Structure and Molecular Dynamics Simulation of Archaeal Prefoldin: The Molecular Mechanism for Binding and Recognition of Nonnative Substrate Proteins

doi:10.1016/j.jmb.2007.12.010

J. Mol. Biol. (2008) 376, 1130–1141

Available online at www.sciencedirect.com

Structure and Molecular Dynamics Simulation of Archaeal Prefoldin: The Molecular Mechanism for Binding and Recognition of Nonnative Substrate Proteins Akashi Ohtaki 1 †, Hiroshi Kida 2 †, Yusuke Miyata 1 †, Naoki Ide 1 †, Akihiro Yonezawa 1 , Takatoshi Arakawa 1 , Ryo Iizuka 1,3 , Keiichi Noguchi 1 , Akiko Kita 2,4 , Masafumi Odaka 1 , Kunio Miki 2,5 and Masafumi Yohda 1 ⁎ 1

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan 2

Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 3 Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 4 Research Reactor Institute, Kyoto University, Kumatori, Osaka 590-0494, Japan 5

RIKEN SPring-8 Center at Harima Institute, Koto 1-1-1, Sayo, Hyogo 679-5148, Japan

Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 Å resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various nonnative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the β subunit is essentially involved in substrate binding and that the α subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the β subunit in the hydrophobic groove have shown that βIle107 has a critical role in forming the hydrophobic groove. © 2007 Elsevier Ltd. All rights reserved.

Received 2 October 2007; received in revised form 28 November 2007; accepted 5 December 2007 Available online 8 December 2007 Edited by R. Huber

Keywords: prefoldin; group II chaperonin; chaperone; archaea; molecular dynamics

*Corresponding author. E-mail address: [email protected]. †These authors equally contributed to this work. Present address: T. Arakawa, Japan Science and Technology Agency, 1-12 Minamiwatarida-cho, Kawasaki, Kanagawa 210-0855, Japan. Abbreviations used: PFD, prefoldin; PhPFD, Pyrococcus horikoshii OT3 prefoldin; MtPFD, Methanobacterium thermoautotrophicum prefoldin; MD, molecular dynamics; CS, citrate synthase; GFP, green fluorescent protein; PDB, Protein Data Bank. 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.

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Structure and MD Simulation of Archaeal Prefoldin

Introduction Molecular chaperones are ubiquitous proteins that are required for the correct folding, transport, and degradation of proteins within the cell.1 Molecular chaperones function by protecting nonnative polypeptides from forming aggregates or by providing them with appropriate environments where they can fold properly. Prefoldin (PFD) stabilizes a nonnative protein and subsequently delivers it to a group II chaperonin to facilitate the proper folding.2–5 Eukaryotic PFD participates in the maturation of actin and members of the tubulin family. It captures actin or

tubulin in the unfolded state and transfers it to the chaperonin-containing TCP-1, the eukaryotic group II chaperonin, for functional folding. Although there is neither actin nor tubulin in archaea, both PFD and group II chaperonins have invariably been identified in all archaeal species. Eukaryotic PFD is a multiplesubunit complex composed of six polypeptides (two α-type and four β-type subunits) in the molecular mass range of 14–23 kDa.2 On the other hand, archaeal PFD is composed of two kinds of subunits, two α and four β subunits. Among archaeal PFDs, Methanobacterium thermoautotrophicum PFD (MtPFD) and Pyrococcus horikoshii

Fig. 1 (legend on next page)

1132

Structure and MD Simulation of Archaeal Prefoldin

Fig. 1. Crystal structure of Pyrococcus PFD. (a) Overall structure of the α2β4 hexameric complex of PhPFD showing side and bottom views as illustrated by the program MOLSCRIPT.14,15 The crystal structure of PhPFD is composed of one α subunit and two β subunits in an asymmetrical unit. The biologically active α2β4 hexamer is derived by applying crystallographic symmetry. The α subunit and two β subunits are shown in red, green (β1 subunit), and blue (β2 subunit). The symmetrical molecules are shown in light colors. (b) The overall structure of each subunit [the coloring is as in (a)]. The hydrophobic residues (αLeu11, αLeu14, αVal131, and αVal138 in the α subunit; βVal8, βLeu12, βLeu15, βLeu103, and βIle107 in the β1 subunit; and βLeu12, βLeu15, βLeu103, and βIle107 in the β2 subunit) at the distal region of each subunit are in CPK (Corey–Pauling–Koltun) representation. (c) The α- and β-subunit hydrophobic groove structure at the distal region of the coiled coil. The hydrophobic residues (αLeu11, αLeu14, αVal131, αVal138, βVal8, βLeu12, βLeu15, βLeu103, and βIle107) are shown by ball-and-stick models.

OT3 PFD (PhPFD) have been characterized in detail. 6–10 The crystal structure of MtPFD has shown that PFD resembles the shape of a jellyfish with six tentacles. This result has shown that the distal regions of the coiled coils that expose hydrophobic patches are required for multivalent binding of nonnative proteins.11 This proposed substratebinding mechanism was supported by the microscopic observation that the substrate interaction sites of PhPFD are at the tips of the coiled coils of each subunit.6 In addition, we have reported that both the N- and C-terminal regions of the PhPFD β subunit are important for its molecular chaperone activity.7 However, the substrate-binding residues that form the hydrophobic patches and molecular dynamics (MD) of each subunit during substrate binding are still unclear. In this study, we determined the crystal structure of PhPFD at a resolution of 3.0 Å, examined the substrate-binding mode by MD simulation, and characterized the substrate binding site of PhPFD.

Results Determination of the crystal structure of PhPFD PhPFD is a hexameric complex composed of two α subunits (151 amino acids) and four β subunits (117 amino acids). The crystal of PhPFD,

with a space group of P21212, contains an αβ2 trimer in the asymmetrical unit. The two β subunits in the asymmetrical unit were designated β1 and β2. We could not determine the structure of the Nand C-terminal residues of each subunit (residues 1–3 of the α subunit, residues 1–4 and 111–117 of the β1 subunit, and residues 1–9 and 111–117 of the β2 subunit) because of poor electron densities probably due to the disordered structure of the regions. In addition, several ambiguous electron densities were observed at residues 4–12, 16, 19, 22, 24, 26, 47, 86, 136–143, 145, and 148 in the α subunit; residues 7, 9, 12, 14, 16, 19, 27, 98, 99, 101, 103–105, and 108 in the β1 subunit; and residues 12, 14, 17, 30, 95, 98, 101, 105, 106, and 108 in the β2 subunit. All atoms of the proteins, except for these residues, were well fitted to the 2Fo − Fc electron density map. Analysis of the stereochemical quality of the models was accomplished using the program PROCHECK.12,13 Finally, the structure was refined to an R-factor of 0.240. Due to the relatively low resolution of 3.0 Å and disordered structure, it was difficult to determine the side chain conformations of the amino acid residues with confidence. However, this resolution range is sufficient to discuss the overall main chain conformation and the orientations of the side chains of the amino acid residues.

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Structure and MD Simulation of Archaeal Prefoldin

Overall structure of PhPFD PhPFD is a hexameric complex of two α subunits and four β subunits. The structure of PhPFD is a double-β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles, and has a large central cavity formed by the six coiled coils (Fig. 1a). The structure is almost the same as that of MtPFD. Each coiled-coil tentacle is fully solvated. Its polar and charged side chains are almost exposed to the solvent. On the other hand, hydrophobic residues form a hydrophobic groove between the two α-helical regions in the same subunit. These hydrophobic grooves in the cavity are likely to be responsible for the interaction with the hydrophobic surface of an unfolded protein. The average B-factor of the coiled-coil region of each subunit is much higher than the β-assembly region, and the coiledcoil tentacles have few intersubunit interactions with other subunits in the hexameric complex, indicating that each individual coiled coil is highly flexible. This flexibility must be a favorable for widening the central cavity and capturing various nonnative proteins. Structures of the α and β subunits The α and β subunits share a similar architecture, as shown in Fig. 1b. In the β subunit, the N- and Cterminal regions (residues 5–47 and 67–110 in β1 and residues 13–47 and 67–110 in β2) form a coiled-coil structure that is connected by a β-hairpin linker (residues 55–66 in both β1 and β2) consisting of two short β strands. The N-terminus of β2 has lost the last turn of the α helix compared with that of β1. The Nterminal region of β2 could not be built due to ambiguous electron density. The electron density shows that the helical structural region of the β2 subunit seems to be deformed by the contact with symmetryrelated molecules in the crystalline state. The Nterminal region of the β2 subunit should take an α-helical conformation from residue 5, like the β1 subunit, in solution. The orientations of the coiled coils relative to the β-hairpin regions of β1 and β2 differ from each other. The α subunit also has a similar basic architecture, containing an α-helical region (residues 4–53 and 97–146), a β hairpin (residues 59–77), and an extra β hairpin (residues 81–95). The extra β hairpin is involved in the dimerization of the α subunits in the α2β4 hexamer. The two α helices of the α subunit are 12 and 14 turns in length. In the longer C-terminal α helix, the α helix is distorted in the middle of the helix around residues 120–130, making a long curved helix that is likely to be induced to avoid steric hindrance with symmetrical molecules. The residues located at the distal region form the hydrogen bond interactions with symmetrical molecules. These observations were also found in the crystal structure of MtPFD. Structure of the hydrophobic groove at the distal region of each subunit The far distal ends of the coiled-coil tentacles of both the α and β subunits seem to take partially

random coil structures. Our previous study using truncated mutants of PhPFD showed that the far distal ends of coiled-coil tentacles of both the α and β subunits were not essential for the chaperone activity.7 Hydrophobic grooves exist in the coiled coils of both the α and β subunits (Fig. 1c). Since the hydrophobic grooves are in the central cavity, these grooves are thought to be involved in binding with a nonnative substrate. These hydrophobic grooves are formed by αLeu11, αLeu14, αVal131, and αVal138 in the α subunit and by βVal8, βLeu12, βLeu15, βLeu103, and βIle107 in the β subunit. In addition, the poor electron density map showed the Cterminal end of the β subunit, where we could not add the model to this determined structure, formed an α-helical structure continuously, suggesting that βLeu111 also formed part of this hydrophobic groove in the β subunit. However, the hydrophobic residues of the α subunit are not orderly facing each other, compared with those of the β subunit. Thus, the hydrophobicity of this groove of the β subunit is higher than that of the α subunit, indicating that the β subunit is essential for binding nonnative proteins. This insight is supported by electron microscopy analyses6 and our previous biochemical studies.7,10 Dynamic structure of PhPFD at 330 K The crystal structure of PhPFD suggested that flexibility of the tentacles is important for capturing and arresting unfolded proteins of various sizes. Therefore, we performed MD simulation of a PhPFD molecule at 330 K, which is close to the temperature for the measurement of chaperone activities. The initial model of PhPFD was constructed by energy minimization from the crystal structure obtained in this study using the program SANDER in the AMBER suite. MD simulation was performed by increasing the temperature gradually from 100 to 330 K to avoid destabilizing the simulated structure. The plots of the r.m.s. fluctuation (i.e., SD) of each residue in the α subunit and two β subunits show the atomic fluctuations along the protein structure during MD simulation at 300 K in the first 200 ps and at 330 K in the next 1 ns (Fig. 2a). The residues of the coiled-coil regions on both the α and β subunits show a high level of flexibility compared with those of the β-assembly region. In addition, short segments composed of residues 54–59 in the α subunit and residues 48–53 in the β subunit exhibit a higher level of flexibility. These regions in the connecting linkers between the α-helical and β-assembly regions seem to play the role of a “hinge” and induce flexible motions of the coiled coils of all subunits. Figure 2b shows the superimposed structure of the initial model and simulated model at 330 K. Although the structure composed of a double-βbarrel assembly and six long coiled coils is kept, the positions of the coiled coils have drastically changed. The coiled-coil regions of the β1′ (β1 in another asymmetrical unit) and β2 subunits move forward toward each other, and the distance between them was reduced by ∼10 Å, resulting in weak van der

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Structure and MD Simulation of Archaeal Prefoldin

Fig. 2. Fluctuation of PhPFD during MD simulation. (a) Fluctuation along the whole trajectory of the Cα atoms of PhPFD during MD simulation at 300 K for 200 ps (solid line) and at 330 K for 1 ns (dashed line). (b) Superimposed structure of the initial model (orange) and simulated model (blue). The simulated model structure is the result of the 1 ns of MD calculation at 330 K.

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Structure and MD Simulation of Archaeal Prefoldin

Waals contacts. A similar change was also observed between the β1 and β2′ subunits. Although the α subunit moves forward toward the β1 subunit, the distance between the two α subunits was kept at ∼50 Å. These results showed that the coiled-coil regions of each subunit tend to move rotationally and that the width of the central cavity was kept open. On the other hand, r.m.s. fluctuations in the βassembly region of both the α and β subunits were less than 1.5 Å, showing a low level of flexibility and a stable structure. Thus, this β-assembly structure contributes to the maintenance of this unique structure and the width of the central cavity. Docking and MD simulation between PhPFD and a nonnative protein Archaeal PFDs have a central role in protecting unfolded proteins from aggregation under various conditions and transferring them to group II chaperonins to facilitate refolding. Previous studies on archaeal PFDs showed that the substrate-binding region is at the distal region of the coiled coil. However, the precise substrate-binding residues and the motion of PFD during the substrate binding were not well known. To address these problems, we performed a docking simulation of denatured insulin into the central cavity of PhPFD. At first, we prepared the denatured structure of insulin by MD simulation. The crystal structure of human insulin [Protein Data Bank (PDB) accession code 2c8r] was used as the initial structure. MD simulation was performed in vacuo at 300 K for 1.0 ns, at 350 K for 1.0 ns, and then at 375 K for 7 ns. In the simulated denatured structure, hydrophobic residues are exposed at the surface. The denatured insulin model was placed in the PhPFD cavity such that the center of the denatured insulin was at the central position of the PhPFD cavity, between the Cα atoms of αLeu11 and βLeu111 of β1′, avoiding unusual short contacts with the PhPFD. After energy minimization for the PhPFD/ nonnative insulin complex using the program SANDER in the AMBER suite, the complex is used for the initial model for the docking MD simulation (Model 1). In addition, we constructed three additional initial models (Models 2, 3, and 4) by changing the orientation of the denatured insulin in the PhPFD/insulin complex at 90, 180, and 270 deg along with the rotation axis, the line between αLeu11 in the α subunit and βLeu111 in the β1′ subunit.

Table 1 shows the numbers of observed interactions in each of the four docking simulations at 1 ns. The β subunit has many interactions with the hydrophobic surface of the denatured insulin compared with the α subunit. Almost all of the interactions in the β subunit are in the hydrophobic groove at the distal region. Since the β subunit showed a similar binding mode in all MD simulations with different initial orientations of the denatured insulin, we analyzed the result of the MD simulation from Model 1 in detail. β subunits maintain their secondary structure with coiled-coil tentacles, although their conformation and orientations are changed. In the MD-simulated structure at 1 ns, the hydrophobic groove at the distal region of the β subunit forms van der Waals contacts with the denatured insulin, as shown in Fig. 3a and b. The distances between Cα atoms of the interacting hydrophobic residues of the β subunits and insulin are plotted in Fig. 3c. The distances decreased in the first 500 ps and then reached constant values in the next 500 ps. This observation indicated that the β subunit makes a noticeable positional change and stable hydrophobic interactions with denatured insulin. On the other hand, with respect to the α subunit, the results changed by the initial models. At 1 ns of MD simulation from Models 2 and 3, the middle of the α-helical structure is loosened and the distal region of the α subunit is detached from insulin to widen its central cavity to form a favorable interaction between insulin and the β subunit. Meanwhile, in the MD simulations from Models 1 and 4, the α subunit maintained its α-helical structure and moved closer to insulin. These observations indicated that the α subunit might demonstrate multiple conformational changes to widen its central cavity for interaction with substrates in compliance with the location, binding site, and molecular size of nonnative substrate proteins. Chaperone activities of PhPFD β-subunit mutants The crystal structure and MD simulation showed that the hydrophobic groove, at the distal region of the β subunit formed by βVal8, βLeu12, βLeu15, βLeu103, βIle107, and βLeu111, is the substrate binding site; three hydrophobic residues located at the N-terminal helix and the other three located at the C-terminal helix are facing each other, as shown in Fig. 1c. The hydrophobic residues of the α subunit

Table 1. The number of observed interactions between PhPFD and the insulin model in the docking MD simulations Hydrophobic interactions

Hydrogen bonds

Initial model

α

α′

β1′

β2

α

α′

β1′

β2

Model 1 Model 2 Model 3 Model 4

– 3 9 –

2 – – –

7 (7) 9 (9) 8 (5) 9 (7)

12 (10) 7 (6) 13 (11) 10 (8)

13 9 15 2

5 – – 3

5 3 1 4

3 3 6 3

Numbers of interactions involved in the hydrophobic groove of the β subunit, as mentioned in the text, are shown in parentheses.

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Structure and MD Simulation of Archaeal Prefoldin

docking simulations. In addition, our previous study7 showed that the effect of deletion of Nterminal 17 residues of the α subunit was relatively marginal compared with the deletion of the βsubunit C-terminus. Therefore, we focused on the β subunit in this study. To elucidate the role of these residues of the β subunit, we constructed mutants in which these residues were replaced by Asn (βV8N, βL12N, βL15N, βL103N, βI107N, and βL111N) and investigated their chaperone activity by measuring their ability to prevent thermal aggregation of insulin and citrate synthase (CS) and spontaneous refolding of acid-denatured green fluorescent protein (GFP). Analyses by analytical size-exclusion chromatography and circular dichroism have shown that the mutants kept their original quaternary structures (data not shown). Figure 4 and Table 2 show the chaperone activities of wild-type and mutant PhPFDs. Wild-type PhPFD efficiently protected insulin and CS from thermal aggregation and prevented the renaturation of GFP. Although the effects of mutations on protection of insulin from aggregation were relatively marginal especially in the N-terminal region, the results showed a similar tendency with the other two substrates. In the results for CS and GFP, βL12N and βI107N exhibited a drastic decrease in chaperone activities. Since these two residues face each other and are located in the middle of this hydrophobic groove, the residues, especially βIle107, are likely to be important for the chaperone activity and hydrophobicity of this groove. We then prepared other mutants in which βIle107 was replaced by other hydrophobic residues, Ala (βI107A) and Trp (βI107W). βI107A retained chaperone activities for both CS and GFP. Unexpectedly, βI107W exhibited partially decreased chaperone activity. Insertion of a bulky amino acid at the central position of the hydrophobic region might induce structural disorder in the hydrophobic region. These results showed that residue β107 is an essential hydrophobic residue for forming a hydrophobic patch at the distal region of the β subunit.

Discussion Substrate binding site of PhPFD

Fig. 3. Details of the interface between the insulin model and the distal region of PhPFD. (a and b) Resulting β1′ subunit (a) and β2 subunit (b) after 1 ns of MDsimulated calculation at 330 K. (c) The distance between the Cα atoms of the interacting hydrophobic residues of the PhPFD β subunits and the insulin model structure.

also interacted with the denatured insulin. However, the residues of the α subunit responsible for binding with the denatured insulin varied among the four

In this study, we determined the crystal structure of PhPFD and examined the interaction between PhPFD and nonnative insulin modeled by MD simulation and mutant analyses. The overall structure of PhPFD is very similar to that of MtPFD. The asymmetrical structural unit of both PFDs consisted of one α subunit (151 amino acid residues in PhPFD and 141 amino acid residues in MtPFD) and two β subunits (117 amino acid residues in PhPFD and 122 amino acid residues in MtPFD) (Fig. 1).9 However, the length of the α-helical region of the β subunit of PhPFD differs from that of MtPFD. The MtPFD crystal was prepared from mutant MtPFD

Structure and MD Simulation of Archaeal Prefoldin

lacking the last seven residues of the β subunit, which is not critical for its biological activity. On the other hand, we used a full-length PhPFD for crystallization and determined its structure. For the Cα atoms, the structure of nearly all of the full-length

1137 α subunit (4–151 amino acid residues) was determined. In the β subunit, the secondary structure of the distal end, which is the truncated region in MtPFD, was disordered because of the symmetrical packing in the crystalline state. These unfavorable contacts at the molecular interface are likely to be the cause of high mosaicity of the crystals. We tried to obtain the high-resolution structure in vain. In addition, the region at the distal end of the β2 subunit contacts that of the symmetrically related β1 subunit. The hydrophobic groove is formed at the distal end, and the grooves of the β2 and symmetryrelated β1 subunits face each other and form hydrophobic interactions. The hydrophobic interactions seem to cause the collapse of the secondary structure at the distal end of the β2 subunit. These interactions may reflect the substrate-binding state of PhPFD, in which the hydrophobic groove of the β subunit interacts with the exposed hydrophobic residues of unfolded proteins. In this observation, the hydrophobic groove is formed by βVal8, βLeu12, βLeu15, βLeu103, βIle107, and βLeu111 of the β subunit. As expected, site-directed mutagenesis analyses showed that these residues of the β subunit are essentially involved in nonnative substrate binding and that βIle107 is a critical residue (Table 2). However, βIle107 is not sufficient to capture the unfolded protein, because a double mutant (βLeu103N/ β111N) showed a drastic decrease in its chaperone activities (Table 2). Replacement of βIle107 to a bulky amino acid, tryptophan, should cause structural disturbance of the region, which results in the decrease of chaperone activity. Thus, βIle107 might serve as a central core to form the hydrophobic region in addition to direct substrate binding. Our previous study, using a 6-amino-acid (residues 112– 117) truncation mutant from the C-terminus of the β

Fig. 4. Chaperone activity of PhPFD mutants with the amino acid replacements in the N-terminal helix (a and b) and C-terminal helix (c and d) of the β subunit. (a and c) Effects of wild-type and mutant PhPFDs on the aggregation of CS (100 nM at a monomer concentration) were monitored by measuring the light scattering at 500 nm with a spectrofluorophotometer at 50 °C with continuous stirring. CS was incubated in the absence (filled circles) or presence [in (a), open circles indicate βL8N; open triangles, βV12N; open squares, βL15N; and filled squares, wild type; in (c), open circles indicate βI103N; open triangles, βI107N; open squares, βL111N; and filled squares, wild type] of PFDs. AU indicates arbitrary units. (b and d) Effects of wild-type and mutant PhPFDs on the refolding of GFP at 60 °C were monitored by the fluorescence at 510 nm with excitation at 396 nm using a spectrofluorophotometer. Acid-denatured GFP was diluted in the folding buffer without (spontaneous, filled circles) or with [in (b), open circles indicate βL8N; open triangles, βV12N; open squares, βL15N; and filled squares, wild type; in (d), open circles indicate βI103N; open triangles, βI107N; open squares, βL111N; and filled squares, wild type] PhPFDs. The amount recovered is expressed as the percentage of the fluorescence intensity of native GFP.

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Structure and MD Simulation of Archaeal Prefoldin

Table 2. Relative activities of the mutated PhPFDs for the protection of insulin and CS from thermal aggregation and prevention of GFP spontaneous refolding Substrates PhPFD Wild type βV8N βL12N βL15N βL103N βI107N βL111N βI107A βI107W βL103N/L111N

Insulin (%)a

CS (%)a

GFP (%)b

100 68.5 106 104 85.4 38.9 80.3 87.3 90.4 65.6

100 49.7 25.7 85.6 94.4 12.1 78.1 95.5 53.2 5.1

100 26.4 49.4 63.4 65.7 22.9 79.2 72.2 63.1 25.6

a Relative suppression of insulin and CS aggregation at the maximum light scattering of insulin and CS against the wild type was calculated. b Relative suppression of fluorescence recovery at 600 s against the wild type was calculated.

subunit, showed no observable effect on CS aggregation or GFP refolding.7 These results provided the convincing evidence for a specific interaction between this hydrophobic groove and unfolded proteins. Roles of the α and β subunits of PhPFD Recently, an electron microscopic study on PhPFD showed that the substrate-binding mode changes with the mass of the substrate unfolded protein; the number of PhPFD coiled coils involved in the interaction with the unfolded substrates increases with the size of the denatured protein.6 With an unfolded protein of a smaller mass as a substrate, only a pair of PhPFD β subunits was required for interaction. Furthermore, the unfolded proteins are not confined inside the cavity formed by PhPFD tentacles and rather protrude from it. In this study, we performed MD simulation analyses of a preliminary rough docking of PhPFD with a nonnative insulin model structure from MD trajectory at 330 K and carried out four runs with different initial models. In all cases, we located the nonnative insulin model in the central cavity of PhPFD. These results indicate that the substrate-binding mode of PhPFD is different from that observed in the electron microscopy study, showing that nonnative insulin was encapsulated inside the central cavity rather than protruding from it. In the electron microscopy study, the smallest substrate protein was a lysozyme with a molecular mass of 14 kDa, while we used insulin of 5.7 kDa (consisting of a 2.4-kDa α chain and a 3.3-kDa β chain) for MD simulation. The difference in binding mode is likely to be due to the difference in the molecular size of unfolded substrate proteins. The small mass protein can move into the cavity, which results in a more stable state as the interaction with PhPFD increases. Therefore, the effects of mutant activities for insulin were relatively marginal, compared with CS and GFP (Table 2). We

are also performing docking simulation with denatured GFP (data not shown). The mobility and binding mode were almost the same as those with insulin, but the binding GFP was protruded from the central cavity as observed in the electron microscopy study.4 In addition, both our results and those from the electron microscopy study showed that the distal region of the β subunit was always involved in substrate binding. The mobile β subunit first interacts with the surface of the unfolded proteins by the hydrophobic interaction to protect them from aggregation. Docking MD simulations at 330 K showed that both α subunits seemed to change the width of the central cavity depending on the binding mode. If the α subunit remains in the same conformation during substrate binding, it causes many unusual contacts between the substrate and PhPFD, even with smaller mass substrates. Then, two α subunits of the hexameric complex need to change their conformations and positions depending on the size and binding mode of the unfolded protein to stabilize the PhPFD/substrate complex. Accompanied by the flexible segments of 54–59 in the α subunit, the α subunit provides the multiplesubstrate-recognition mechanism of PhPFD and the wide substrate specificities that arrest various unfolded proteins. Further functional and MD comparative studies on PhPFD interaction with other unfolded proteins and mutational analyses in the α subunit could help us understand the distribution of function of each subunit.

Materials and Methods Crystallization and X-ray data collection The purification of wild-type PhPFD has been already reported.10 A crystal of PhPFD was grown by the vapor diffusion method using a protein solution (20 mg/ml) and a reservoir solution [30% (v/v) polyethylene glycol 400, 100 mM sodium chloride, and 100 mM lithium sulfate in 100 mM 4-morpholineethanesulfonic acid–NaOH, pH 6.5] at 18 °C. The diffraction data for PhPFD were collected at 100 K using an ADSC CCD detector system on the NW12 beam line in the Photon Factory and the BL44B2 beam line in the SPring-8 Center. Diffraction data were processed using the program HKL2000.16 The collected data and scaling results are listed in Table 3. Structure determination and refinements The structure of PhPFD was determined by the molecular replacement method using the structure of MtPFD as a probe model with the program Molrep in the CCP4 program suite.14 Models were corrected on the 2Fo − Fc electron density map using the program Coot,17 and the structure without solvent molecules was constructed using the maximum-resolution data. Calculations of structural refinements were carried out by the programs REFMAC518 and PHENIX.19 After several cycles of positional and temperature factor refinements, three sulfate ions were found and added to the structure. Finally,

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Structure and MD Simulation of Archaeal Prefoldin Table 3. Data collection and refinement statistics PhPFD Temperature (K) Resolution (Å) No. of measured reflections No. of unique reflections Completeness (%) Rmergeb Io/σ(Io) Space group Cell dimensions (Å) a b c Structure refinement Resolution range (Å) No. of reflections Rc Rfreed r.m.s.d. bond length (Å) r.m.s.d. bond angle (°) No. of protein atoms No. of ligand atoms (SO4)

100 3.0 61,014 10,215 94.1 (77.9)a 5.5 (43.4)a 25.2 (3.6)a P21212 65.17 98.75 78.62 50–3.0 9396 0.240 0.277 0.002 0.43 2651 15

a The values for the highest-resolution shell are given in parentheses (3.11–3.00-Å resolution). b Rmerge = ∑hkl∑i|Ii(hkl) − 〈I(hkl)〉|/∑hkl∑Ii(hkl), where Ii(hkl) is the ith intensity measurement of reflection hkl, including symmetryrelated reflections, and 〈I(hkl)〉 is its average. c R = ∑hkl(|Fo|−|Fc|)/Σhkl|Fo|. d Rfree was calculated on 5% of the data omitted randomly.

refinement of the structure converged at an R-factor of 0.240 (Rfree = 0.277)20 with good chemical geometry, as listed in Table 3. Solvent molecules were not included in the refined structure due to the relatively low-resolution range of the diffraction data. The refinement statistics are listed in Table 3. Preliminary modeling of the PhPFD complex with nonnative insulin for MD simulation At first, we prepared the PhPFD model. The possible side chain positions of the residues of poor electron density were placed by manual fitting, avoiding unusual intramolecular short contacts. The coiled-coil structure of the β2 subunit at the distal region was replaced by an α helix, which is the same as the β1 subunit. The structures of both the β1 and β2 subunits at the C-terminal distal region (residues βLeu111 and βArg112) were added to the crystal structure as α helix. The N- and C-terminal residues (residues 1–4 and 113–117 in the β subunit) were not added to the model structure because these regions are not critical for its biological activity, as previously described.7 After the generation of the PhPFD model, structure energy minimization calculation was carried out with the program SANDER in the AMBER suite22 without X-ray data. Second, we prepared the random structure of insulin showing a nonnative-like structure. The crystal structure of human insulin (PDB accession code 2c8r) was used as the initial structure. The insulin molecule was subjected to restrained MD calculations with parm96 of the AMBER 8 force field in vacuo, where the total number of atoms was 756. The van der Waals and electrostatic interactions were calculated using the MD Server (NEC Corporation, Japan), a computer designed for MD calculation.23 A cutoff distance of 1000 Å was applied to evaluate the van der

Waals and electrostatic interactions. The potential energy minimization of the initial MD system was carried out using the conjugated gradient method. Under the restrained conditions that only the water molecules were allowed to move and the proteins were kept frozen, MD simulation was started at 100 K for 100 ps using the energy-minimized structure. The system was equilibrated by gradually increasing the temperature from 100 to 250 K for 300 ps in total, where all atoms were allowed to move. After equilibration, the system was heated to 300 K and then to 350 K for 1.0 ns at each temperature and to 375 K for 7 ns. After generating the denatured insulin model, we defined the center position of PhPFD between the Cα atoms of αLeu11 in the α subunit and βLeu111 in the β1′ subunit, and the line between them was defined as a rotation axis. We placed the center position of the nonnative insulin at the center position of PhPFD, avoiding unusual short contacts with PhPFD. The initial model of the complex was denoted as Model 1, and energy minimization for the PhPFD/nonnative insulin complex was performed using the program SANDER in the AMBER suite. This complex was used as the initial model for docking MD simulation. In addition, we calculated three other initial models. The orientation of nonnative insulin in the PhPFD/insulin complex was rotated by 90, 180, and 270 deg along the rotation axis, denoted as Models 2, 3, and 4, respectively. MD simulation The solvent water molecules within 40 Å of the atoms of the PhPFD/insulin complex were removed to avoid close atomic contacts. A half-harmonic potential22 was applied around the spherical water droplet to prevent the water molecules from evaporating. The ff96 parameter24,25 in the AMBER force field, along with the TIP3P water model,26 was used in this study. The van der Waals and electrostatic interactions were calculated using the MD Server (NEC Corporation).23 A cutoff distance of 1000 Å was applied to evaluate the van der Waals and electrostatic interactions. The potential energy minimization of the initial MD system was carried out using the conjugated gradient method. Under the restrained conditions that only the water molecules were allowed to move and the proteins were kept frozen, MD simulation was started at 100 K for 100 ps using the energy-minimized structure. The system was equilibrated by gradually increasing the temperature from 100 to 250 K for 300 ps in total and to 300 K for 200 ps, where all atoms were allowed to move. After equilibration, the system was heated to 330 K for 1.0 ns. An additional 1.0-ns run was performed for detailed analyses at 330 K, which is close to the measurement temperature of the PhPFD chaperone activities. The trajectories of the atoms above 300 K were stored every 1 ps. Visual Molecular Dynamics27 was used for the analyses of structure and molecular motions and for the preparation of graphical representations. Preparation of PhPFD mutants The single and double mutants of PhPFD (βV8N, βL12N, βL15N, βL103N, βI107N, βI107A, βI107W, βL111N, and βL103N/L111N) were constructed with a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the plasmid pPhPFD as a template.7 All constructs were verified by DNA sequencing. The purification of these mutants was the same as for the wildtype PhPFD.7

1140 Insulin from bovine serum and CS from porcine heart were purchased from Sigma. GFP was purified as previously described.28,29 Thermal aggregation of CS was monitored by measuring the light scattering at 500 nm with a fluorophotometer (FP-6500, JASCO) at 50 °C. GFP refolding was monitored by measuring the fluorescence of GFP at 510 nm with excitation at 396 nm. The measurement of PhPFD activities using CS and GFP as substrates was done as previously described.7,10 Thermal aggregation of insulin was monitored by measuring the light scattering at 500 nm with a fluorophotometer (FP6500) at 60 °C with continuous stirring. Monitoring started after the addition of insulin (15 μM) to 50 mM Tris–HCl buffer (pH 8.0) containing 20 mM DTT with 1.5 μM wild-type PhPFD or mutant PhPFDs preincubated at 60 °C. PDB accession number The atomic coordinates and structure factors of PhPFD have been deposited in the PDB21 under accession code 2ZDI.

Acknowledgements The work reported here is part of the 21st Century Center of Excellence Program of “Future NanoMaterials” research and education project, which is financially supported by the Ministry of Education, Science, Sports, Culture, and Technology through the Tokyo University of Agriculture and Technology. This work was also supported by Grants-in-Aid for Scientific Research on Priority Areas (17028013, 17066005, and 18031010) 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 K.M. and M.Y. We thank the beam line scientists of the Photon Factory and SPring-8 Center for their help with data collection.

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