- Email: [email protected]
Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin
doi:10.1016/j.jmb.2010.04.046
J. Mol. Biol. (2010) 399, 628–636
Available online at www.sciencedirect.com
Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin Muhamad Sahlan 1 , Tamotsu Zako 2 , Phan The Tai 1 , Akashi Ohtaki 1 , Keiichi Noguchi 1 , Mizuo Maeda 2 , Hideyuki Miyatake 3 , Naoshi Dohmae 3 and Masafumi Yohda 1 ⁎ 1
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan 2
Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
3
Biomolecular Characterization Team, Advanced Development and Supporting Center, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Received 22 February 2010; received in revised form 20 April 2010; accepted 23 April 2010 Available online 29 April 2010
Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit and characterized the interactions between T. KS-1 CPNs (CPNα and CPNβ) and T. KS-1 PFDs (PFDα1–β1 and PFDα2–β2). As predicted from its amino acid sequence, the PFDβ2 subunit conforms to a structure similar to those of the PFDβ1 subunit and the Pyrococcus horikoshii OT3 PFDβ subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNα and PFDα1–β1; CPNα and PFDα2–β2; CPNβ and PFDα1–β1; and CPNβ and PFDα2–β2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 ºC. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD–CPN interactions, the entropy difference in binding between CPNβ and PFDα2–β2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDα2–β2 and CPNβ subunit is induced upon heat shock, our results suggest that PFDα1–β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2–β2 is specific for CPNβ. © 2010 Elsevier Ltd. All rights reserved.
Edited by K. Morikawa
Keywords: prefoldin; group II chaperonin; crystal structure; surface plasmon resonance; thermodynamics
Introduction Prefoldin (PFD) is a jellyfish-shaped heterohexameric cochaperone that captures an unfolded protein substrate and transfers it to group II chaperonin (CPN) to complete protein folding.1–3 It has been shown that PFD participates in the maturation of actin and tubulin members in collaboration with the eukaryotic CPN, CCT (cytosolic chaperonin containing TCP-1).1,4,5 Although archaea do not possess *Corresponding author. E-mail address: [email protected]. Abbreviations used: PFD, prefoldin; CPN, group II chaperonin; T. KS-1, Thermococcus strain KS-1; PhPFD, Pyrococcus horikoshii OT3 PFD; GFP, green fluorescence protein; SPR, surface plasmon resonance.
actin or tubulin, both PFD and CPN have invariably been identified in all archaeal species.1,6 The PFD– CPN system functions as a general molecular chaperone in archaea because only limited members of chaperones exist.6 The archaeal PFD is composed of two α subunits and four β subunits (α2β4). The structures of Methanobacterium thermoautotrophicum PFD and Pyrococcus horikoshii OT3 PFD (PhPFD) have been characterized.3,7 Archaeal PFD consists of a double β-barrel assembly with six coiled-coil domains that protrude from the center like a jellyfish with six tentacles. The eukaryotic PFD is composed of six different subunits (two α-type subunits and four β-type subunits), and it is thought to have a structure similar to those of archaeal PFDs.8 Biochemical studies have shown that PFDs bind and stabilize unfolded target polypeptides and subsequently deliver them to CPNs to complete
0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.
Prefoldin–Group II Chaperonin Interaction
folding.9–11 The transfer of a substrate from PFD to CPN involves a direct interaction.11,12 We have studied the mechanism of protein folding mediated by the archaeal PFD–CPN system.9,10,13 PhPFD and PFDs of Thermococcus strain KS-1 (T. KS-1) can capture an acid-denatured green fluorescence protein (GFP) and transfer it to T. KS-1 CPN for ATPdependent folding. The release of a substrate protein from PhPFD is facilitated by T. KS-1 CPN.10 We also identified the regions in PFD and CPN that are responsible for their interaction.11,14 The tips of the coiled coils of PFD and the helical protrusion region in the apical domain of CPN are thought to interact with substrate proteins and CPNs.3,5,7,8,11,12,14,15 T. KS-1 expresses two different CPN subunits (α and β).16,17 Recombinant α and β subunits assemble to form a double-ring homo-oligomer: CPNα and CPNβ. Both of them have high ATP-dependent protein folding activity toward denatured GFP and citrate synthase.18 The expression of α subunit and β subunit is regulated differently, and only the β subunit is heat inducible.16 The subunit composition of natural T. KS-1 CPN is variable, with an increase in the proportion of β subunits at higher temperatures. Indeed, the thermostability of the CPN
629 complex is correlated with the number of β subunits in the complex. Thermostability is thought to arise from the differences in 20 amino acids at the Cterminal end.19 CPNβ interacts with PFD more strongly than CPNα.11 Comparing the interaction of CPNα, CPNβ, and their mutants with PhPFD, we have previously shown that two acidic amino acids in the helical protrusion of the β subunit (E250 and E256) are responsible for the strong interaction with PhPFD.11 Thus, the interaction between PFD and CPN is partly driven by electrostatic interactions. T. KS-1 expresses two pairs of PFD subunits genes: two α-subunit genes pfdα1 and pfdα2, and two βsubunit genes pfdβ1 and pfdβ2.13 We have functionally characterized four recombinant T. KS-1 PFD complexes (PFDα1–β1, PFDα1–β2, PFDα2–β1, and PFDα2–β2).13 All four complexes make similar heterohexameric structures and exhibit chaperone activity to suppress thermal aggregation. Moreover, the transfer of a denatured GFP to CPNα for folding was demonstrated for PFDα1–β1. The affinities of PFDα1–β1 and PFDα2–β1 for CPNα were almost identical, whereas CPNβ exhibited a relatively strong affinity for PFD complexes. PFD complexes with β2 subunit exhibited only marginal interactions
Fig. 1. The crystal structure of the PFDβ2 subunit. (a) The monomer structure of the PFDβ2 subunit. Hydrophobic residues (Ala6, Leu9, Leu13, Ile87, Leu91, and Leu94) at the distal region are presented as stick models. (b) Superposition of Cα atoms of the coiled-coil regions (residues 26–33 and 59–67; PFDβ2 numbering) of PFDβ1 and PFDβ2 subunits. Hydrophobic residues (PFDβ2), basic residues (K93 in PFDβ2), and basic residues (K101 and R112 in PFDβ1) are shown as stick models. (c) Bottom view and (d) side view of the jellyfish-like structure of the PFDβ2 subunit tetramer. In the β2 tetramer complex structure, the β2 subunit is shown in green, and symmetric molecules are shown in gray. (e) The dimer of the PFDβ2 subunit tetramer. Each tetramer is shown in green and gray. These figures were created with the program MOLSCRIPT.
630 with CPNβ. Therefore, we hypothesized that PFD complexes containing the β1 subunit cooperate with both CPNα and CPNβ, whereas PFD complexes with the β2 subunit cooperate only with CPNβ. This hypothesis coincides well with the fact that the CPNβ and PFDβ2 subunits are expressed at elevated temperatures. A previous study on the cognate Thermococcus species Thermococcus kodakaraensis showed that PFDα1–β1 and PFDα2–β2 complexes are dominantly expressed in cells.20 Transcriptional and translational analyses showed that the β1 subunit of T. kodakaraensis is constitutively expressed under normal and heat-stressed conditions, and that β2 expression is highly induced at elevated temperatures. We reported the crystal structure of T. KS-1 PFDβ1 subunit at 1.7 Å resolution and found that T. KS-1 PFDβ1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFDs. The β-hairpin linkers of β1 subunits assemble to form a β-barrel “body” around a central 4-fold axis. As our previous results showed that the difference in interactions between T. KS-1 PFDs and CPNs is caused by differences in the PFDβ subunit, we attempted to determine the crystal structure of the PFDβ2 subunit. In this study, we investigate the crystal structure of the T. KS-1 PFDβ2 subunit and examine the thermodynamic properties of the interaction between CPNs and PFDs. The results clearly demon-
Prefoldin–Group II Chaperonin Interaction
strated that the interaction between CPNs and PFDs is driven by entropy changes and that the contribution of electrostatic interactions is marginal.
Results Crystal structure of the T. KS-1 PFDβ2 subunit To elucidate the difference in affinity between T. KS-1 PFDs and CPNs caused by the difference in PFDβ subunit, we determined the crystal structure of the PFDβ2 subunit. The crystal of the β2 subunit with space group P4212 has two molecules in the asymmetric unit. Both structures have 94 (residues 1–94) of the 99 sequenced residues. The monomer structure of PFDβ2 is divided into three regions: an N-terminal helix (residues 1–38), a β-hairpin linker (residues 46–58), and a C-terminal helix (residues 59–94), as shown in Fig. 1a. The N-terminal and Cterminal helices form an anti-parallel helical coiledcoil assembly. The β-hairpin linker contributes to the formation of the tetramer, as mentioned below. At the distal region of the coiled-coil structure, the hydrophobic groove, a putative substrate-binding site, is formed by Ala6, Leu9, Leu13, Ile87, Leu91, and Leu94. The structure fits well with the structure of the PFDβ1 subunit (Fig. 1b).
Fig. 2. Comparison of the interaction between T. KS-1 PFDs and CPNs, and the effect of two amino acid substitutions (K250E and K256E) on the CPNα subunit. SPR sensorgrams of the interactions of PFDα1–β1 with CPNα, CPNβ, and CPNαK250E/K256E (a), and SPR sensorgrams of the interactions of PFDα2–β2 with CPNα, CPNβ, and CPNαK250E/ K256E (b). The concentrations of analytes (CPNs) were fixed at 50 nM (a) and 100 nM (b). (c) Sequence alignment of PFDβ subunits. Identical, highly similar, and poorly similar amino acids are shown as (⁎), (:), and (.), respectively. K101 and R112 of PhPFDβ subunit and T. KS-1 PFDβ1 subunit are marked by (#). Acidic residues and basic residues are shown in red and blue, respectively. PH_b, PhPFDβ subunit; KS_b1, T. KS-1 PFDβ1 subunit; KS_b2, T. KS-1 PFDβ2 subunit.
Prefoldin–Group II Chaperonin Interaction
The two molecules in the asymmetric unit are of the same structure, with an r.m.s.d. of 1.52 Å. Both molecules form a tetramer related by the crystallographic 4-fold axis, as shown in Fig. 1c and d. The tetramer forms a jellyfish-like structure (Fig. 1c and d), which is similar to the structures of the PFDβ1 tetramer and the hexameric PhPFD and M. thermoautotrophicum PFD. Four tentacles emanate from the body of the jellyfish, forming a cavity at their center. In the crystalline state, two PFDβ2 tetramers face each other, resulting in a dimer of tetramers (Fig. 1e). One tentacle of the tetramer interacts with only one tentacle of the other tetramer through hydrogen bonds. The hydrophobic groove of the tentacles does not interact with any other PFDβ2 subunit. Each tentacle fits tightly between the tentacles of the other tetramer, forming a highly stable structure. These interactions between tentacles seem to open the cavity. This crystal structure of the PFDβ2 tetramer showed that each coiled-coil domain of the PFDβ2 tetramer is more highly flexible and changes various conformations when in solution. Although we tried to crystallize PFDα2–β2, we could not obtain crystals. The crystals obtained from the crystallization of PFDα2–β2 yielded only crystals of PFDβ2, similar to the case of PFDα2–β1.21 Thus, the complexes likely dissociated in the crystallization solution.
631 PFDα2–β2 lacks the interaction site for the two acidic residues on the helical protrusions of the CPNβ subunit We showed that the affinity of CPNβ for PhPFD is stronger than that of CPNα.11 The difference is attributed to two amino acid residues at the helical protrusion. The 250th and 256th amino acid residues of CPNβ and Pyrococcus horikoshii CPN are acidic glutamates, but those of CPNα are basic residues. These residues are located on the inner surface of the helical protrusion. Single and double mutants of these residues in the CPNα subunit, namely CPNαK250E, CPNαK256E, and CPNαK250E/ K256E, exhibited a stronger interaction with PhPFD than CPNα. Next, we compared the interactions of CPNαK250E/K256E with T. KS-1 PFDs (PFDα1–β1 and PFDα2–β2) by surface plasmon resonance (SPR) using the Biacore J system (Biacore AB, Uppsala, Sweden) at room temperature (Fig. 2). As previously reported, the affinity of PFDα1–β1 for CPNα was weaker than that for CPNβ. The affinity for the double mutant CPNαK250E/K256E was significantly increased and was larger than that for CPNβ. Since PFDα1–β1 is similar to PhPFD, PFDα1–β1 should have conserved CPN interaction sites as PhPFD. However, no change in the interaction between PFDα2–β2 and CPNα was observed
Fig. 3. Interaction models between T. KS-1 PFD and T. KS-1 CPN subunits. The prediction of the model of interactions between the T. KS-1 CPNαK250E/K256E subunit and the PFDβ1 and PFDβ2 subunits, two lysine amino acids at the 250th and 256th positions in the apical domain of CPNα, was substituted by glutamic acids and shown as stick model (orange). Basic residues in PFD [lysine 101 and arginine 112 in PFDβ1 (gray) and lysine 93 in PFDβ2 (green)] are shown in stick model: overall (a) and in detail (b).
632
Prefoldin–Group II Chaperonin Interaction
Fig. 4. Temperature dependence of the SPR profiles of the interaction between T. KS-1 PFDs and CPNs. SPR sensorgrams of the interaction between T. KS-1 CPNs and T. KS-1 PFDs at 20, 30, 25, 35, 40, and 45 °C were obtained by the Biacore T100 system. (a) CPNα and PFDα1–β1; (b) CPNα and PFDα2–β2; (c) CPNβ and PFDα1–β1; (d) CPNβ and PFDα2–β2. The concentrations of analytes (PFDs) were 400 nM (a and b) and 200 nM (c and d).
with the double mutation. A comparison of sequence and structure between the PFDβ1 subunit and the PFDβ2 subunit showed that the tips of the coiled-coil domain are missing in the PFDβ2 subunit (Fig. 2c). A conserved arginine residue exists in the PhPFDβ and PFDβ1 subunits. The arginine residue R112 is likely to be the interaction site for E250 or E256. Considering the location of E250 and E256 in the helical protrusion, E250 of CPNβ is thought to interact electrostatically with R112 of the PhPFDβ and PFDβ1 subunits. The interaction site for E256 is assumed to be K101 within the PhPFDβ and PFDβ1 subunits. The point mutations of these amino acids in the PFDβ1 subunit resulted in a weak interaction between PFDα1–β1 and CPNβ (unpublished data). Although basic residues (K93 and R95) exist in the vicinity corresponding to the same region in PFDβ2, they do not appear to interact with E256. Based on the crystal structures of the CPNα, PFDβ1, and PFDβ2 subunits, we constructed interaction models between CPNαK250E/K256E and PFDβ1 or PFDβ2 subunits (Fig. 3). The side chains of K101 and R112 in PFDβ1 are oriented to interact with those of E256 and E250 in CPNαK250E/K256E, respectively. On the contrary, it seems that K93 does not interact with E256 in the CPN, since the position of K93 in PFDβ2 is near but not fit with K101 in PFDβ1.
Kinetic and thermodynamic features of the interaction between PFD and CPN We analyzed the interaction between T. KS-1 CPNs and PFDs (CPNα and PFDα1–β1; CPNα and PFDα2–β2; CPNβ and PFDα1–β1; and CPNβ and
Fig. 5. Association constants between CPNs and PFDs at various temperatures. Association constants (Ka) between CPNs (CPNα and CPNβ) and PFDs (PFDα1–β1 and PFDα2–β2) at 20, 25, 30, 35, 40, and 45 °C were calculated from the interaction data obtained by the Biacore T100 system (see Materials and Methods).
633
Prefoldin–Group II Chaperonin Interaction Table 1. Thermodynamic parameters of the interaction between CPNs and PFDs CPN CPNα CPNα CPNβ CPNβ
PFD
ΔSo (J/K)
ΔHo (kJ)
ΔG (kJ) at 25 °C
ΔG (kJ) at 45 °C
PFDα1–β1 PFDα2–β2 PFDα1–β1 PFDα2–β2
219 183 290 448
29.8 19.7 49.7 98.6
− 35.4 − 34.9 − 36.7 − 34.9
− 39.8 − 38.5 − 42.5 − 43.8
PFDα2–β2) using the Biacore T100 system (Biacore AB) at various temperatures ranging from 20 to 45 ºC. The sensorgrams of the interaction of PFDs with 400 nM CPNα and 200 nM CPNβ are shown in Fig. 4. Both PFDs showed weaker interactions with CPNα than with CPNβ. The affinities between CPNs and PFDs increased with temperature. In particular, the interaction between CPNβ and PFDα2–β2 was increased significantly at elevated temperatures (Fig. 4d). The interaction between T. KS-1 PFDs and CPNs was analyzed using the Biacore T100 evaluation software, version 2.0. The association constants (Ka) between CPNα and PFDα1–β1, between CPNα and PFDα2–β2, between CPNβ and PFDα1–β1, and between CPNβ and PFDα2–β2 at 20, 25, 30, 35, 40, and 45 ºC were calculated (Fig. 5). As observed previously, PFDα2–β2 showed a weak interaction with both CPNα and CPNβ at temperatures lower than 30 °C. However, the affinity between PFDα2–β2 and CPNβ increased significantly at temperatures higher than 30 °C. At relatively high temperatures, CPNβ showed higher interaction affinities with both types of PFDs compared to CPNα, and the interaction affinities increased with temperature. To study the thermodynamic properties of the interactions between CPNs and PFDs, we fitted association constants to the van't Hoff equation: ln (Ka) = ΔSo/R − ΔHo/RT. The integrated form of the van't Hoff equation used as ln(Ka) exhibited a linear correlation with 1/T. The calculated ΔHo and ΔSo values are shown in Table 1. The results clearly show that the interaction between CPNs and PFDs is entropy driven. In contrast, the enthalpy changes (ΔHo) during the interaction between PFDs and CPNs are positive. Among the four combinations of CPNs and PFDs, ΔHo for the interaction between CPNβ and PFDα2–β2 was the largest. Moreover, ΔSo was also the largest; thus, Ka increased significantly with an increase in temperature. We initially thought that the difference between CPNα and CPNβ in the interaction with PFDα1–β1 is caused by the difference in ΔHo, as electrostatic interactions are thought to be responsible for binding. However, during the interaction between PFDα1–β1 and CPNβ, ΔHo was larger than that for CPNα. The relatively high affinity between PFDα1–β1 and CPNβ is also entropy driven, explaining the larger affinity difference at high temperatures.
Discussion T. KS-1 cells express two different subunits of CPN, α and β, that constitute functional homo-
oligomers (CPNα and CPNβ). Among them, the expression of the β subunit is induced at heat shock temperatures. T. KS-1 also expresses two pairs of PFD subunits genes: pfdα1 and pfdα2, and pfdβ1 and pfdβ 2 . 13 All four complexes (PFDα1–β1, PFDα1–β2, PFDα2–β1, and PFDα2–β2) take on similar heterohexameric structures and exhibit chaperone activity to suppress thermal aggregation. Among them, PFDα1–β1 and PFDα2–β2 are thought to exist and function mainly in T. KS-1 cells. At moderate temperatures, CPNα and PFDα1–β1 are expressed. As the expression of CPNβ and PFDα2–β2 is induced at elevated temperatures, they are thought to cooperate in folding thermally denatured proteins. However, it remained unclear whether PFDα2–β2 really cooperates with CPNβ, as the affinity between them was estimated to be marginal. The tip of the coiled-coil domain of the PhPFDβ subunit was proposed to be the interaction site for CPN from our previous study.14 However, PFDβ2 does not contain the corresponding region. In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit. As predicted from its amino acid sequence, the PFDβ2 subunit exhibits a structure identical to those of the PFDβ1 and PhPFDβ subunits, with the exception of the absence of the tip of the coiled coil.7,21 Although PFDα1–β1 showed elevated affinities with CPNα containing double mutations at the 250th and 256th lysine residues, the affinity of PFDα2–β2 for the double mutant was almost the same as that for wild-type CPNα. Thus, the difference in interaction affinity is likely caused by the loss of the tip of the coiled coil. We then examined the interaction between two PFDs with two CPNs at various temperatures by SPR using the Biacore T100 system. Interestingly, the affinities between PFDs with CPNs increased with an increase in temperature. The thermodynamic parameters calculated from the association constants showed that the interaction of PFDs with CPN is entropy driven. Among the four combinations of PFD–CPN interactions, the entropy difference in binding between CPNβ and PFDα2–β2 was the largest, and the affinity significantly increased at higher temperatures. Although the analysis was performed up to 45 °C, the data reinforced the idea that CPNβ functions and cooperates with PFDs at higher temperatures, as compared to CPNα.16,17 Considering that the expression of PFDα2–β2 and CPNβ subunits is induced under heat shock conditions, our results support our previous hypothesis that PFDα1–β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2–β2 is specific for CPNβ. Our previous hypothesis that CPN–PFD is driven by
634 electrostatic interaction is based on the difference in the affinities of PhPFD–CPNβ and PhPFD–CPNα at room temperature. However, even though the affinity is weak, CPNα exhibited interaction with PFDs at room temperature. The fact suggested the existence of another interaction site between CPN and PFD. This study clearly shows the existence of another interaction that is probably driven by hydrophobic interaction. This interaction is significant at elevated temperatures compared with electrostatic interaction. The tips of the coiled coils of the PhPFDβ and PFDβ1 subunits are relatively rich in hydrophobic amino acid residues. It was previously speculated that they are responsible for the interaction with denatured proteins. It is plausible that the interaction is partly driven by hydrophobic interactions between the tip of coiled coils and helical protrusions. However, PFDβ2 does not contain the corresponding hydrophobic region, suggesting the existence of other regions that contribute to hydrophobic interactions between CPNs and PFDs. The possibility of the contribution of CPN helical protrusions is also excluded, since the CPNα and CPNβ subunits are almost identical in the helical protrusion, with the exception of two charged residues, E250 and E256, in the CPNβ subunit irrespective of entropy changes. The largest change between the CPNα subunit and the CPNβ subunit exists in the C-terminus. Previously, we obtained data suggesting the contribution of the C-terminal region of CPN to the interaction with PFD. Interestingly, the PFDβ subunit, especially the PFDβ2 subunit, is too short to interact with the CPN C-terminal region. Thus, the only possible interaction site in PFD is the tip of the coiled coils of the α subunit. The interaction between the long coiled coils of the PFDα subunit and the CPN Cterminal region might trigger a conformational change in CPN to facilitate the interaction between CPN and PFD. Thermodynamic characterization from the interaction data obtained by the Biacore T100 system clearly revealed the nature of the interaction between PFDs and CPNs. As the contribution of hydrophobic interactions is not easily observed using point mutants, thermodynamics characterization of the interaction will be very useful to the study of the interactions driven by entropy changes. Although this study deals with the interaction between archaeal PFDs and CPNs, our data should be valid for the eukaryotic PFD–CPN system.
Materials and Methods Protein expression and purification Recombinant α and β subunits of T. KS-1 CPN were overexpressed in Escherichia coli strain BL21(DE3) with the expression vectors pET9a and pET23b, respectively. Expressed CPNα and CPNβ were purified as described previously. 1 8 The plasmids pTKS1PFDα1–β1, pTKS1PFDα2–β2, and pTKS1PFDβ2 were used for the
Prefoldin–Group II Chaperonin Interaction expression of PFDα1–β1, PFDα2–β2, and PFDβ2, respectively. Recombinant PFDα1–β1, PFDα2–β2, and PFDβ2 were purified as described previously.13 Crystallization and X-ray data collection Crystals for the T. KS-1 PFDβ2 subunit were grown by vapor diffusion using a protein solution (20 mg/ml) and a reservoir solution of 500 mM ammonium sulfate, 500 mM ammonium chloride, and 100 mM sodium citrate (pH 4.5) at 20 °C. The diffraction data were collected at 95 K using an ADSC CCD detector system at beamline NW12 in the Photon Factory Advanced Ring and at beamline BL26B1 in SPring-8. Diffraction data were processed using the program HKL2000.22 Effective derivative crystals were prepared by soaking the wild-type crystals in the reservoir solution containing 1 mM OsCl2 for 24 h. The data collected and the scaling results are listed in Table 2.
Table 2. Data collection and refinement statistics T. KS-1 PFDβ2 Data collection Temperature (K) Resolution (Å) Number of measured reflections Number of unique reflections Rmergea Io/σ(Io) Completeness (%) Space group Cell dimensions a (Å) b (Å) c (Å) Derivative Concentration (mM) Soaking time (h) Phasing Resolution range (Å) Phasing powerc Mean figure of meritd (centric/acentric) Number of sites Structure refinement Resolution range (Å) Number of reflections Re Rfreef r.m.s.d. bond lengths (Å) r.m.s.d. bond angles (°) Number of protein atoms Number of solvent atoms Number of ligand atoms
Os derivative
Wild type
100 2.10 339,121 23,355 0.061 (0.236)b 15.9 99.8 (100)b P4212
100 1.83 307,371 24,056 0.051 (0.313)b 17.7 99.7 (100)b P4212
66.96 66.96 91.71
67.32 67.32 92.41
2 24 45.83–2.10 2.37 0.106/0.418 4 50.0–1.70 24,056 0.210 0.243 0.006 1.3 1596 160 18
a Rmerge = ∑hkl∑i|Ii(hkl) − 〈I(hkl)〉|/∑hkl∑Ii(hkl), where Ii(hkl) is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl)〉 is its average. b The values for the highest-resolution shell are given in parentheses (2.10–2.15 Å resolution for Os derivative T. KS-1 PFDβ2; 1.70–1.76 Å resolution for wild-type T. KS-1 PFDβ2). c Phasing power = 〈F(H)〉/E, where 〈F(H)〉 is the r.m.s. amplitude of the heavy-atom structure factor, and E is the lack of closure error. d Mean figure of merit = 〈∑P(α)exp(iα)/∑P(α)〉, where P(α) is the phase probability at angle α. e R = ∑hkl(|Fo| − |Fc|)/∑hkl|Fo|. f Rfree was calculated on 5% of the data omitted randomly.
635
Prefoldin–Group II Chaperonin Interaction Structure determination and refinements
Accession number
Initial single-wavelength anomalous diffraction phasing at 2.1 Å for the Os-derived T. KS-1 PFDβ2 subunit was performed by locating two Os sites in the peak data set using the program SOLVE.23 After electron density modification, 70% of amino acid residues could be located in the resultant electron density map using the program RESOLVE.24 Further automated model building was performed using ARP/wARP.25 Model building was continued manually using the program Xfit in the XtalView program system.26 The structure was refined using the program CNS.27 After several cycles of refinement and model building, 94 of 99 sequenced residues with an R-factor of 0.285 were obtained. Using the structure of the Os-derived PFDβ2 subunit, we determined the structure of the wild-type PFDβ2 subunit by molecular replacement and refined it at a resolution of 1.7 Å (Table 2).
Coordinates and structure factors have been deposited in the Protein Data Bank28 with accession number 3AEI.
Surface plasmon resonance SPR experiments were performed using Biacore J at room temperature and the Biacore T100 system at various sensor temperatures (20, 25, 30, 35, 40, and 45 °C). Analysis at temperatures higher than 45 °C could not be performed due to the temperature limit of this instrument. T. KS-1 PFDs was coupled to the sensor chip (CM5 research grade) via standard N-hydroxysuccinimide and N-ethyl-N-(dimethylaminopropyl) carbodiimide activation. To immobilize T. KS-1 PFD, we injected T. KS-1 PFD which dissolved in 10 mM sodium phosphate buffer (pH 5.0), on the sensor surface, with HBS EP buffer [10 mM Hepes (pH 7.4), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid, and 0.05% surfactant P20] employed as mobile phase buffer during the immobilization process. Tris–HCl buffer (50 mM, pH 7.5) was then injected to quench the unreacted N-hydroxysuccinimide groups. TKM buffer [50 mM Tris– HCl (pH 7.5), 100 mM KCl, and 25 mM MgCl2] was used as mobile phase buffer. T. KS-1 CPN samples at various concentrations were injected as analytes, and bound analytes were subsequently removed by washing with the mobile phase buffer at 600 s after the injection. Regeneration buffer [50 mM Tris–HCl (pH 9.0)] was injected prior to each analyte injection. Kinetic constants were calculated from the sensorgrams using the Biacore T100 evaluation software, version 2.0 (Biacore AB). Association constants (Ka) were calculated by the resonance unit at equilibrium using the following equation: Req ¼Rmax C=ðCþ1=Ka Þ where Req represents equilibrium resonance units, Rmax is the resonance signal at saturation, and C is the concentration of free analyte. The thermodynamic parameters were calculated by the van't Hoff equation: DGo ¼ − RTlnKa ¼DH o − TDSo or lnKa ¼DSo = R − DHo = RT Therefore, a plot of the natural logarithm of the association constant versus the reciprocal temperature gives a straight line. The slope of the line is equal to minus the standard enthalpy change divided by the gas constant −ΔHo/R, and the intercept is equal to the standard entropy change divided by the gas constant ΔSo/R.
Acknowledgements The work reported here is part of the support program for improving the graduate school education of the “Human Resource Development Program for Scientific Powerhouse,” which is financially supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan through the Tokyo University of Agriculture and Technology. This study was also supported by grants-in-aids for scientific research (19370038, 20059013, and 21370067) and by a grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports, and Culture of Japan (to M.Y.).
References 1. Vainberg, I. E., Lewis, S. A., Rommelaere, H., Ampe, C., Vandekerckhove, J., Klein, H. L. & Cowan, N. J. (1998). Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin. Cell, 93, 863–873. 2. Hartl, F. U. & Hayer-Hartl, M. (2002). Molecular chaperones in the cytosol: from nascent chain to folded protein. Science, 295, 1852–1858. 3. Siegert, R., Leroux, M. R., Scheufler, C., Hartl, F. U. & Moarefi, I. (2000). Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins. Cell, 103, 621–632. 4. Hansen, W. J., Cowan, N. J. & Welch, W. J. (1999). Prefoldin–nascent chain complexes in the folding of cytoskeletal proteins. J Cell Biol. 145, 265–277. 5. Leroux, M. R., Fandrich, M., Klunker, D., Siegers, K., Lupas, A. N., Brown, J. R. et al. (1999). MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin. EMBO J. 18, 6730–6743. 6. Macario, A. J. & Conway De Macario, E. (2001). The molecular chaperone system and other anti-stress mechanisms in archaea. Front. Biosci. 6, D262–D283. 7. Ohtaki, A., Kida, H., Miyata, Y., Ide, N., Yonezawa, A., Arakawa, T. et al. (2008). Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins. J. Mol. Biol. 376, 1130–1141. 8. Martin-Benito, J., Gomez-Reino, J., Stirling, P. C., Lundin, V. F., Gomez-Puertas, P., Boskovic, J. et al. (2007). Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart. Structure, 15, 101–110. 9. Okochi, M., Yoshida, T., Maruyama, T., Kawarabayasi, Y., Kikuchi, H. & Yohda, M. (2002). Pyrococcus prefoldin stabilizes protein-folding intermediates and transfers them to chaperonins for correct folding. Biochem. Biophys. Res. Commun. 291, 769–774.
636 10. Zako, T., Iizuka, R., Okochi, M., Nomura, T., Ueno, T., Tadakuma, H. et al. (2005). Facilitated release of substrate protein from prefoldin by chaperonin. FEBS Lett. 579, 3718–3724. 11. Zako, T., Murase, Y., Iizuka, R., Yoshida, T., Kanzaki, T., Ide, N. et al. (2006). Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate. J. Mol. Biol. 364, 110–120. 12. Martin-Benito, J., Boskovic, J., Gomez-Puertas, P., Carrascosa, J. L., Simons, C. T., Lewis, S. A. et al. (2002). Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT. EMBO J. 21, 6377–6386. 13. Iizuka, R., Sugano, Y., Ide, N., Ohtaki, A., Yoshida, T., Fujiwara, S. et al. (2008). Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1. J. Mol. Biol. 377, 972–983. 14. Okochi, M., Nomura, T., Zako, T., Arakawa, T., Iizuka, R., Ueda, H. et al. (2004). Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin. J. Biol. Chem. 279, 31788–31795. 15. Simons, C. T., Staes, A., Rommelaere, H., Ampe, C., Lewis, S. A. & Cowan, N. J. (2004). Selective contribution of eukaryotic prefoldin subunits to actin and tubulin binding. J. Biol. Chem. 279, 4196–4203. 16. Yoshida, T., Ideno, A., Hiyamuta, S., Yohda, M. & Maruyama, T. (2001). Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric chaperonin with variable subunit composition. Mol. Microbiol. 39, 1406–1413. 17. Yoshida, T., Ideno, A., Suzuki, R., Yohda, M. & Maruyama, T. (2002). Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS-1. Mol. Microbiol. 44, 761–769. 18. Yoshida, T., Kawaguchi, R., Taguchi, H., Yoshida, M., Yasunaga, T., Wakabayashi, T. et al. (2002). Archaeal
Prefoldin–Group II Chaperonin Interaction
19.
20.
21.
22. 23. 24. 25.
26. 27.
28.
group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin. J. Mol. Biol. 315, 73–85. Yoshida, T., Kanzaki, T., Iizuka, R., Komada, T., Zako, T., Suzuki, R. et al. (2006). Contribution of the Cterminal region to the thermostability of the archaeal group II chaperonin from Thermococcus sp. strain KS1. Extremophiles, 10, 451–459. Danno, A., Fukuda, W., Yoshida, M., Aki, R., Tanaka, T., Kanai, T. et al. (2008). Expression profiles and physiological roles of two types of prefoldins from the hyperthermophilic archaeon Thermococcus kodakaraensis. J. Mol. Biol. 382, 298–311. Kida, H., Sugano, Y., Iizuka, R., Fujihashi, M., Yohda, M. & Miki, K. (2008). Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1. J. Mol. Biol. 383, 465–474. Otwinowski, Z. & Minor, W. (1997). Processing of Xray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Terwilliger, T. C. & Berendzen, J. (1999). Automated MAD and MIR structure solution. Acta Crystallogr. Sect. D, 55, 849–861. Terwilliger, T. C. (2002). Automated structure solution, density modification and model building. Acta Crystallogr. Sect. D, 58, 1937–1940. Perrakis, A., Morris, R. & Lamzin, V. S. (1999). Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463. McRee, D. E. (1999). XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. Sect. D, 54, 905–921. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H. et al. (2000). The Protein Data Bank. Nucleic Acids Res. 28, 235–242.
J. Mol. Biol. (2010) 399, 628–636
Available online at www.sciencedirect.com
Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin Muhamad Sahlan 1 , Tamotsu Zako 2 , Phan The Tai 1 , Akashi Ohtaki 1 , Keiichi Noguchi 1 , Mizuo Maeda 2 , Hideyuki Miyatake 3 , Naoshi Dohmae 3 and Masafumi Yohda 1 ⁎ 1
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan 2
Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
3
Biomolecular Characterization Team, Advanced Development and Supporting Center, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Received 22 February 2010; received in revised form 20 April 2010; accepted 23 April 2010 Available online 29 April 2010
Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit and characterized the interactions between T. KS-1 CPNs (CPNα and CPNβ) and T. KS-1 PFDs (PFDα1–β1 and PFDα2–β2). As predicted from its amino acid sequence, the PFDβ2 subunit conforms to a structure similar to those of the PFDβ1 subunit and the Pyrococcus horikoshii OT3 PFDβ subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNα and PFDα1–β1; CPNα and PFDα2–β2; CPNβ and PFDα1–β1; and CPNβ and PFDα2–β2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 ºC. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD–CPN interactions, the entropy difference in binding between CPNβ and PFDα2–β2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDα2–β2 and CPNβ subunit is induced upon heat shock, our results suggest that PFDα1–β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2–β2 is specific for CPNβ. © 2010 Elsevier Ltd. All rights reserved.
Edited by K. Morikawa
Keywords: prefoldin; group II chaperonin; crystal structure; surface plasmon resonance; thermodynamics
Introduction Prefoldin (PFD) is a jellyfish-shaped heterohexameric cochaperone that captures an unfolded protein substrate and transfers it to group II chaperonin (CPN) to complete protein folding.1–3 It has been shown that PFD participates in the maturation of actin and tubulin members in collaboration with the eukaryotic CPN, CCT (cytosolic chaperonin containing TCP-1).1,4,5 Although archaea do not possess *Corresponding author. E-mail address: [email protected]. Abbreviations used: PFD, prefoldin; CPN, group II chaperonin; T. KS-1, Thermococcus strain KS-1; PhPFD, Pyrococcus horikoshii OT3 PFD; GFP, green fluorescence protein; SPR, surface plasmon resonance.
actin or tubulin, both PFD and CPN have invariably been identified in all archaeal species.1,6 The PFD– CPN system functions as a general molecular chaperone in archaea because only limited members of chaperones exist.6 The archaeal PFD is composed of two α subunits and four β subunits (α2β4). The structures of Methanobacterium thermoautotrophicum PFD and Pyrococcus horikoshii OT3 PFD (PhPFD) have been characterized.3,7 Archaeal PFD consists of a double β-barrel assembly with six coiled-coil domains that protrude from the center like a jellyfish with six tentacles. The eukaryotic PFD is composed of six different subunits (two α-type subunits and four β-type subunits), and it is thought to have a structure similar to those of archaeal PFDs.8 Biochemical studies have shown that PFDs bind and stabilize unfolded target polypeptides and subsequently deliver them to CPNs to complete
0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.
Prefoldin–Group II Chaperonin Interaction
folding.9–11 The transfer of a substrate from PFD to CPN involves a direct interaction.11,12 We have studied the mechanism of protein folding mediated by the archaeal PFD–CPN system.9,10,13 PhPFD and PFDs of Thermococcus strain KS-1 (T. KS-1) can capture an acid-denatured green fluorescence protein (GFP) and transfer it to T. KS-1 CPN for ATPdependent folding. The release of a substrate protein from PhPFD is facilitated by T. KS-1 CPN.10 We also identified the regions in PFD and CPN that are responsible for their interaction.11,14 The tips of the coiled coils of PFD and the helical protrusion region in the apical domain of CPN are thought to interact with substrate proteins and CPNs.3,5,7,8,11,12,14,15 T. KS-1 expresses two different CPN subunits (α and β).16,17 Recombinant α and β subunits assemble to form a double-ring homo-oligomer: CPNα and CPNβ. Both of them have high ATP-dependent protein folding activity toward denatured GFP and citrate synthase.18 The expression of α subunit and β subunit is regulated differently, and only the β subunit is heat inducible.16 The subunit composition of natural T. KS-1 CPN is variable, with an increase in the proportion of β subunits at higher temperatures. Indeed, the thermostability of the CPN
629 complex is correlated with the number of β subunits in the complex. Thermostability is thought to arise from the differences in 20 amino acids at the Cterminal end.19 CPNβ interacts with PFD more strongly than CPNα.11 Comparing the interaction of CPNα, CPNβ, and their mutants with PhPFD, we have previously shown that two acidic amino acids in the helical protrusion of the β subunit (E250 and E256) are responsible for the strong interaction with PhPFD.11 Thus, the interaction between PFD and CPN is partly driven by electrostatic interactions. T. KS-1 expresses two pairs of PFD subunits genes: two α-subunit genes pfdα1 and pfdα2, and two βsubunit genes pfdβ1 and pfdβ2.13 We have functionally characterized four recombinant T. KS-1 PFD complexes (PFDα1–β1, PFDα1–β2, PFDα2–β1, and PFDα2–β2).13 All four complexes make similar heterohexameric structures and exhibit chaperone activity to suppress thermal aggregation. Moreover, the transfer of a denatured GFP to CPNα for folding was demonstrated for PFDα1–β1. The affinities of PFDα1–β1 and PFDα2–β1 for CPNα were almost identical, whereas CPNβ exhibited a relatively strong affinity for PFD complexes. PFD complexes with β2 subunit exhibited only marginal interactions
Fig. 1. The crystal structure of the PFDβ2 subunit. (a) The monomer structure of the PFDβ2 subunit. Hydrophobic residues (Ala6, Leu9, Leu13, Ile87, Leu91, and Leu94) at the distal region are presented as stick models. (b) Superposition of Cα atoms of the coiled-coil regions (residues 26–33 and 59–67; PFDβ2 numbering) of PFDβ1 and PFDβ2 subunits. Hydrophobic residues (PFDβ2), basic residues (K93 in PFDβ2), and basic residues (K101 and R112 in PFDβ1) are shown as stick models. (c) Bottom view and (d) side view of the jellyfish-like structure of the PFDβ2 subunit tetramer. In the β2 tetramer complex structure, the β2 subunit is shown in green, and symmetric molecules are shown in gray. (e) The dimer of the PFDβ2 subunit tetramer. Each tetramer is shown in green and gray. These figures were created with the program MOLSCRIPT.
630 with CPNβ. Therefore, we hypothesized that PFD complexes containing the β1 subunit cooperate with both CPNα and CPNβ, whereas PFD complexes with the β2 subunit cooperate only with CPNβ. This hypothesis coincides well with the fact that the CPNβ and PFDβ2 subunits are expressed at elevated temperatures. A previous study on the cognate Thermococcus species Thermococcus kodakaraensis showed that PFDα1–β1 and PFDα2–β2 complexes are dominantly expressed in cells.20 Transcriptional and translational analyses showed that the β1 subunit of T. kodakaraensis is constitutively expressed under normal and heat-stressed conditions, and that β2 expression is highly induced at elevated temperatures. We reported the crystal structure of T. KS-1 PFDβ1 subunit at 1.7 Å resolution and found that T. KS-1 PFDβ1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFDs. The β-hairpin linkers of β1 subunits assemble to form a β-barrel “body” around a central 4-fold axis. As our previous results showed that the difference in interactions between T. KS-1 PFDs and CPNs is caused by differences in the PFDβ subunit, we attempted to determine the crystal structure of the PFDβ2 subunit. In this study, we investigate the crystal structure of the T. KS-1 PFDβ2 subunit and examine the thermodynamic properties of the interaction between CPNs and PFDs. The results clearly demon-
Prefoldin–Group II Chaperonin Interaction
strated that the interaction between CPNs and PFDs is driven by entropy changes and that the contribution of electrostatic interactions is marginal.
Results Crystal structure of the T. KS-1 PFDβ2 subunit To elucidate the difference in affinity between T. KS-1 PFDs and CPNs caused by the difference in PFDβ subunit, we determined the crystal structure of the PFDβ2 subunit. The crystal of the β2 subunit with space group P4212 has two molecules in the asymmetric unit. Both structures have 94 (residues 1–94) of the 99 sequenced residues. The monomer structure of PFDβ2 is divided into three regions: an N-terminal helix (residues 1–38), a β-hairpin linker (residues 46–58), and a C-terminal helix (residues 59–94), as shown in Fig. 1a. The N-terminal and Cterminal helices form an anti-parallel helical coiledcoil assembly. The β-hairpin linker contributes to the formation of the tetramer, as mentioned below. At the distal region of the coiled-coil structure, the hydrophobic groove, a putative substrate-binding site, is formed by Ala6, Leu9, Leu13, Ile87, Leu91, and Leu94. The structure fits well with the structure of the PFDβ1 subunit (Fig. 1b).
Fig. 2. Comparison of the interaction between T. KS-1 PFDs and CPNs, and the effect of two amino acid substitutions (K250E and K256E) on the CPNα subunit. SPR sensorgrams of the interactions of PFDα1–β1 with CPNα, CPNβ, and CPNαK250E/K256E (a), and SPR sensorgrams of the interactions of PFDα2–β2 with CPNα, CPNβ, and CPNαK250E/ K256E (b). The concentrations of analytes (CPNs) were fixed at 50 nM (a) and 100 nM (b). (c) Sequence alignment of PFDβ subunits. Identical, highly similar, and poorly similar amino acids are shown as (⁎), (:), and (.), respectively. K101 and R112 of PhPFDβ subunit and T. KS-1 PFDβ1 subunit are marked by (#). Acidic residues and basic residues are shown in red and blue, respectively. PH_b, PhPFDβ subunit; KS_b1, T. KS-1 PFDβ1 subunit; KS_b2, T. KS-1 PFDβ2 subunit.
Prefoldin–Group II Chaperonin Interaction
The two molecules in the asymmetric unit are of the same structure, with an r.m.s.d. of 1.52 Å. Both molecules form a tetramer related by the crystallographic 4-fold axis, as shown in Fig. 1c and d. The tetramer forms a jellyfish-like structure (Fig. 1c and d), which is similar to the structures of the PFDβ1 tetramer and the hexameric PhPFD and M. thermoautotrophicum PFD. Four tentacles emanate from the body of the jellyfish, forming a cavity at their center. In the crystalline state, two PFDβ2 tetramers face each other, resulting in a dimer of tetramers (Fig. 1e). One tentacle of the tetramer interacts with only one tentacle of the other tetramer through hydrogen bonds. The hydrophobic groove of the tentacles does not interact with any other PFDβ2 subunit. Each tentacle fits tightly between the tentacles of the other tetramer, forming a highly stable structure. These interactions between tentacles seem to open the cavity. This crystal structure of the PFDβ2 tetramer showed that each coiled-coil domain of the PFDβ2 tetramer is more highly flexible and changes various conformations when in solution. Although we tried to crystallize PFDα2–β2, we could not obtain crystals. The crystals obtained from the crystallization of PFDα2–β2 yielded only crystals of PFDβ2, similar to the case of PFDα2–β1.21 Thus, the complexes likely dissociated in the crystallization solution.
631 PFDα2–β2 lacks the interaction site for the two acidic residues on the helical protrusions of the CPNβ subunit We showed that the affinity of CPNβ for PhPFD is stronger than that of CPNα.11 The difference is attributed to two amino acid residues at the helical protrusion. The 250th and 256th amino acid residues of CPNβ and Pyrococcus horikoshii CPN are acidic glutamates, but those of CPNα are basic residues. These residues are located on the inner surface of the helical protrusion. Single and double mutants of these residues in the CPNα subunit, namely CPNαK250E, CPNαK256E, and CPNαK250E/ K256E, exhibited a stronger interaction with PhPFD than CPNα. Next, we compared the interactions of CPNαK250E/K256E with T. KS-1 PFDs (PFDα1–β1 and PFDα2–β2) by surface plasmon resonance (SPR) using the Biacore J system (Biacore AB, Uppsala, Sweden) at room temperature (Fig. 2). As previously reported, the affinity of PFDα1–β1 for CPNα was weaker than that for CPNβ. The affinity for the double mutant CPNαK250E/K256E was significantly increased and was larger than that for CPNβ. Since PFDα1–β1 is similar to PhPFD, PFDα1–β1 should have conserved CPN interaction sites as PhPFD. However, no change in the interaction between PFDα2–β2 and CPNα was observed
Fig. 3. Interaction models between T. KS-1 PFD and T. KS-1 CPN subunits. The prediction of the model of interactions between the T. KS-1 CPNαK250E/K256E subunit and the PFDβ1 and PFDβ2 subunits, two lysine amino acids at the 250th and 256th positions in the apical domain of CPNα, was substituted by glutamic acids and shown as stick model (orange). Basic residues in PFD [lysine 101 and arginine 112 in PFDβ1 (gray) and lysine 93 in PFDβ2 (green)] are shown in stick model: overall (a) and in detail (b).
632
Prefoldin–Group II Chaperonin Interaction
Fig. 4. Temperature dependence of the SPR profiles of the interaction between T. KS-1 PFDs and CPNs. SPR sensorgrams of the interaction between T. KS-1 CPNs and T. KS-1 PFDs at 20, 30, 25, 35, 40, and 45 °C were obtained by the Biacore T100 system. (a) CPNα and PFDα1–β1; (b) CPNα and PFDα2–β2; (c) CPNβ and PFDα1–β1; (d) CPNβ and PFDα2–β2. The concentrations of analytes (PFDs) were 400 nM (a and b) and 200 nM (c and d).
with the double mutation. A comparison of sequence and structure between the PFDβ1 subunit and the PFDβ2 subunit showed that the tips of the coiled-coil domain are missing in the PFDβ2 subunit (Fig. 2c). A conserved arginine residue exists in the PhPFDβ and PFDβ1 subunits. The arginine residue R112 is likely to be the interaction site for E250 or E256. Considering the location of E250 and E256 in the helical protrusion, E250 of CPNβ is thought to interact electrostatically with R112 of the PhPFDβ and PFDβ1 subunits. The interaction site for E256 is assumed to be K101 within the PhPFDβ and PFDβ1 subunits. The point mutations of these amino acids in the PFDβ1 subunit resulted in a weak interaction between PFDα1–β1 and CPNβ (unpublished data). Although basic residues (K93 and R95) exist in the vicinity corresponding to the same region in PFDβ2, they do not appear to interact with E256. Based on the crystal structures of the CPNα, PFDβ1, and PFDβ2 subunits, we constructed interaction models between CPNαK250E/K256E and PFDβ1 or PFDβ2 subunits (Fig. 3). The side chains of K101 and R112 in PFDβ1 are oriented to interact with those of E256 and E250 in CPNαK250E/K256E, respectively. On the contrary, it seems that K93 does not interact with E256 in the CPN, since the position of K93 in PFDβ2 is near but not fit with K101 in PFDβ1.
Kinetic and thermodynamic features of the interaction between PFD and CPN We analyzed the interaction between T. KS-1 CPNs and PFDs (CPNα and PFDα1–β1; CPNα and PFDα2–β2; CPNβ and PFDα1–β1; and CPNβ and
Fig. 5. Association constants between CPNs and PFDs at various temperatures. Association constants (Ka) between CPNs (CPNα and CPNβ) and PFDs (PFDα1–β1 and PFDα2–β2) at 20, 25, 30, 35, 40, and 45 °C were calculated from the interaction data obtained by the Biacore T100 system (see Materials and Methods).
633
Prefoldin–Group II Chaperonin Interaction Table 1. Thermodynamic parameters of the interaction between CPNs and PFDs CPN CPNα CPNα CPNβ CPNβ
PFD
ΔSo (J/K)
ΔHo (kJ)
ΔG (kJ) at 25 °C
ΔG (kJ) at 45 °C
PFDα1–β1 PFDα2–β2 PFDα1–β1 PFDα2–β2
219 183 290 448
29.8 19.7 49.7 98.6
− 35.4 − 34.9 − 36.7 − 34.9
− 39.8 − 38.5 − 42.5 − 43.8
PFDα2–β2) using the Biacore T100 system (Biacore AB) at various temperatures ranging from 20 to 45 ºC. The sensorgrams of the interaction of PFDs with 400 nM CPNα and 200 nM CPNβ are shown in Fig. 4. Both PFDs showed weaker interactions with CPNα than with CPNβ. The affinities between CPNs and PFDs increased with temperature. In particular, the interaction between CPNβ and PFDα2–β2 was increased significantly at elevated temperatures (Fig. 4d). The interaction between T. KS-1 PFDs and CPNs was analyzed using the Biacore T100 evaluation software, version 2.0. The association constants (Ka) between CPNα and PFDα1–β1, between CPNα and PFDα2–β2, between CPNβ and PFDα1–β1, and between CPNβ and PFDα2–β2 at 20, 25, 30, 35, 40, and 45 ºC were calculated (Fig. 5). As observed previously, PFDα2–β2 showed a weak interaction with both CPNα and CPNβ at temperatures lower than 30 °C. However, the affinity between PFDα2–β2 and CPNβ increased significantly at temperatures higher than 30 °C. At relatively high temperatures, CPNβ showed higher interaction affinities with both types of PFDs compared to CPNα, and the interaction affinities increased with temperature. To study the thermodynamic properties of the interactions between CPNs and PFDs, we fitted association constants to the van't Hoff equation: ln (Ka) = ΔSo/R − ΔHo/RT. The integrated form of the van't Hoff equation used as ln(Ka) exhibited a linear correlation with 1/T. The calculated ΔHo and ΔSo values are shown in Table 1. The results clearly show that the interaction between CPNs and PFDs is entropy driven. In contrast, the enthalpy changes (ΔHo) during the interaction between PFDs and CPNs are positive. Among the four combinations of CPNs and PFDs, ΔHo for the interaction between CPNβ and PFDα2–β2 was the largest. Moreover, ΔSo was also the largest; thus, Ka increased significantly with an increase in temperature. We initially thought that the difference between CPNα and CPNβ in the interaction with PFDα1–β1 is caused by the difference in ΔHo, as electrostatic interactions are thought to be responsible for binding. However, during the interaction between PFDα1–β1 and CPNβ, ΔHo was larger than that for CPNα. The relatively high affinity between PFDα1–β1 and CPNβ is also entropy driven, explaining the larger affinity difference at high temperatures.
Discussion T. KS-1 cells express two different subunits of CPN, α and β, that constitute functional homo-
oligomers (CPNα and CPNβ). Among them, the expression of the β subunit is induced at heat shock temperatures. T. KS-1 also expresses two pairs of PFD subunits genes: pfdα1 and pfdα2, and pfdβ1 and pfdβ 2 . 13 All four complexes (PFDα1–β1, PFDα1–β2, PFDα2–β1, and PFDα2–β2) take on similar heterohexameric structures and exhibit chaperone activity to suppress thermal aggregation. Among them, PFDα1–β1 and PFDα2–β2 are thought to exist and function mainly in T. KS-1 cells. At moderate temperatures, CPNα and PFDα1–β1 are expressed. As the expression of CPNβ and PFDα2–β2 is induced at elevated temperatures, they are thought to cooperate in folding thermally denatured proteins. However, it remained unclear whether PFDα2–β2 really cooperates with CPNβ, as the affinity between them was estimated to be marginal. The tip of the coiled-coil domain of the PhPFDβ subunit was proposed to be the interaction site for CPN from our previous study.14 However, PFDβ2 does not contain the corresponding region. In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit. As predicted from its amino acid sequence, the PFDβ2 subunit exhibits a structure identical to those of the PFDβ1 and PhPFDβ subunits, with the exception of the absence of the tip of the coiled coil.7,21 Although PFDα1–β1 showed elevated affinities with CPNα containing double mutations at the 250th and 256th lysine residues, the affinity of PFDα2–β2 for the double mutant was almost the same as that for wild-type CPNα. Thus, the difference in interaction affinity is likely caused by the loss of the tip of the coiled coil. We then examined the interaction between two PFDs with two CPNs at various temperatures by SPR using the Biacore T100 system. Interestingly, the affinities between PFDs with CPNs increased with an increase in temperature. The thermodynamic parameters calculated from the association constants showed that the interaction of PFDs with CPN is entropy driven. Among the four combinations of PFD–CPN interactions, the entropy difference in binding between CPNβ and PFDα2–β2 was the largest, and the affinity significantly increased at higher temperatures. Although the analysis was performed up to 45 °C, the data reinforced the idea that CPNβ functions and cooperates with PFDs at higher temperatures, as compared to CPNα.16,17 Considering that the expression of PFDα2–β2 and CPNβ subunits is induced under heat shock conditions, our results support our previous hypothesis that PFDα1–β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2–β2 is specific for CPNβ. Our previous hypothesis that CPN–PFD is driven by
634 electrostatic interaction is based on the difference in the affinities of PhPFD–CPNβ and PhPFD–CPNα at room temperature. However, even though the affinity is weak, CPNα exhibited interaction with PFDs at room temperature. The fact suggested the existence of another interaction site between CPN and PFD. This study clearly shows the existence of another interaction that is probably driven by hydrophobic interaction. This interaction is significant at elevated temperatures compared with electrostatic interaction. The tips of the coiled coils of the PhPFDβ and PFDβ1 subunits are relatively rich in hydrophobic amino acid residues. It was previously speculated that they are responsible for the interaction with denatured proteins. It is plausible that the interaction is partly driven by hydrophobic interactions between the tip of coiled coils and helical protrusions. However, PFDβ2 does not contain the corresponding hydrophobic region, suggesting the existence of other regions that contribute to hydrophobic interactions between CPNs and PFDs. The possibility of the contribution of CPN helical protrusions is also excluded, since the CPNα and CPNβ subunits are almost identical in the helical protrusion, with the exception of two charged residues, E250 and E256, in the CPNβ subunit irrespective of entropy changes. The largest change between the CPNα subunit and the CPNβ subunit exists in the C-terminus. Previously, we obtained data suggesting the contribution of the C-terminal region of CPN to the interaction with PFD. Interestingly, the PFDβ subunit, especially the PFDβ2 subunit, is too short to interact with the CPN C-terminal region. Thus, the only possible interaction site in PFD is the tip of the coiled coils of the α subunit. The interaction between the long coiled coils of the PFDα subunit and the CPN Cterminal region might trigger a conformational change in CPN to facilitate the interaction between CPN and PFD. Thermodynamic characterization from the interaction data obtained by the Biacore T100 system clearly revealed the nature of the interaction between PFDs and CPNs. As the contribution of hydrophobic interactions is not easily observed using point mutants, thermodynamics characterization of the interaction will be very useful to the study of the interactions driven by entropy changes. Although this study deals with the interaction between archaeal PFDs and CPNs, our data should be valid for the eukaryotic PFD–CPN system.
Materials and Methods Protein expression and purification Recombinant α and β subunits of T. KS-1 CPN were overexpressed in Escherichia coli strain BL21(DE3) with the expression vectors pET9a and pET23b, respectively. Expressed CPNα and CPNβ were purified as described previously. 1 8 The plasmids pTKS1PFDα1–β1, pTKS1PFDα2–β2, and pTKS1PFDβ2 were used for the
Prefoldin–Group II Chaperonin Interaction expression of PFDα1–β1, PFDα2–β2, and PFDβ2, respectively. Recombinant PFDα1–β1, PFDα2–β2, and PFDβ2 were purified as described previously.13 Crystallization and X-ray data collection Crystals for the T. KS-1 PFDβ2 subunit were grown by vapor diffusion using a protein solution (20 mg/ml) and a reservoir solution of 500 mM ammonium sulfate, 500 mM ammonium chloride, and 100 mM sodium citrate (pH 4.5) at 20 °C. The diffraction data were collected at 95 K using an ADSC CCD detector system at beamline NW12 in the Photon Factory Advanced Ring and at beamline BL26B1 in SPring-8. Diffraction data were processed using the program HKL2000.22 Effective derivative crystals were prepared by soaking the wild-type crystals in the reservoir solution containing 1 mM OsCl2 for 24 h. The data collected and the scaling results are listed in Table 2.
Table 2. Data collection and refinement statistics T. KS-1 PFDβ2 Data collection Temperature (K) Resolution (Å) Number of measured reflections Number of unique reflections Rmergea Io/σ(Io) Completeness (%) Space group Cell dimensions a (Å) b (Å) c (Å) Derivative Concentration (mM) Soaking time (h) Phasing Resolution range (Å) Phasing powerc Mean figure of meritd (centric/acentric) Number of sites Structure refinement Resolution range (Å) Number of reflections Re Rfreef r.m.s.d. bond lengths (Å) r.m.s.d. bond angles (°) Number of protein atoms Number of solvent atoms Number of ligand atoms
Os derivative
Wild type
100 2.10 339,121 23,355 0.061 (0.236)b 15.9 99.8 (100)b P4212
100 1.83 307,371 24,056 0.051 (0.313)b 17.7 99.7 (100)b P4212
66.96 66.96 91.71
67.32 67.32 92.41
2 24 45.83–2.10 2.37 0.106/0.418 4 50.0–1.70 24,056 0.210 0.243 0.006 1.3 1596 160 18
a Rmerge = ∑hkl∑i|Ii(hkl) − 〈I(hkl)〉|/∑hkl∑Ii(hkl), where Ii(hkl) is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl)〉 is its average. b The values for the highest-resolution shell are given in parentheses (2.10–2.15 Å resolution for Os derivative T. KS-1 PFDβ2; 1.70–1.76 Å resolution for wild-type T. KS-1 PFDβ2). c Phasing power = 〈F(H)〉/E, where 〈F(H)〉 is the r.m.s. amplitude of the heavy-atom structure factor, and E is the lack of closure error. d Mean figure of merit = 〈∑P(α)exp(iα)/∑P(α)〉, where P(α) is the phase probability at angle α. e R = ∑hkl(|Fo| − |Fc|)/∑hkl|Fo|. f Rfree was calculated on 5% of the data omitted randomly.
635
Prefoldin–Group II Chaperonin Interaction Structure determination and refinements
Accession number
Initial single-wavelength anomalous diffraction phasing at 2.1 Å for the Os-derived T. KS-1 PFDβ2 subunit was performed by locating two Os sites in the peak data set using the program SOLVE.23 After electron density modification, 70% of amino acid residues could be located in the resultant electron density map using the program RESOLVE.24 Further automated model building was performed using ARP/wARP.25 Model building was continued manually using the program Xfit in the XtalView program system.26 The structure was refined using the program CNS.27 After several cycles of refinement and model building, 94 of 99 sequenced residues with an R-factor of 0.285 were obtained. Using the structure of the Os-derived PFDβ2 subunit, we determined the structure of the wild-type PFDβ2 subunit by molecular replacement and refined it at a resolution of 1.7 Å (Table 2).
Coordinates and structure factors have been deposited in the Protein Data Bank28 with accession number 3AEI.
Surface plasmon resonance SPR experiments were performed using Biacore J at room temperature and the Biacore T100 system at various sensor temperatures (20, 25, 30, 35, 40, and 45 °C). Analysis at temperatures higher than 45 °C could not be performed due to the temperature limit of this instrument. T. KS-1 PFDs was coupled to the sensor chip (CM5 research grade) via standard N-hydroxysuccinimide and N-ethyl-N-(dimethylaminopropyl) carbodiimide activation. To immobilize T. KS-1 PFD, we injected T. KS-1 PFD which dissolved in 10 mM sodium phosphate buffer (pH 5.0), on the sensor surface, with HBS EP buffer [10 mM Hepes (pH 7.4), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid, and 0.05% surfactant P20] employed as mobile phase buffer during the immobilization process. Tris–HCl buffer (50 mM, pH 7.5) was then injected to quench the unreacted N-hydroxysuccinimide groups. TKM buffer [50 mM Tris– HCl (pH 7.5), 100 mM KCl, and 25 mM MgCl2] was used as mobile phase buffer. T. KS-1 CPN samples at various concentrations were injected as analytes, and bound analytes were subsequently removed by washing with the mobile phase buffer at 600 s after the injection. Regeneration buffer [50 mM Tris–HCl (pH 9.0)] was injected prior to each analyte injection. Kinetic constants were calculated from the sensorgrams using the Biacore T100 evaluation software, version 2.0 (Biacore AB). Association constants (Ka) were calculated by the resonance unit at equilibrium using the following equation: Req ¼Rmax C=ðCþ1=Ka Þ where Req represents equilibrium resonance units, Rmax is the resonance signal at saturation, and C is the concentration of free analyte. The thermodynamic parameters were calculated by the van't Hoff equation: DGo ¼ − RTlnKa ¼DH o − TDSo or lnKa ¼DSo = R − DHo = RT Therefore, a plot of the natural logarithm of the association constant versus the reciprocal temperature gives a straight line. The slope of the line is equal to minus the standard enthalpy change divided by the gas constant −ΔHo/R, and the intercept is equal to the standard entropy change divided by the gas constant ΔSo/R.
Acknowledgements The work reported here is part of the support program for improving the graduate school education of the “Human Resource Development Program for Scientific Powerhouse,” which is financially supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan through the Tokyo University of Agriculture and Technology. This study was also supported by grants-in-aids for scientific research (19370038, 20059013, and 21370067) and by a grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports, and Culture of Japan (to M.Y.).
References 1. Vainberg, I. E., Lewis, S. A., Rommelaere, H., Ampe, C., Vandekerckhove, J., Klein, H. L. & Cowan, N. J. (1998). Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin. Cell, 93, 863–873. 2. Hartl, F. U. & Hayer-Hartl, M. (2002). Molecular chaperones in the cytosol: from nascent chain to folded protein. Science, 295, 1852–1858. 3. Siegert, R., Leroux, M. R., Scheufler, C., Hartl, F. U. & Moarefi, I. (2000). Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins. Cell, 103, 621–632. 4. Hansen, W. J., Cowan, N. J. & Welch, W. J. (1999). Prefoldin–nascent chain complexes in the folding of cytoskeletal proteins. J Cell Biol. 145, 265–277. 5. Leroux, M. R., Fandrich, M., Klunker, D., Siegers, K., Lupas, A. N., Brown, J. R. et al. (1999). MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin. EMBO J. 18, 6730–6743. 6. Macario, A. J. & Conway De Macario, E. (2001). The molecular chaperone system and other anti-stress mechanisms in archaea. Front. Biosci. 6, D262–D283. 7. Ohtaki, A., Kida, H., Miyata, Y., Ide, N., Yonezawa, A., Arakawa, T. et al. (2008). Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins. J. Mol. Biol. 376, 1130–1141. 8. Martin-Benito, J., Gomez-Reino, J., Stirling, P. C., Lundin, V. F., Gomez-Puertas, P., Boskovic, J. et al. (2007). Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart. Structure, 15, 101–110. 9. Okochi, M., Yoshida, T., Maruyama, T., Kawarabayasi, Y., Kikuchi, H. & Yohda, M. (2002). Pyrococcus prefoldin stabilizes protein-folding intermediates and transfers them to chaperonins for correct folding. Biochem. Biophys. Res. Commun. 291, 769–774.
636 10. Zako, T., Iizuka, R., Okochi, M., Nomura, T., Ueno, T., Tadakuma, H. et al. (2005). Facilitated release of substrate protein from prefoldin by chaperonin. FEBS Lett. 579, 3718–3724. 11. Zako, T., Murase, Y., Iizuka, R., Yoshida, T., Kanzaki, T., Ide, N. et al. (2006). Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate. J. Mol. Biol. 364, 110–120. 12. Martin-Benito, J., Boskovic, J., Gomez-Puertas, P., Carrascosa, J. L., Simons, C. T., Lewis, S. A. et al. (2002). Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT. EMBO J. 21, 6377–6386. 13. Iizuka, R., Sugano, Y., Ide, N., Ohtaki, A., Yoshida, T., Fujiwara, S. et al. (2008). Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1. J. Mol. Biol. 377, 972–983. 14. Okochi, M., Nomura, T., Zako, T., Arakawa, T., Iizuka, R., Ueda, H. et al. (2004). Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin. J. Biol. Chem. 279, 31788–31795. 15. Simons, C. T., Staes, A., Rommelaere, H., Ampe, C., Lewis, S. A. & Cowan, N. J. (2004). Selective contribution of eukaryotic prefoldin subunits to actin and tubulin binding. J. Biol. Chem. 279, 4196–4203. 16. Yoshida, T., Ideno, A., Hiyamuta, S., Yohda, M. & Maruyama, T. (2001). Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric chaperonin with variable subunit composition. Mol. Microbiol. 39, 1406–1413. 17. Yoshida, T., Ideno, A., Suzuki, R., Yohda, M. & Maruyama, T. (2002). Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS-1. Mol. Microbiol. 44, 761–769. 18. Yoshida, T., Kawaguchi, R., Taguchi, H., Yoshida, M., Yasunaga, T., Wakabayashi, T. et al. (2002). Archaeal
Prefoldin–Group II Chaperonin Interaction
19.
20.
21.
22. 23. 24. 25.
26. 27.
28.
group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin. J. Mol. Biol. 315, 73–85. Yoshida, T., Kanzaki, T., Iizuka, R., Komada, T., Zako, T., Suzuki, R. et al. (2006). Contribution of the Cterminal region to the thermostability of the archaeal group II chaperonin from Thermococcus sp. strain KS1. Extremophiles, 10, 451–459. Danno, A., Fukuda, W., Yoshida, M., Aki, R., Tanaka, T., Kanai, T. et al. (2008). Expression profiles and physiological roles of two types of prefoldins from the hyperthermophilic archaeon Thermococcus kodakaraensis. J. Mol. Biol. 382, 298–311. Kida, H., Sugano, Y., Iizuka, R., Fujihashi, M., Yohda, M. & Miki, K. (2008). Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1. J. Mol. Biol. 383, 465–474. Otwinowski, Z. & Minor, W. (1997). Processing of Xray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Terwilliger, T. C. & Berendzen, J. (1999). Automated MAD and MIR structure solution. Acta Crystallogr. Sect. D, 55, 849–861. Terwilliger, T. C. (2002). Automated structure solution, density modification and model building. Acta Crystallogr. Sect. D, 58, 1937–1940. Perrakis, A., Morris, R. & Lamzin, V. S. (1999). Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463. McRee, D. E. (1999). XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. Sect. D, 54, 905–921. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H. et al. (2000). The Protein Data Bank. Nucleic Acids Res. 28, 235–242.