (FP6500, JASCO). The emission spectra of the Cy3-IPMDH/PFD and Cy5-CPN only samples were also obtained. 18
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FRET was estimated by the decrease in donor fluorescence, and FRET dissolution by the increase in donor fluorescence, respectively. FRET efficiency was calculated as
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where Fd represents the donor fluorescence in the presence or absence of the acceptor dye. The FRET dissolution efficiency following the addition of CPN was determined as the
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increase in donor fluorescence with the added CPNs. The FRET efficiency and FRET
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dissolution efficiency in the presence of the wild-type CPN was normalized to 100%.
Acknowledgments
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The work reported here was supported in part by a program intended to improve the graduate school education provided by the "Human Resource Development Program for
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Scientific Powerhouse," which is financially supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan through Tokyo University of Agriculture &
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Technology. This study was also supported by grants-in-aid for scientific research (21370067, 22020011, 24370064 and 26102511 (MY), and 24570143 (TZ)), and a grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports, and Culture of Japan to MY, and RIKEN.
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ACCEPTED MANUSCRIPT terminal region to the thermostability of the archaeal group II chaperonin from Thermococcus sp. strain KS-1. Extremophiles. 2006;10:451-9. [15] Tang YC, Chang HC, Roeben A, Wischnewski D, Wischnewski N, Kerner MJ, et al. Structural
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Figure Legends
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Fig. 1 Functional characterization of the various CPN mutants.
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A. The effect of C-terminal truncations of CPN on the thermal aggregation of CS. CS (120 nM) was incubated in the absence (“without CPN”) or presence of chaperonins (120 nM
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CPNβWT, CPNβTc2 and CPNβTc6) at 50°C.
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B. Refolding of GFP by CPNs. The folding mixture was incubated at 60°C, and the recovery of native GFP was continuously monitored by measurement of the fluorescence emission at
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510 nm. The fluorescence intensity of native GFP at the same concentration was set to 100%. At 0 min of incubation, acid-denatured GFP (5 M) was diluted 100-fold into the folding
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buffer containing 100 nM CPNs (CPNβWT, CPNβTc2, or CPNβTc6). At 5 min after dilution, 1 mM ATP was added. The spontaneous refolding of GFP was observed upon dilution of the
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“spontaneous”.
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denatured GFP into the folding buffer without chaperonins or ATP and is marked as
C. Protease sensitivity assay demonstrating the conformational changes in CPNs. CPNs (CPNβWT, CPNβTc2, or CPNβTc6) were incubated with thermolysin (1 ng/L) in the presence or absence of ATP (1 mM) and then analyzed by SDS-PAGE. The details are provided in the Materials and Methods section.
Fig. 2 Comparison of interaction between the CPN variants and PFD. Sensorgrams of the interactions of PFD with the CPNs (CPNβWT, CPNβTc1, CPNβTc2, and CPNβTc6) analyzed with the Biacore J system are shown. PFDα1β1 was immobilized on a Biacore CM biosensor chip at 5000 RU, and CPNs at various concentrations were injected as the analyte. In the figure, sensorgrams of 4 analyte concentrations were shown for comparison.
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Fig. 3 Substrate transfer from PFD to CPN examined by FRET
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A Transfer of denatured Cy3-IPMDH captured by 488-PFD to non-labeled CPNs. 488PFD (100 nM) was incubated with denatured Cy3-IPMDH (100 nM) in TKM buffer for 5 min
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at 60°C, and non-labeled CPNs (100 nM, CPNβWT, red; CPNβTc6, blue) were then added and incubated for 5 min. The emission spectra were recorded upon excitation at 460 nm with
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a spectrofluorometer. The emission spectra of 488-PFD (black), the 488-PFD/Cy3-IPMDH complex (green) and Cy3-IPMDH (yellow) were also shown. The FRET dissolution
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efficiency was obtained by the increase in the peak intensity of the donor fluorescence with the addition of CPNs from that of the 488-PFD/Cy3-IPMDH complex. The FRET dissolution
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efficiency of the wild-type CPN was normalized to 100%. The average value of two
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independent experiments is shown.
B Transfer of denatured Cy3-IPMDH captured by PFD to Cy5-CPNs. PFD (100 nM) was
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incubated with denatured Cy3-IPMDH (100 nM, black) in TKM buffer for 5 min at 60°C, and Cy5-CPNs (100 nM, CPNβWT, red; CPNβTc6, blue) were then added and incubated for 5 min. The emission spectra were recorded upon excitation at 520 nm with a spectrofluorometer. The emission spectra of Cy5-CPNβWT (green) and Cy5-Tc6 (yellow) were also shown. The FRET efficiency was calculated from the decrease in the donor fluorescent intensity, as described in the text. The FRET efficiency of the wild-type CPN was normalized to 100%. The average value of two independent experiments is shown.
Fig. 4 IPMDH refolding by CPN transferred from PFD A, IPMDH refolding by CPN. IPMDH (4 M) that had been unfolded in 6 M GdnHCl (dIPMDH) was diluted to 25 nM in an assay buffer (TKM buffer including 0.4 mM
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isopropylmalate acid (IPMA) and 1 mM NAD) in the presence or absence of 100 nM CPNs (CPNβWT, red; CPNβTc6, blue). dIPMDH refolding was started with the addition of 1 mM
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ATP. IMPDH refolding was measured as the rate of increase in absorbance at 340 nm and 60°C of NADH, which was converted from NAD by the refolded IMPDH, using a
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spectrophotometer (V-570, JASCO). The increase in the refolding rate of the native IPMDH was set to 100%. Spontaneous refolding without chaperones was performed as a negative
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control experiment (green).
B, ATP-dependent refolding of IPMDH by CPN after the transfer from PFD. Twenty-five
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nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer without ATP for 300 sec at 60ºC, and 100 nM CPNs (CPNβWT, red; CPNβTc6, blue) were then added. After a 300
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sec incubation, 1 mM ATP was added to initiate refolding IPMDH by CPNs. IPMDH
shown in green.
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refolding was monitored as described above. Spontaneous refolding without chaperones is
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C, Transfer of IPMDH from PFD and refolding by CPN. Twenty-five nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer with 1 mM ATP for 300 sec at 60ºC, and 100 nM CPNs (CPNβWT, red; CPNβTc6, blue) were then added for transfer and CPNdependent refolding of dIPMDH. IPMDH refolding was monitored as described above. Spontaneous refolding without chaperones is shown in green. D, Refolding of IPMDH by CPN at various concentrations transferred from PFD. Twenty-five nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer with 1 mM ATP for 300 sec at 60ºC, and CPNs (CPNβWT or CPNβTc6) at various concentrations (25, 50, 100, 150 and 200 nM) were then added for transfer and CPN-dependent refolding of dIPMDH. Ratio of refolding yields after 900 sec incubation by CPNβWT against CPNβTc6 was shown in the figure. The average values of three independent experiments were shown. 25
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Fig. 5 Effect of the C-terminal truncation of the PFD α subunit on the interaction with
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CPN and cooperative IPMDH refolding
A, Interaction between PFDs and CPN. Sensorgrams of the interactions between CPNβWT
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and PFDs (PFDα1β1 and PFDα1tcβ1) analyzed with the Biacore T-100 system are shown. PFDα1β1 (red) and PFDα1tcβ1 (blue) were immobilized on a Biacore CM biosensor chip
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(10700 RU for PFDα1β1 and 9700 RY for PFDα1tcβ1, respectively). The sensorgram of CPNβWT injected at 20 nM as an analyte is shown. The KD values were calculated to be
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28.3±4.7 nM for PFDα1β1 and 37.9 ± 5.8 nM for PFDα1tcβ1 (averaged values of three
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independent runs).
B, Refolding of IPMDH by CPN transferred from PFD truncation mutant. Twenty-five
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nM dIMPDH was pre-incubated with 100 nM PFDs (PFDα1β1 (red) and PFDα1tcβ1 (blue)) in the assay buffer with 1 mM ATP for 300 sec at 60ºC, and 100 nM CPNβWT was then
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added for transfer and CPN-dependent refolding of dIPMDH. IPMDH refolding was monitored as the rate of increase in absorbance at 340 nm and 60°C using a spectrophotometer (V-650, JASCO), as described above. Spontaneous refolding without chaperones is shown in green.
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Table 1. Primer sequences used in this study Name
Sequence
Restriction site
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Primers for the construction of the truncated mutants
5’-CGCGGATCCGCGTCAGAGATCGCTTCCGAAGTC-3’
BamH I
CPNβTc2 Rv
5’-CGGGATCCCGTCAATCGCTTCCGAAGTC-3’
BamH I
CPNβTc6 Rv
5’-CGGGATCCCGTCAGTCCTCGCTACCGCCCTT-3’
BamH I
CPNβ Fw
5’-GGAATTCCATATGGCCCAGCTTGCAGGCCAG-3’
PFDα1tc Fw
5'-CAGGCTCAGGAAATCCAGCAGTGACAGGCGATGGGCTTCAGCG-3'
PFDα1tc Rv
5'-CGCTGAAGCCCATCGCCTGTCACTGCTGGATTTCCTGAGCCTG-3'
Nde I
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CPNβTc1 Rv
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Primers for the construction of the cysteine mutants
5'-GATCTTCGTCGAGGGCTCCAAGAACCCGAAG-3'
CPNβC366S Rv
5'-CTTCGGGTTCTTGGAGCCCTCGACGAAGATC-3'
CPNβ_Cys Fw
5'-GAAGCGATCTCGACTGCTGAAGCTTGCGGCC-3'
CPNβ_Cys Rv
5'-GGCCGCAAGCTTCAGCAGTCGAGATCGCTTC-3'
CPNβTc6_Cys Fw
5'-GTAGCGAGGACTGCTAATTCGGAAGCGATCTC-3'
CPNβTc6_Cys Rv
5'-GAGATCGCTTCCGAATTAGCAGTCCTCGCTAC-3'
PFDα1_Cys Fw
5'-GGGCTTCAGCGTTAAAAAGTGCTGAGAATTCGAG-3'
PFDα1_Cys Rv
5'-CTCGAATTCTCAGCACTTTTTAACGCTGAAGCCC-3'
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CPNβC366S Fw
The restriction enzyme sites used for cloning are underlined. The stop codon is shown in bold.
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Table 2. Dissociation constants between PFD11 and CPN variants
Relative KD
5.0± 0.53 × 10-8
CPNβTc1
7.1± 2.2 × 10-8
CPNβTc2
1.1± 0.27 × 10-7
CPNβTc6
1.4± 0.31 × 10-7
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Wild type
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KD (M)
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Analyte
1.0 1.4 2.1 2.8
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The dissociation constants (KD) were calculated by steady-state affinity determination
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Graphical abstract
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Highlights - Prefoldin (PFD) interacts with Group II chaperonin (CPN) for substrate transfer.
- Truncation of the CPN C-terminal region decreases affinity with PFD.
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- The flexible C-terminal region of CPN protrudes into the cavity of CPN.
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- Truncation also decreases substrate transfer from PFD and folding by CPN.
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- The C-terminal region of CPN is newly found interaction site with PFD.
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S0022-2836(16)30050-X doi: 10.1016/j.jmb.2016.04.006 YJMBI 65057
To appear in:
Journal of Molecular Biology
Received date: Revised date: Accepted date:
19 February 2016 23 March 2016 4 April 2016
Please cite this article as: Zako, T., Sahlan, M., Fujii, S., Yamamoto, Y.Y., Tai, P.T., Sakai, K., Maeda, M. & Yohda, M., Contribution of the C-terminal region of a group II chaperonin to its interaction with prefoldin and substrate transfer, Journal of Molecular Biology (2016), doi: 10.1016/j.jmb.2016.04.006
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Contribution of the C-terminal region of a group II chaperonin to its
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interaction with prefoldin and substrate transfer
Tamotsu Zako1, $, &, Muhamad Sahlan2,3, $, Sayaka Fujii2, $, Yohei Y. Yamamoto2$,
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Phan The Tai2, Kotaro Sakai1,2, Mizuo Maeda1 and Masafumi Yohda2*
1 2
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Bioengineering Laboratory, RIKEN Institute, Saitama, Japan.
Department of Biotechnology and Life Science, Tokyo University of Agriculture and
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Department of Chemical Engineering, University of Indonesia, Depok, Indonesia. Equal contribution
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$
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Technology, Tokyo, Japan.
&
Current address: Department of Chemistry and Biology, Graduate School of Science and
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Engineering, Ehime University
Correspondence: Masafumi Yohda, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-4-16 Naka-cho, Koganei-shi, Tokyo, Japan 1848588; Tel/fax: +81-420-388-7479; email: [email protected]
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Abbreviations CPN, chaperonin; T.KS-1, hyperthermophilic archaeon Thermococcus sp. strain KS-1; PFD,
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prefoldin; PFD11, prefoldin of Thermococcus sp. strain KS-1 composed of 1 and 1
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subunits; PFD22, prefoldin of Thermococcus sp. strain KS-1 composed of 2 and 2
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subunits; CS, citrate synthase from porcine heart; GFP, green fluorescent protein; SPR, surface plasmon resonance; PAGE, polyacrylamide gel electrophoresis; CPN, T.KS-1
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chaperonin homo-oligomer; CPNTc1, T.KS-1 chaperonin mutant with the truncation of one amino acid from the C-terminus; CPNTc2, T.KS-1 chaperonin mutant with the
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truncation of two amino acids from the C-terminus; CPNTc6, T.KS-1 chaperonin truncation mutant with the truncation of six amino acids from the C-terminus; IPMDH, 3-
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Isopropylmalate dehydrogenase from Thermus thermophilus HB8; dIPMDH, denatured IPMDH; IPMA, isopropylmalate acid; Cy3-dIPMDH, Cy3-labeled dIPMDH; 488-PFDα1β1,
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AlexaFluor 488-labeled PFDα1β1; Cy5-CPNβWT, Cy5-labeled CPNβW; Cy5-CPNβTc6,
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Cy5-labeled CPNTc6; Cy5-CPNβs, Cy5-labeled CPNβs; PFD1tc1, prefoldin of Thermococcus sp. strain KS-1 composed of two 1 subunits with truncation from the Cterminus and four 1 subunits; RU, Resonance Unit; TKM buffer, 50 mM Tris-HCl pH 7.5, 100 mM KCl and 25 mM MgCl2.
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Abstract Prefoldin is a molecular chaperone that captures an unfolded protein substrate and transfers it
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to a group II chaperonin. Previous studies have shown that the interaction sites for prefoldin are located in the helical protrusions of group II chaperonins. However, it does not exclude
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the possibility of the existence of other interaction sites. In this study, we constructed Cterminal truncation mutants of a group II chaperonin and examined the effects of these
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mutations on the chaperone’s function and interaction with prefoldin. Whereas the mutants with up to six amino acids truncation from the C-terminus retained more than 90% chaperone
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activities for protecting citrate synthase from thermal aggregation and refolding of green fluorescent protein and isopropylmalate dehydrogenase, the truncation mutants showed
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decreased affinities for prefoldin. Consequently, the truncation mutants showed reduced transfer efficiency of the denatured substrate protein from prefoldin and subsequent
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chaperonin-dependent refolding. The results clearly show that the C-terminal region of group
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II chaperonins contributes to their interactions with prefoldin, the transfer of the substrate protein from prefoldin and its refolding.
Keywords
Chaperone; Chaperonin; Prefoldin; Protein interaction; Protein folding
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Introduction Chaperonins (CPNs) are ubiquitous, double ring-shaped molecular chaperones that
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capture unfolded proteins in their cavities and assist in protein folding in an ATP-dependent manner [1, 2]. CPNs are subdivided into group I and group II [2, 3]. Group I CPNs are
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present in bacteria and in the organelles of eukaryotes. Group II CPNs exist in the cytosol of archaea and eukaryotes. Studies of the reaction cycle of a group I CPN using Escherichia coli
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GroEL have advanced our knowledge [1, 4, 5]. The heptameric ring-shaped complex of a cochaperonin, GroES, acts as a lid for the central cavity. Group II CPNs exist in archaea and the
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eukaryotic cytosol [3, 6, 7]. The group II CPNs do not require a GroES-like co-chaperonin but have a built-in lid that is composed of a helical protrusion in the apical domain [8-11].
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We have been studying the molecular mechanisms of group II CPNs using the chaperonin from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1 (T. KS-1).
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There are two CPN subunits (α and β) in T. KS-1 [12]. Their amino acid sequences are highly
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similar throughout their entire length, except for approximately 20 amino acid residues at the C-terminus. The T. KS1 CPN α subunit contains the GGM repeat, which is also observed in the C-terminal region in the group II CPNs. However, the GGM repeat does not exist in the C-terminal region of the β subunit. Both subunits can assemble into functional double ring complexes. The stoichiometry of the natural complex varies with temperature [13]. The β subunit content increases with temperature. We have shown that the C-terminal segment correlates with the thermal stability of the complex [14]. The C-terminal segments of the CPNs protrude from the bottom into the cavity but are not resolved in the crystal structure [8]. Tang et al. postulated that the C-terminal GGM repeats of GroEL facilitate the rearrangement of certain folding intermediates by providing a mildly hydrophobic interactive surface [15]. We have determined the crystal structure of the T. KS1 CPN α subunit homo-oligomer [11]. Similar to those of other CPNs, the structure of the 19 C-terminal amino acid residues was 4
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disordered. The high mobility of this region was confirmed by NMR analysis [16]. The fact that the T. KS-1 CPN with a His-tag sequence at the C-terminus can be purified by a nickel
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chelating column also supports the idea that the C-terminal residues protrude into the cavity. Prefoldin (PFD) is a jellyfish-like hexameric protein that is exclusively found in
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archaea and eukaryotes [17-19]. Biochemical studies have shown that PFDs bind to and stabilize unfolded target polypeptides and subsequently deliver them to group II CPNs to
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complete the folding process [20-22]. The transfer of a substrate from PFD to CPN involves a direct interaction [21, 23]. The three-dimensional reconstructed image of a complex of a
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eukaryotic CPN and PFD showed that PFD interacts with the apical domain of CPN [23]. We have investigated the protein-folding mechanism of PFD and CPN using the
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PFD-CPN systems of the hyperthermophilic archaea, T. KS-1 and Pyrococcus horikoshii [16, 20-22, 24-26]. We have shown that both the N- and C-terminal regions of the β subunit of
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PFD are important for its interaction with CPN [16, 26]. The rate of transfer of a substrate
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protein from PFD to CPN correlated with the strength of the PFD-CPN interaction [22]. We have also determined the location of the PFD interaction site that lies on the helical protrusion of CPN. However, based on our study using NMR, we speculated that the C-terminus CPN might also contributes to the interaction with PFD [16]. There are two pairs of PFD subunits (α1, α2 and β1, β2) in T. KS1 [24]. PFDα1β1 and PFDα2β2 are thought to be the physiological complexes. T. KS-1 PFDα1β1 binds to the T.KS-1 CPN β homo-oligomer (CPNβ) at high affinity [24]. In this study, we constructed Cterminal truncation mutants of CPNβ and examined the effects of the truncations on their chaperone functions and interactions with PFDα1β1. The results indicate that the C-terminal region of CPN also influences the transfer efficiency and CPN-mediated refolding of the substrate proteins. These results also imply a novel role for the CPN C-terminal region, which is buried in the cavity, in the interaction with PFD binding and substrate transfer from PFD. 5
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Results Construction and functional characterization of the CPN C-terminal truncation mutants
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In this study, we used CPNβ and PFDα1β1 from T. KS1, because these isoforms interact with each other at high affinity [24]. We expressed and purified three CPNβ mutants,
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with the truncation of one, two and six amino acids from the C-terminus, named CPNβTc1, CPNβTc2, and CPNβTc6, respectively. CPNβTc2 and CPNβTc6 were designed to delete two
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hydrophobic amino acid residues from the CPNβ C-terminus (Gly-Ser-Glu-Asp-/Phe-Gly-SerAsp-/Leu-Asp, (/, Tc6 and Tc2 truncation positions)). The mutants formed hexadecameric
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homo-oligomers, similar to the wild type CPNβ, CPNβWT (data not shown). First, we investigated the effects of the truncations on the ability of CPNβ to protect
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porcine citrate synthase (CS) from thermal aggregation (Fig. 1A). The thermal aggregation of CS was completely suppressed by the presence of an equimolar amount of the CPNβ variants.
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The truncation mutants exhibited the same protection abilities as CPNβWT. The protein
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folding activities of the CPNβ variants were examined using acid-denatured GFP as a substrate. The acid-denatured GFP refolds in a neutral pH environment to emit fluorescence. The amount of spontaneous refolding reached a maximum of approximately 28% at 400 sec after dilution and gradually decreased to approximately 24% at 1200 sec. When CPNβWT is present in the folding mixture, the spontaneous refolding of GFP is inhibited. The addition of ATP at 300 sec induced productive refolding of GFP, and the refolding yield reached approximately 35% at 900 sec after the addition of ATP (Fig. 1B). The refolding yields of the truncated CPNβ variants were more than 90% of as CPNβWT (102% for CPNβTc2 and 91% for CPNβTc6, respectively) (Fig. 1B). The ATP-dependent conformational changes of the truncation mutants were also investigated using a protease sensitivity assay (Fig. 1C). In the open state, the built-in lid is sensitive to protease digestion, while in the closed state, it is not [27]. The CPNβ variants 6
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were subjected to thermolysin digestion in the presence or absence of ATP at 60ºC. In the presence of ATP, all CPNβ variants were relatively resistant to thermolysin. In contrast, the
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CPNβ variants were nearly completely degraded in the absence of ATP. Therefore, like the wild-type protein, the confirmation of the truncated mutants can change to the closed state in
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an ATP-dependent manner.
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Interaction affinities of the CPNβ variants for PFDα1β1
To assess the interaction between PFDα1β1 and the CPNβ variants, we monitored
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their association and dissociation by surface plasmon resonance using Biacore. PFDα1β1 was immobilized on the sensor chip and the CPNβ variants were applied as analytes. The
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sensorgrams of these CPNβ variants injected at various concentrations are shown in Fig. 2. The dissociation constants (KD) for the interaction between the mutants and PFDα1β1 were
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calculated by steady-state affinity determination method (Fig. S1, Supplementary
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information). The KD values for the CPNβ variants are represented in Table 2. Interestingly, the KD values increased in a stepwise manner with the removal of 1, 2, and 6 residues from the C-terminus of CPNβ. These data demonstrated that the affinities of the CPNβ truncation mutants for PFDα1β1 decreased relative to CPNβWT and that the mutant of CPNβ lacking the 6 terminal amino acids showed the weakest affinity for PFDα1β1.
The C-terminal region contributes to the functional cooperation between PFDα1β1 and CPNβ In an aim to clarify the effect of the affinity between PFDα1β1 and the CPNβ variants (CPNβWT and CPNβTc6) on cooperative substrate refolding, substrate transfer and the subsequent refolding were examined. Substrate transfer was examined with FRET using fluorescently labeled PFDα1β1, the CPNβ variants and denatured 3-Isopropylmalate 7
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dehydrogenase (IPMDH) from Thermus thermophilus HB8 (dIPMDH). Substrate release from PFDα1β1 was estimated by dissolution of FRET between Cy3-labeled dIPMDH (Cy3-
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dIPMDH) captured by AlexaFluor 488-labeled PFDα1β1 (488-PFDα1β1) by the addition of non-labeled CPNβ variants. First, FRET between 488-PFDα1β1 and Cy3-dIPMDH was
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confirmed by a decrease in donor fluorescence (AlexaFluor 488) (Fig. 3A). The donor fluorescence increased with the addition of CPNβWT, indicating that the FRET between 488-
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PFDα1β1 and Cy3-dIPMDH was canceled by the substrate release from PFDα1β1. Although the FRET dissolution was also observed by the addition of CPNβTc6, the efficiency decreased
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to 49% of that with CPNβWT. FRET dissolution efficiency by CPNβWT was higher than that by CPNβTc6 at various CPN concentrations (Fig. S2, Supplementary information). This result
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clearly indicates that affinity between PFD and CPNβ correlates with the substrate transfer efficiency.
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To further confirm substrate transfer from PFD to CPNβ, the capture of the released
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substrate by CPN was examined by FRET between Cy3-labeled dIPMDH and Cy5-labeled CPNβs (Cy5-CPNβs). Cy5-CPNβs (Cy5-CPNβWT and Cy5-CPNβTc6) were added to the solution containing a complex of Cy3-dIPMDH and PFDα1β1. As shown in Fig. 3B, the Cy3 fluorescence decreased with the addition of Cy5-CPNβs, indicating the capture of the released substrate by the CPNβs. The FRET efficiency of CPNβTc6 was 64% of that of CPNβWT, supporting a correlation between affinity and substrate transfer. Finally, we examined the effect of the C-terminal truncations on the cooperative refolding of dIPMDH. First, the ATP-dependent refolding of IPMDH by the CPNβ variants was confirmed (Fig. 4A). Both CPNβWT and CPNβTc6 were able to refold dIPMDH in an ATP-dependent manner with the same efficiency, which is consistent with the results for GFP (Fig. 1B). Then, the refolding of dIPMDH captured by PFDα1β1 was applied to CPNβdependent folding. The dIPMDH-PFD complex was added to CPNβWT or CPNβTc6. After 8
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incubation for 300 sec, ATP was added to induce refolding of IPMDH by CPNs (Fig. 4B). The results clearly illustrate the role of PFD in transferring denatured proteins to CPN for
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productive folding. The refolding yields were significantly increased in this experiment compared with those without PFD. The refolding yield by CPNβTc6 was approximately 80%
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of that by CPNβWT. The difference in the refolding yield should be due to the difference in the affinities with PFD between CPNβWT and CPNβTc6. In both cases, initial refolding rate
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is significantly high, which is probably due to the accumulation of CPN-IPMDH complex by substrate transfer from PFD to CPN during the incubation before ATP addition. To observe the
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effect of the difference in the affinities between PFDα1β1 and CPNβ variants more clearly, the dIPMDH-PFD complex was prepared in the buffer containing ATP and was mixed with
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CPNβWT or CPNβTc6 in the presence of ATP (Fig. 4C). Addition of CPNs initiates transfer of dIPMDH from PFDα1β1 to CPNs and refolding of IPMDH by CPNs. In this condition, the
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transferred dIPMDH is immediately subjected to the refolding process by CPN. Thus, the
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yield of IPMDH refolding directly correlates with the interaction efficiency. The refolding yield of CPNβTc6 was approximately 55% of that of CPNβWT. Then, we compared the refolding yields of IPMDH by CPNβWT or CPNβTc6 at various CPN concentrations (Fig. 4D). As the increase of CPN concentration, the difference of the refolding yield increased and reached saturation. At high CPN concentrations, the difference should be determined by the difference of affinity between CPN and PFD. Taken together, the C-terminal region of CPNβ, which is located inside the cavity, affects the interaction affinity with PFD, substrate transfer efficiency and subsequent refolding of substrate proteins by CPNβ.
The PFD α subunit is not responsible for the interaction with the C-terminus of CPN Regarding the possible binding site in PFD for the C-terminal region in the cavity of CPN, we hypothesized that the PFD α subunit, which is relatively long, could contribute to 9
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the interaction. Although the tip of the PFD α subunit appears disordered in the crystal structure, the model of the complex structure indicates that the tip of the αsubunit may be
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deeply inserted into the cavity [23]. In particular, the C-terminal segment of the T. KS1-PFD α1 subunit is long and possesses three Lys residues, and it was likely that this region could be
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important for the interaction with the CPN C-terminal region, which has three Asp residues in the last 7 residues. To confirm this hypothesis, we constructed a PFD α1 subunit C-terminal
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truncation mutant (PFDα1tcβ1), where all three Lys residues were deleted (10 amino acid residues in total), and examined the interaction with CPNβWT. However, the result
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contradicted the hypothesis. The C-terminal truncation of the PFD α subunit did not decrease the interaction with CPNβWT (Fig. 5A), indicating that the C-terminal region of the PFD α
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subunit is not important for the interaction with the CPN C-terminal region. The effect of PFD α1 subunit C-terminal truncation on the cooperative folding of
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IPMDH with CPNβWT was also examined. As shown in Figure 5B, there was no significant
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difference between PFDα1delβ1 and the wild type PFDα1β1 on cooperative IPMDH refolding, supporting the result above. These results indicate that the C-terminus of the PFD α subunit is not important for the interaction with CPN and substrate transfer.
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Discussion Previous studies have shown that the C-terminus of the PFD β subunit interacts with
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the helical protrusions of CPNs via electrostatic interactions [22]. However, the thermodynamic parameters calculated from the association constants indicated that the
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interaction between PFD and CPN is entropy driven [28]. There are two pairs of prefoldin subunits (α1, α2 and β1, β2) in T. KS-1, and PFDα1β1 and PFDα2β2 are thought to be the
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physiological complexes. We could construct four different PFD complexes (PFDα1β1, PFDα1β2, PFDα2β1 and PFDα2β2) from them, and CPNβ exhibited higher affinities to all of
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them compared to CPNα [24]. Among all of the PFD complexes, PFD complexes containing the β1 subunit (PFDα1β1 and PFDα2β1) exhibited relatively higher affinity to CPNβ than
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those with the β2 subunit (PFDα1β2 and PFDα2β2), possibly because the PFD β2 subunit has shorter N- and C-termini compared to the β1 subunit and lacks one Lys residue that is thought
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to interact with the Glu residue on the CPN helical protrusion [22, 28].
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However an unexplained result remained. PFD complexes containing the β1 subunit have higher affinities to CPNα, which contains basic residues in the helical protrusion, than those with the β2 subunit [24]. This result suggests the existence of another interaction site. Our previous NMR-based study also demonstrated that the C-terminal region of the T.KS-1 CPN subunits is highly flexible in the large PFD-CPN complex, suggesting that it plays a role in the interaction between T.KS-1 CPN and PFD [16]. Thus, we postulated that the CPN Cterminal region would be another important site for the interaction with PFD. In this study, we constructed CPNβ with C-terminal truncations and examined the effects of these truncations on the interaction with PFD. The mutants exhibited the similar chaperone activity and ability to undergo a conformational change as the wild-type protein. We then examined the interaction between PFD and the CPNβ variants by surface plasmon resonance using the Biacore system. The comparison of the sensorgrams derived from the 11
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wild-type protein and the mutants showed that the affinities of all of the C-terminal truncation mutants for PFD were significantly decreased compared to the wild-type protein. The mutant
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with a 6 amino acid truncation from C-terminus had the lowest affinity of any mutant examined. In addition, the dissociation constants for the C-terminal truncation mutants with 2
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and 6 amino acid truncations were not significantly different (Fig. 2, Table 2). These results clearly demonstrate that the C-terminal segment of CPNβ contributes to the interaction of
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CPNβ with PFD. Of the 6 C-terminal amino acids (Phe-Gly-Ser-Asp-Leu-Asp), the last two, Asp and Leu, appear to be the most important for binding to PFD.
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Importantly, the C-terminal truncation of CPNβ also affected substrate transfer from PFD. The efficiency of substrate transfer, as estimated by FRET, and CPN-dependent
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refolding were decreased for the C-terminal truncation mutants. This result indicates that affinity between PFD and CPN correlates with substrate transfer efficiency and supports the
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importance of the CPN C-terminal region for cooperative refolding by PFD and CPNβ.
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Both the N- and C-termini of CPNβ are located at the bottom of the CPNβ cavity in the crystal structure of the T. KS-1 CPN αhomo-oligomer [11]. However, the N-terminal 8 and the C-terminal 19 residues are disordered and were therefore excluded from the final model. Combined with our previous NMR data, this observation suggests that the C-terminal segment might not be located at the bottom of the cavity and therefore may contribute to protein folding and interaction with PFD in the cavity. Tang et al. have reported that the Cterminal segment of GroEL contributes to the rapid folding of certain proteins encapsulated in the central cavity, supporting the flexible movement of the C-terminal segment [15]. We hypothesized that the C-terminal region of the PFD α subunit might be responsible for the interaction, because it is long and possesses three Lys residues. However, this was clearly not the case in the experiment using PFD containing a truncation mutant of the α1 subunit. 12
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Taking all of the results into account, it is plausible that the PFD β subunit could interact with the CPN C-terminal region; i.e., the PFD β subunit simultaneously interacts with
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the apical domain [23] and C-terminal region of CPNβ. This idea could be supported by our previous observation showing that T. KS1 PFD variants, including the β1 subunit, showed
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stronger affinity to CPNβWT than those including the β2 subunit [24], possibly because the longer β1 subunit (117 a.a.) could interact more effectively with the flexible C-terminal
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segment in the cavity than the β2 subunit (99 a.a.). Our other previous studies showing that truncation of PhPFD β subunit reduced its affinity to T. KS1 CPNs [16, 26] also support this
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idea. In addition, this is consistent with our previous finding showing that the interaction between PFD and T. KS1 CPN is also entropy-driven [28]. It is plausible that the disordered
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regions of the T. KS1 CPN C-terminus and PFD interact with each other. We have postulated that the difference in the interaction of CPN and the PFD complexes containing the β1 and β2
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subunits is caused by the difference in the interaction with helical protrusion, as β1 and lacks
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one Lys residue that is thought to interact with Glu residue. However, there are no basic residues in the extended region of the β1 subunit, and the β2 subunit retained some basic residues. Thus, it is reasonable to think that the difference between the β1 and β2 subunits should be caused by the differences in interaction with the C-terminal region and not with the helical protrusion. Studies on the PFD β subunit disruptants have shown that PFDα1β1 (PfdA/PfdB complex) plays a crucial role at all growth temperatures and PFDα2β2 (PfdC/PfdD complex) contributes to survival in a high-temperature [29]. The C-terminal region seems to be responsible for discrimination between PFDα1β1 and PFDα2β2. Disruption of CPNβ gene significantly affected the growth at the elevated temperatures [30]. We have thought that the effect is due to relatively low thermal stability of the remaining CPN, CPNα. However, it might be partly due to the lack of cooperation between CPNα and PFDα2β2 [24]. 13
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In conclusion, we demonstrated that in addition to the group II CPN apical domain, the C-terminal region in the cavity of the group II CPN is important for the interaction with
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PFD and their cooperative folding. Our results also suggested that the PFD β subunit would interact with both the CPN apical domain and C-terminal region simultaneously. Further
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studies, such as detailed cryo-EM observations, would be necessary to confirm this idea.
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Materials and Methods Proteins
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The genes for the truncation mutant CPNβ subunits were constructed by polymerase
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chain reaction (PCR) using the primers shown in Table 1. The expression vector pET9a (Merck Millipore, Billerica, MA, USA) was used to express the CPNβs. The CPNβ
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complexes with the wild-type and C-terminal truncation mutants were expressed and purified as described previously [31]. The gene for the truncation mutant (10 amino acid residues from
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the C-terminus) of the T. KS-1 PFD α1 subunit (PFDα1tc) was constructed with the
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QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) by replacing the corresponding sequence with a stop codon. The primers used are shown in Table 1. PFDα1β1
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and PFDα1tcβ1 were expressed and purified as described [24]. IPMDH was expressed in E. coli and purified as described [25].
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For site-specific fluorescent dye labeling of CPN, the intrinsic cysteine residue of CPNβ was replaced with serine (CPNβC366S), and cysteine was introduced in the C-terminal
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end of CPNβC366S (CPNβ_Cys) and C-terminal 6 amino acid truncation mutant of CPNβC366S (CPNβTc6_Cys) using the QuikChange Site-directed Mutagenesis Kit. The Cterminal end of the α subunit of PFDα1β1 was also replaced with cysteine (PFD_Cys). PFDα1β1 has no intrinsic cysteine residues. The primers used are shown in Table 1.
Characterization of T. KS1 chaperonins Thermal aggregation of citrate synthase from porcine heart (CS) was monitored using light scattering at 500 nm in a fluorophotometer (FP-6500, JASCO, Tokyo, Japan) at 50ºC. CS (120 nM, as monomer) was incubated in TKM buffer (50 mM Tris-HCl pH 7.5, 100 mM KCl and 25 mM MgCl2) with or without each CPN variant (120 nM). The assay buffer was pre-incubated at 50ºC and continuously stirred throughout the measurements.
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For the protease sensitivity assay, the CPNs were incubated with or without ATP (1 mM) at 60ºC in TKM buffer under continuous mixing. Digestion with thermolysin (1 ng/L)
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was then performed for 5 min at 60ºC. The proteins in the reaction mixture were precipitated by the addition of trichloroacetic acid and then analyzed by SDS polyacrylamide gel
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electrophoresis.
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Refolding assay
The CPN-dependent green fluorescence protein (GFP) refolding reactions were
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performed at 60ºC, as previously described [22, 31]. In brief, the acid-denatured GFP was diluted into TKM buffer with 5 mM DTT in the presence or absence of the CPNs. The
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fluorescence of GFP at 510 nm with excitation at 396 nm was monitored using a spectrofluorometer (FP-6500, JASCO). As a control, the native GFP protein was diluted into
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the folding buffer. The fluorescence intensity of native GFP was set to 100%.
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IMPDH refolding was performed as described, with slight modifications [25]. IPMDH (4 M) was unfolded in 6 M GdnHCl and 50 mM TrisHCl, pH 7.5, overnight at room temperature. The denatured IPMDH (dIPMDH) was diluted to 25 nM in an assay buffer (TKM buffer including 0.4 mM isopropylmalate acid (IPMA) and 1 mM NAD) in the presence or absence of 100 nM CPN. dIPMDH refolding was started with the addition of 1 mM ATP. IMPDH refolding was measured as the rate of increase in absorbance at 340 nm of NADH, which was converted from NAD by the refolded IMPDH, using a spectrophotometer (V-570 or V-650 JASCO). The increase in the rate of the native IPMDH was set to 100%. For ATP-dependent refolding experiment of IPMDH by CPN after the transfer from PFD, 25 nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer without ATP for 300 sec at 60ºC, and 100 nM CPN were then added. After a 300 sec incubation, 1 mM ATP was added to initiate refolding IPMDH by CPN. For the substrate transfer experiment from PFD to CPN, 16
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25 nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer with 1 mM ATP for 300 sec at 60ºC, and 100 nM CPN was then added for transfer and CPN-dependent refolding
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of dIPMDH. IPMDH refolding was monitored as described above. Spontaneous refolding
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without chaperones was performed as a negative control experiment.
Analysis of the PFD-CPN interaction by surface plasmon resonance
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The surface plasmon resonance (SPR) experiments were performed using the Biacore J or Biacore T-100 systems (GE Healthcare, Buckinghamshire, UK) at a sensor temperature
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of 25ºC, as previously described [26, 28]. The dissociation constants (KD) were calculated by steady-state affinity determination method. In brief, each CPN mutant was injected on
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immobilized PFD at various concentrations (at least 5 concentrations). Then KD was obtained by fitting to the SPR signal data with a recursive least-squares algorithm by KaleidaGraph
Req = Rmax ·C/(C + KD)
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(Synergy Software, Reading, PA, USA) using the following equation:
where Req represents the resonance units at equilibrium, Rmax is the resonance signal at saturation, and C is the injected CPN concentration. Req values were obtained by fitting to the sensorgrams using Langmuir binding model with BIAevaluation software (GE Healthcare). Relative KD values were calculated by dividing the KD values of CPN mutants by the KD value of the wild-type CPN.
Estimation of substrate transfer from PFD to CPN with fluorescent resonance energy transfer (FRET) Substrate transfer from PFD to CPN was also examined with FRET using fluorescently labeled dIPMDH, PFD and CPNβ. IPMDH was incubated with the Cy3 mono-reactive dye (GE Healthcare) (Cy3-IPMDH) overnight at 4°C. The labeled substrate proteins were 17
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separated from the unreacted dyes using a NAP5 gel filtration column (GE Healthcare) equilibrated with TKM buffer. For site-specific labeling of PFD and CPN, the C-terminal
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residues of the α subunit of PFDα1β1 and cysteine-free CPN mutant were replaced with cysteine residues, as described above (PFD_Cys and CPNβ_Cys). PFD_Cys and CPNβ_Cys
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were labeled with AlexaFluor 488-C5-maleimide (AlexaFluor 488, Invitrogen, OR, 488-PFD) and Cy5-maleimide (GE Healthcare, Cy5-CPN), as described above. The molar ratios of
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AlexaFluor 488 to PFDα1β1, Cy5 to CPNβWT/CPNβTc6 and Cy3 to IPMDH were 0.6 (per PFD complex), 3.3/2.8 (per CPN oligomer) and 2.5, respectively. The labeled PFD and CPN
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proteins showed similar chaperone activity as the unlabeled proteins (data not shown). Substrate transfer was estimated with two different FRET phenomena, as follows:
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1) denatured Cy3-IPMDH captured by 488-PFD was transferred to the non-labeled CPNs and 2) denatured Cy3-IPMDH captured by non-labeled PFD was transferred to Cy5-CPNs
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(CPNβWT and CPNβTc6). In the former case, FRET between AlexaFluor 488 and Cy3 was
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canceled by substrate release from PFD. Cy3-IPMDH was denatured as described above (Cy3-dIPMDH). 488-PFD (100 nM) was incubated with denatured Cy3-IPMDH (100 nM) in TKM buffer for 5 min at 60°C, and the non-labeled CPNs (100 nM) were then added and incubated for 5 min. The emission spectra were recorded upon excitation at 460 nm with a spectrofluorometer (FP-6500, JASCO). The emission spectra of the same concentrations of 488-PFD only, Cy3-dIPMDH only and 488-PFD/Cy3-dIPMDH without CPN were also obtained. In the latter case, FRET between the Cy3 and Cy5 dyes was observed by substrate capture by CPN. Denatured Cy3-dIPMDH (100 nM) was incubated with non-labeled PFD for 5 min at 60°C, and Cy5-CPNs (100 nM) were then added and incubated for 5 min. The emission spectra were recorded upon excitation at 540 nm with a spectrofluorometer (FP6500, JASCO). The emission spectra of the Cy3-IPMDH/PFD and Cy5-CPN only samples were also obtained. 18
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FRET was estimated by the decrease in donor fluorescence, and FRET dissolution by the increase in donor fluorescence, respectively. FRET efficiency was calculated as
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follows: FRET efficiency=1 – Fd(+acceptor)/Fd (-acceptor),
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where Fd represents the donor fluorescence in the presence or absence of the acceptor dye. The FRET dissolution efficiency following the addition of CPN was determined as the
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increase in donor fluorescence with the added CPNs. The FRET efficiency and FRET
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dissolution efficiency in the presence of the wild-type CPN was normalized to 100%.
Acknowledgments
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The work reported here was supported in part by a program intended to improve the graduate school education provided by the "Human Resource Development Program for
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Scientific Powerhouse," which is financially supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan through Tokyo University of Agriculture &
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Technology. This study was also supported by grants-in-aid for scientific research (21370067, 22020011, 24370064 and 26102511 (MY), and 24570143 (TZ)), and a grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports, and Culture of Japan to MY, and RIKEN.
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protein. Science. 2002;295:1852-8.
[2] Horwich AL, Fenton WA, Chapman E, Farr GW. Two families of chaperonin: physiology and mechanism. Annu Rev Cell Dev Biol. 2007;23:115-45. protein folding machine. J Mol Biol. 1999;293:295-312.
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[3] Gutsche I, Essen LO, Baumeister W. Group II chaperonins: new TRiC(k)s and turns of a [4] Saibil HR, Fenton WA, Clare DK, Horwich AL. Structure and allostery of the chaperonin
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GroEL. J Mol Biol. 2013;425:1476-87.
[5] Taguchi H. Reaction Cycle of Chaperonin GroEL via Symmetric "Football" Intermediate. J Mol
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Figure Legends
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Fig. 1 Functional characterization of the various CPN mutants.
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A. The effect of C-terminal truncations of CPN on the thermal aggregation of CS. CS (120 nM) was incubated in the absence (“without CPN”) or presence of chaperonins (120 nM
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CPNβWT, CPNβTc2 and CPNβTc6) at 50°C.
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B. Refolding of GFP by CPNs. The folding mixture was incubated at 60°C, and the recovery of native GFP was continuously monitored by measurement of the fluorescence emission at
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510 nm. The fluorescence intensity of native GFP at the same concentration was set to 100%. At 0 min of incubation, acid-denatured GFP (5 M) was diluted 100-fold into the folding
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buffer containing 100 nM CPNs (CPNβWT, CPNβTc2, or CPNβTc6). At 5 min after dilution, 1 mM ATP was added. The spontaneous refolding of GFP was observed upon dilution of the
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“spontaneous”.
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denatured GFP into the folding buffer without chaperonins or ATP and is marked as
C. Protease sensitivity assay demonstrating the conformational changes in CPNs. CPNs (CPNβWT, CPNβTc2, or CPNβTc6) were incubated with thermolysin (1 ng/L) in the presence or absence of ATP (1 mM) and then analyzed by SDS-PAGE. The details are provided in the Materials and Methods section.
Fig. 2 Comparison of interaction between the CPN variants and PFD. Sensorgrams of the interactions of PFD with the CPNs (CPNβWT, CPNβTc1, CPNβTc2, and CPNβTc6) analyzed with the Biacore J system are shown. PFDα1β1 was immobilized on a Biacore CM biosensor chip at 5000 RU, and CPNs at various concentrations were injected as the analyte. In the figure, sensorgrams of 4 analyte concentrations were shown for comparison.
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Fig. 3 Substrate transfer from PFD to CPN examined by FRET
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A Transfer of denatured Cy3-IPMDH captured by 488-PFD to non-labeled CPNs. 488PFD (100 nM) was incubated with denatured Cy3-IPMDH (100 nM) in TKM buffer for 5 min
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at 60°C, and non-labeled CPNs (100 nM, CPNβWT, red; CPNβTc6, blue) were then added and incubated for 5 min. The emission spectra were recorded upon excitation at 460 nm with
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a spectrofluorometer. The emission spectra of 488-PFD (black), the 488-PFD/Cy3-IPMDH complex (green) and Cy3-IPMDH (yellow) were also shown. The FRET dissolution
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efficiency was obtained by the increase in the peak intensity of the donor fluorescence with the addition of CPNs from that of the 488-PFD/Cy3-IPMDH complex. The FRET dissolution
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efficiency of the wild-type CPN was normalized to 100%. The average value of two
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B Transfer of denatured Cy3-IPMDH captured by PFD to Cy5-CPNs. PFD (100 nM) was
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incubated with denatured Cy3-IPMDH (100 nM, black) in TKM buffer for 5 min at 60°C, and Cy5-CPNs (100 nM, CPNβWT, red; CPNβTc6, blue) were then added and incubated for 5 min. The emission spectra were recorded upon excitation at 520 nm with a spectrofluorometer. The emission spectra of Cy5-CPNβWT (green) and Cy5-Tc6 (yellow) were also shown. The FRET efficiency was calculated from the decrease in the donor fluorescent intensity, as described in the text. The FRET efficiency of the wild-type CPN was normalized to 100%. The average value of two independent experiments is shown.
Fig. 4 IPMDH refolding by CPN transferred from PFD A, IPMDH refolding by CPN. IPMDH (4 M) that had been unfolded in 6 M GdnHCl (dIPMDH) was diluted to 25 nM in an assay buffer (TKM buffer including 0.4 mM
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isopropylmalate acid (IPMA) and 1 mM NAD) in the presence or absence of 100 nM CPNs (CPNβWT, red; CPNβTc6, blue). dIPMDH refolding was started with the addition of 1 mM
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ATP. IMPDH refolding was measured as the rate of increase in absorbance at 340 nm and 60°C of NADH, which was converted from NAD by the refolded IMPDH, using a
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spectrophotometer (V-570, JASCO). The increase in the refolding rate of the native IPMDH was set to 100%. Spontaneous refolding without chaperones was performed as a negative
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control experiment (green).
B, ATP-dependent refolding of IPMDH by CPN after the transfer from PFD. Twenty-five
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nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer without ATP for 300 sec at 60ºC, and 100 nM CPNs (CPNβWT, red; CPNβTc6, blue) were then added. After a 300
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sec incubation, 1 mM ATP was added to initiate refolding IPMDH by CPNs. IPMDH
shown in green.
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refolding was monitored as described above. Spontaneous refolding without chaperones is
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C, Transfer of IPMDH from PFD and refolding by CPN. Twenty-five nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer with 1 mM ATP for 300 sec at 60ºC, and 100 nM CPNs (CPNβWT, red; CPNβTc6, blue) were then added for transfer and CPNdependent refolding of dIPMDH. IPMDH refolding was monitored as described above. Spontaneous refolding without chaperones is shown in green. D, Refolding of IPMDH by CPN at various concentrations transferred from PFD. Twenty-five nM dIMPDH was pre-incubated with 100 nM PFD in the assay buffer with 1 mM ATP for 300 sec at 60ºC, and CPNs (CPNβWT or CPNβTc6) at various concentrations (25, 50, 100, 150 and 200 nM) were then added for transfer and CPN-dependent refolding of dIPMDH. Ratio of refolding yields after 900 sec incubation by CPNβWT against CPNβTc6 was shown in the figure. The average values of three independent experiments were shown. 25
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Fig. 5 Effect of the C-terminal truncation of the PFD α subunit on the interaction with
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CPN and cooperative IPMDH refolding
A, Interaction between PFDs and CPN. Sensorgrams of the interactions between CPNβWT
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and PFDs (PFDα1β1 and PFDα1tcβ1) analyzed with the Biacore T-100 system are shown. PFDα1β1 (red) and PFDα1tcβ1 (blue) were immobilized on a Biacore CM biosensor chip
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(10700 RU for PFDα1β1 and 9700 RY for PFDα1tcβ1, respectively). The sensorgram of CPNβWT injected at 20 nM as an analyte is shown. The KD values were calculated to be
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28.3±4.7 nM for PFDα1β1 and 37.9 ± 5.8 nM for PFDα1tcβ1 (averaged values of three
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independent runs).
B, Refolding of IPMDH by CPN transferred from PFD truncation mutant. Twenty-five
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nM dIMPDH was pre-incubated with 100 nM PFDs (PFDα1β1 (red) and PFDα1tcβ1 (blue)) in the assay buffer with 1 mM ATP for 300 sec at 60ºC, and 100 nM CPNβWT was then
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added for transfer and CPN-dependent refolding of dIPMDH. IPMDH refolding was monitored as the rate of increase in absorbance at 340 nm and 60°C using a spectrophotometer (V-650, JASCO), as described above. Spontaneous refolding without chaperones is shown in green.
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Table 1. Primer sequences used in this study Name
Sequence
Restriction site
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Primers for the construction of the truncated mutants
5’-CGCGGATCCGCGTCAGAGATCGCTTCCGAAGTC-3’
BamH I
CPNβTc2 Rv
5’-CGGGATCCCGTCAATCGCTTCCGAAGTC-3’
BamH I
CPNβTc6 Rv
5’-CGGGATCCCGTCAGTCCTCGCTACCGCCCTT-3’
BamH I
CPNβ Fw
5’-GGAATTCCATATGGCCCAGCTTGCAGGCCAG-3’
PFDα1tc Fw
5'-CAGGCTCAGGAAATCCAGCAGTGACAGGCGATGGGCTTCAGCG-3'
PFDα1tc Rv
5'-CGCTGAAGCCCATCGCCTGTCACTGCTGGATTTCCTGAGCCTG-3'
Nde I
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CPNβTc1 Rv
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Primers for the construction of the cysteine mutants
5'-GATCTTCGTCGAGGGCTCCAAGAACCCGAAG-3'
CPNβC366S Rv
5'-CTTCGGGTTCTTGGAGCCCTCGACGAAGATC-3'
CPNβ_Cys Fw
5'-GAAGCGATCTCGACTGCTGAAGCTTGCGGCC-3'
CPNβ_Cys Rv
5'-GGCCGCAAGCTTCAGCAGTCGAGATCGCTTC-3'
CPNβTc6_Cys Fw
5'-GTAGCGAGGACTGCTAATTCGGAAGCGATCTC-3'
CPNβTc6_Cys Rv
5'-GAGATCGCTTCCGAATTAGCAGTCCTCGCTAC-3'
PFDα1_Cys Fw
5'-GGGCTTCAGCGTTAAAAAGTGCTGAGAATTCGAG-3'
PFDα1_Cys Rv
5'-CTCGAATTCTCAGCACTTTTTAACGCTGAAGCCC-3'
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CPNβC366S Fw
The restriction enzyme sites used for cloning are underlined. The stop codon is shown in bold.
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Table 2. Dissociation constants between PFD11 and CPN variants
Relative KD
5.0± 0.53 × 10-8
CPNβTc1
7.1± 2.2 × 10-8
CPNβTc2
1.1± 0.27 × 10-7
CPNβTc6
1.4± 0.31 × 10-7
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Wild type
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KD (M)
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Analyte
1.0 1.4 2.1 2.8
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The dissociation constants (KD) were calculated by steady-state affinity determination
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Graphical abstract
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Highlights - Prefoldin (PFD) interacts with Group II chaperonin (CPN) for substrate transfer.
- Truncation of the CPN C-terminal region decreases affinity with PFD.
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- The flexible C-terminal region of CPN protrudes into the cavity of CPN.
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- Truncation also decreases substrate transfer from PFD and folding by CPN.
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- The C-terminal region of CPN is newly found interaction site with PFD.
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