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The Structure of Rat Liver Vault at 3.5 Angstrom Resolution

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Science  16 Jan 2009:
Vol. 323, Issue 5912, pp. 384-388
DOI: 10.1126/science.1164975

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Abstract

Vaults are among the largest cytoplasmic ribonucleoprotein particles and are found in numerous eukaryotic species. Roles in multidrug resistance and innate immunity have been suggested, but the cellular function remains unclear. We have determined the x-ray structure of rat liver vault at 3.5 angstrom resolution and show that the cage structure consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain, and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The shoulder domain is structurally similar to a core domain of stomatin, a lipid-raft component in erythrocytes and epithelial cells.

Vaults are large barrel-shaped ribonucleoprotein particles that are highly conserved in a wide variety of eukaryotes (1). Although several functions have been proposed for vaults since their discovery in 1986 (210), including roles in multidrug resistance, cell signaling, and innate immunity, their cellular function remains unclear. Most vault particles are present in the cytoplasm, but a few of them localize to the nucleus (11,12). Rat liver vault comprises a small untranslated RNA consisting of 141 bases (vRNA) (13) and three proteins. The sequences of these proteins are known for the human vault. The 99-kD major vault protein (MVP, Swiss-Prot entry Q14764) is the major structural protein and can self-assemble to form vault-like particles (14); the 193-kD vault poly(adenosine diphosphate–ribose) polymerase (VPARP, Swiss-Prot entry Q9UKK3) presumably ribosylates substrates (15); and the 290-kD telomerase-associated protein 1 (TEP1, Swiss-Prot entry Q99973) (16) is important for stabilization of vRNA (17). Molecular composition of the vault has been roughly estimated as 96 MVPs, eight VPARPs, two TEP1s, and at least six copies of vRNA (18).

Electron microscopy and x-ray crystal structural analysis at 9 Å resolution (19,20) have revealed a cagelike structure with two protruding caps and an invaginated waist. On the basis of the x-ray structure, which was of an empty vault built from a cysteine-tagged construct of MVP [termed cpMVP vaults; Protein DataBank entry (PDB) 2QZV], the cpMVP vault was proposed to comprise 96 MVP molecules, each folding into 14 domains. Forty-eight MVP molecules were proposed to extend between the middle and each tip of the vault, so that the cpMVP vault exhibited 48-fold dihedral symmetry. The resolution, however, was too low to determine the main chain fold. Nuclear magnetic resonance (NMR) spectroscopy of an MVP fragment from human (domains 3 to 4: PDB 1Y7X) reveals that each domain contains a three-stranded antiparallel β sheet and loops (21). Our previously published crystallographic analysis of rat liver vault, at 10 Å resolution, demonstrated that the particle exhibits 39-fold dihedral symmetry, instead of 48-fold dihedral symmetry (22). Here, we report the determination of the x-ray crystal structure of rat liver vault at 3.5 Å resolution.

Vault complexes containing all components were purified and crystallized as described previously (22,23). The initial electron density of a vault was obtained by the molecular replacement (MR) method, by using the cryo-EM (cryo-EM) map (22) as a starting model of noncrystallographic symmetry (NCS) averaging. Phase extension was performed by NCS averaging about the 3-fold axis contained within the 39-fold symmetry, by using a mask generated by the EM structure (22). Judging from the R factor, correlation coefficient between observed and calculated structure factors (Fobs and Fcalc), and the soundness of the electron density distributions of helical region and β barrels, a phase extension by the 3-fold axis at (ω = 19.83°, φ = 0.00°) resulted in the best electron density map of the crystal. The R factor, Σ∥Fobs| – |Fcalc∥/Σ|Fobs|, and the correlation coefficient of structure factors, Σ(|Fobs| – <|Fobs|>)(|Fcalc| – <|Fcalc|>)/[Σ(|Fobs| – <|Fobs|>)2 Σ(|Fcalc| – <|Fcalc|>)2]1/2, were 0.137 and 0.971, respectively, where <|Fobs|> and <|Fcalc|> were the averaged values of |Fobs| and |Fcalc|, respectively. The 3.5 Å resolution map showed that a vault comprises 78 MVP chains with 39-fold dihedral symmetry coming from the 39 MVP chains in each half-vault (Fig. 1). Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain, and a cap-ring domain (Fig. 2).

Fig. 1.

Overall structure of the vault shell. One molecule of MVP is colored in tan, and the others are colored in purple. (Left) Side view of the ribbon representation. The whole vault shell comprises a 78-oligomer polymer of MVP molecules. The size of the whole particle is ∼670 Å from the top to the bottom and ∼400 Å in maximum diameter. The particle has two protruding caps, two shoulders, and a body with an invaginated waist. Two half-vaults are associated at the waist with N-terminal domains of MVP. (Right) Top view of the ribbon representation. The maximum diameter of the cap is ∼200 Å. The outer and the inner diameters of the cap-ring are shown.

Fig. 2.

Stereoscopic ribbon drawing of the overall fold of an MVP monomer. The MVP monomer is folded into nine structural repeat domains, a shoulder domain, a cap-helix domain, and a cap-ring domain. Each domain is depicted in a different color: domain 1 (Met1-Pro55), purple; domain 2 (Arg56-Thr110), pink; domain 3 (Pro111-Ile163), light green; domain 4 (Gln164-Val216), coral; domain 5 (Asp217-Val271), light blue; domain 6 (Pro272-Asp322), magenta; domain 7 (Val323-Gln378), yellow; domain 8 (Ala379-Arg456), red; domain 9 (Val457-Gly519), cyan; shoulder domain (Pro520-Val646), green; cap-helix domain (Asp647-Leu802), purple; and cap-ring domain (Gly803-Ala845), dark red.

After preparing a molecular mask from the atomic model of MVP, we performed further phase refinement by 39-fold NCS averaging. NCS parameters of each MVP domain were refined during the phase extension. The averaged value for the correlation coefficient of electron density for domains 3 to 9, the shoulder domain, and the cap-helix domain were ∼0.95, whereas domain 1, domain 2, and the cap-ring domain had smaller correlation coefficients, ∼0.92. The electron density distribution was significantly improved in all regions of the MVP molecule compared with densities obtained by NCS averaging of three-fold symmetries. Bulky electron density maps of aromatic residues are clearly shown in the regions with higher correlation coefficients (∼0.95). An electron density map of each domain, together with the structural model, is depicted in fig. S1, A to D.

Refinement statistics of the whole structure are summarized in Table 1. The final round of refinement, using the program CNS (24), reduced the R factor to 31.1% and Rfree factor to 33.0% for reflections in the resolution range 204.0 to 3.5 Å. The refined vault structure converged well to root-mean-square deviations (RMSDs) from the ideal bond lengths and angles of 0.010 Å and 1.48°, respectively. Of the nonglycine residues, 80.0% were in the most favorable region of the Ramachandran plot (25), 19.7% in the allowed region, and 0.3% in the disallowed region. Out of 861 total amino acid residues, we determined the tertiary structures, including side-chain structures, of 782 residues from Met1 to Pro815. Residues Leu429-Pro448, Met608-Pro620, and Phe846-Lys861 could not be determined, and only the Cα trace could be determined for the residues Glu816-Ala845.

Table 1.

Refinement statistics.

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Although the vault sample prepared for crystallization contained all the protein components and vRNA as shown in fig. S2, only MVP could be definitively assigned in the electron density map. A cylindrical region with ∼25 Å diameter and ∼25 Å height at the top center of the cap exhibited significant electron density both in the NCS averaged map (fig. S1D) and a σ-A weighted 2FobsFcalc map, but was unlikely to be part of MVP, because it did not have 39-fold symmetry. The probable candidate for this density is TEP1, because it is located in the cap (18), and the size is reasonable for its molecular weight of 290 kD. Molar ratios of VPARP, TEP1, and vRNA to MVP are less than one to eight (18), so these proteins would likely not have 39-fold symmetry and, thus, may not be observed in the region exhibiting 39-fold symmetry.

The vault shell measures ∼670 Å in length and ∼400 Å in maximum diameter (Fig. 1). The barrel wall of only 15 to 25 Å in thickness encloses an internal cavity with the length of ∼620 Å, and the maximum diameter of ∼350 Å, large enough to enclose most objects found within the cell. The body consists of 78 copies of the nine MVP structural repeat domains (39 copies in each half-vault), with the waist formed by end-to-end association of structural domain 1. The nine structural repeat domains can be classified into two subgroups by their topological arrangements of antiparallel β strands. Structural repeat domains 8 and 9 consist of five antiparallel β strands termed S1, S2, S3, S4, and S5 (fig. S3, A and C). The other seven structural repeats have two antiparallel β strands (S2a and S2b) inserted between S2 and S3 (fig. S3, B and D). The latter structure is consistent with the domain structure determined by the NMR method (21).

The shoulder region is ∼25 Å in height along the 39-fold axis and ∼315 Å in diameter. Each shoulder domain (Pro520 to Val646) folds into a single α/β globular domain with a four-stranded antiparallel β sheet on one side and four α helices on the other side (Fig. 3). The ∼155 Å high cap is formed by the cap-helix domains (Asp647 to Leu802) that form a 42-turn-long α helix that exhibits a quarter turn of superhelical structure (Fig. 2) and the cap-ring domains (Gly803 to Ala845) that form a U-shaped structure at the top of the cap. The inner and outer diameters of the cap-ring are ∼50 Å and ∼130 Å, respectively (Fig. 1).

Fig. 3.

A stereoscopic ribbon representation of the shoulder domain, along with secondary structure notations. The secondary structures are β1, β2, α1, α2, α3, α4, β3, and β4 from the N-terminal side to the C-terminal side.

Details of intersubunit interactions are provided in the supplementary online text and table S1 (A, B, and C). Out of 74 total interactions observed between two adjacent MVP subunits, 41 interactions were between cap-helix domains; this suggests that these interactions promote self-assembly of the particle. In the cap-helix domain, most polar residues are exposed to inner or outer surfaces of the particle, and hydrophobic residues appear on the interface between two helices to form hydrophobic interactions (fig. S4). Specific ionic pairs and hydrophobic interactions for the cap-helix domain are depicted in fig. S5 and Fig. 4A, respectively. N-terminal residues of MVP domain 1 form an intermolecular antiparallel β sheet around the two-fold axis (Fig. 4B). Using an electron microscopic method, Kedersha et al. (26) have observed flowerlike structures, each of which is composed of petals surrounding the central ring. This observation is consistent with our structure. Because interactions stabilizing the dimer of half-vaults are weaker than interactions stabilizing the cap structure, it is reasonable that the particle may separate into half-vaults with MVP structural repeat domains separated and appearing as petals around the central ring formed by the cap structure.

Fig. 4.

(A) Hydrophobic interactions for the cap-helix domain. In the cap-helix domain most of the hydrophobic residues appear at the interface between two helices to form hydrophobic interactions. These side-by-side interactions of the cap-helix domain play key roles in self-assembly of the particle. (B) Intermolecular interactions between two half-vaults. N-terminal residues of domain 1, Met1-Glu4, formed an intermolecular antiparallel β sheet with those of the two-fold symmetry–related molecule. An ionic bond of Glu4-Arg42 was another specific interaction between two half-vaults. In contrast to the association of C-terminal cap domains, N-terminal associations were abundant in hydrophilic interactions.

A search for three-dimensional structures similar to the shoulder domain using the DALI server (27) revealed that the shoulder domain is structurally similar to the core domain of stomatin from Pyrococcus horikoshii (PhStoCD) (28) (PDB 3BK6) and the flotillin-2 band-7 domain (FlotBD7) (PDB 1WIN). The structure of the shoulder domain is superimposed on those of PhStoCD and FlotBD7 with RMSDs of 2.1 and 2.2 Å, respectively (fig. S6, A and B). The core domain of stomatin is evolutionarily conserved and falls within the stomatin-prohibitin-flotillin-HflK-C (SPFH) domain family (29). Although the physiological function of stomatin is not yet clearly understood, the SPFH domain is known to be involved in lipid raft association (30). Human stomatin, which has 40.3% and 18.4% sequence identities with PhStoCD and FlotBD7, respectively, is a major integral membrane protein of human erythrocytes. Podocin from mouse and mechano-sensory protein 2 from Caenorhabditis elegans have SPFH domains that bind cholesterol but not phosphatidylcholine (31). Cholesterol binding of the SPFH domain is likely an important factor in lipid raft association. Thus, the structural similarity between the shoulder domain and SPFH domain family supports the report proposing that MVP is recruited to lipid rafts, e.g., when human lung epithelial cells are infected with Pseudomonas aeruginosa (10).

Supporting Online Material

www.sciencemag.org/cgi/content/full/323/5912/384/DC1

Materials and Methods

Figs. S1 to S11

Tables S1 and S2

References and Notes

References and Notes

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