Abstract
The Vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here, we present a 3.6 Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high affinity and five low affinity binding sites in vitro, consistent with conformational asymmetry induced upon ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring-structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.
Introduction
The ESCRT (Endosomal Sorting Complex Required for Transport) machinery composed of ESCRT-0, -I, -II, -III and the Vps4 (Vacuolar protein sorting 4) complex was originally discovered to catalyze plasma membrane receptor down regulation via multivesicular body formation1,2. Part of the ESCRT machinery including ESCRT-III and Vps4 are also recruited during cytokinesis3,4 and budding of some enveloped viruses to catalyze membrane fission5–7. ESCRT-III is thought to assemble into helical filaments within membrane necks of budding vesicles or viruses or the cytokinetic midbody in order to constrict it and set the stage for membrane fission8–10. Vps4, a member of the type-I or meiotic clade of AAA family of proteins (ATPase associated with various activities)11 disassembles ESCRT-III polymers from membranes12 which might be coupled to the membrane fission reaction itself13.
Proteins homologous to Vps4 and ESCRT-III are also present in the Crenarchaeota phylum of archaea where they catalyze final steps in cell division1415, virus budding16 and vesicle release17.
Vps4 contains an N-terminal MIT (Microtubule Interacting and Trafficking) domain that interacts with C-terminal peptide motifs termed MIM (MIT domain interacting motif) in activated eukaryotic and archaeal ESCRT-III proteins4. A linker region that varies in length between archaeal, yeast and mammalian Vps4 regulates its assembly and activity18 and connects to the large ATPase domain, followed by the small ATPase domain and a C-terminal helix that packs against the large subdomain19–21, thereby adopting a structure closely related to p9722 and spastin23. Nucleotide binds into a cleft between the large and the small ATPase domains and induces conformational changes24,25 associated with the ATPase reaction cycle26. Eukaryotic VPS4 has a β-domain inserted into the small ATPase domain19,20,24 that recruits the VPS4 activator Vta1p/LIP5 implicated in assembly and ATPase activity21,27–30. Crenarchaeal Vps4 lacks the β-domain29 and the regulatory factors are absent in Crenarcheota31. While human cells have two paralogs of vps4 (VPS4A and VPS4B)32, a single gene encoding Vps4 is present in archaea and yeast2.
Vps4 oligomerizes upon nucleotide binding12 like other AAA-type ATPases33. Exceptions are crenarchaeal Vps4, which can form dodecamers in the absence of nucleotides at non physiological protein concentrations29,34. Three cryo-electron microscopy structures of yeast Vps4 revealed two stacked ring structures with 12 or 14 subunits with substantial differences that might be attributed to different conformational states35–37. However, these structures were obtained with a catalytic inactive mutant (Glu233Gln) or in the presence of non-hydrolyzable ATP analogues. Recent work on archaeal and yeast Vps4 showed that wild-type proteins form hexamers in the presence of ATP, suggesting that the physiological oligomer is hexameric29. To date however only models of hexameric Vps4 exist that are based on the p97 hexamer structure22 and crystal packing contacts derived from 6-fold screw axis crystal packing interactions19,20,24,25.
Here, we characterized the oligomeric state of the Vps4 homologue from Metallosphera sedula (MsVps4) that belongs to the phylum Crenarchaeota. While MsVps4 forms dodecamers at non-physiological protein concentrations in the absence of nucleotides, a MIT domain deletion mutant (MsVps4ΔMIT) assembles only into hexamers in solution. The catalytic activity of full length MsVps4 is around 10 times higher than that of MsVps4ΔMIT at physiological concentrations, consistent with a role for the MIT domain in ring assembly. The crystal structure of MsVps4ΔMIT revealed an asymmetric hexameric ring structure present in the asymmetric crystal unit. Further conformational changes occur upon nucleotide binding as revealed by the structure in complex with ADP and Mg2+. The physiological relevance of two variable protomer interfaces was confirmed by site-directed mutagenesis in vitro and in vivo by probing oligomerization, ATPase activity, ESCRT-III disassembly and HIV-1 budding. We suggest that the structure may present a Vps4 intermediate state that is consistent with the conformational flexibility and asymmetry important for ATP-driven ESCRT-III disassembly.
Results
Oligomeric state of MsVps4 ΔMIT and enzymatic activity
Full length MsVps4 as well as Vps4 from Sulfolobus solfataricus (SsoVps4) and Acidianus hospitalis (AhosVps4) form concentration dependent dodecamers in the absence of nucleotides29,34 but their oligomeric state is reduced to hexamers in the presence of ATP or ADP29. In order to determine the role of the N-terminal MIT domain in oligomerization, we purified a MIT domain deletion mutant that retained the linker region connecting the MIT domain to the ATPase domain (Figure 1a and Supplementary Fig. 1). As for full length protein, MsVps4ΔMIT (residues 75 to 369) oligomerizes in a concentration-dependent manner (Supplementary Fig. 2) without a 75°C heating step applied during SsoVps4 and AhosVps4 purification29. The largest oligomers elute at 14.1 ± 0.1 ml from a Superose 6 size exclusion chromatography (SEC) column, at a position slightly bigger than the marker protein catalase (Fig. 1b). Chemical cross linking of MsVps4ΔMIT in the absence and the presence of AMP-PNP and Mg2+ revealed a band migrating at ~ 200 kDa indicating hexamer formation (Fig. 1b, right panel). This was confirmed by native mass spectrometry that produced a molecular weight (MW) of 201.56 ± 0.01 kDa (calculated MW 201.36 kDa) and a slightly larger MW (211.77 ± 0.01 kDa) in the presence of AMP-PNP and Mg2+(Fig. 1c). Although MsVps4ΔMIT hexamer formation can be observed at concentrations higher than 5–10 µM (Supplementary Fig. 2), no dodecamers are detected at concentrations up to 0.5 mM (20 mg/ml), corroborating the importance of the MIT domain for dodecamer assembly in vitro.
Comparison of the ATPase activity of full length MsVps4 versus MsVps4ΔMIT demonstrated that MsVps4ΔMIT is enzymatically active at 60 °C but requires a 40 times higher protein concentration. While full length MsVps4 was fully active at 0.25 µM with a turnover rate of ~16 ATP per minute, MsVps4ΔMIT attained similar activity at 10 µM (Fig. 1d). From these data we conclude that the presence of the MIT domain most likely favors ATP dependent oligomer formation and/or stabilization, but is not essential for ATPase activity.
Crystal structure of Vps4ΔMIT
The structure of MsVps4ΔMIT was solved by molecular replacement and SAD techniques and the model was refined to a resolution of 3.6 Å (Table 1; Supplementary Fig. 3a). The asymmetric unit of the P212121 space group contained one hexameric ring structure composed of the typical large ATPase domain and the small ATPase domain with its C-terminal helix packed against the large domain of the neighboring protomer (Fig. 2a). The β-domain inserted into a loop of the small ATPase domain in eukaryotic Vps4 (Figure 1a) is missing as in the related archaeal SsoVps4 homologue29. The 13 amino acid linker sequence plus 6 amino terminal residues that form a short β-strand (β’) in mammalian VPS4A and B are disordered in all protomers. The orientation of the large ATPase domain with respect to the small one is the same in all six protomers and their Cα atoms superimpose with a r.m.s.d. of 0.62 Å. The ring structure has two-fold symmetry where opposing protomers make similar interactions (Fig. 2a). Three different protomer-protomer interfaces were identified between molecules A–B, B–C and C–D (interface D–E is the same as A–B, E–F corresponds to B–C and F–A to C–D), which are generated by a 10 and 15° rotation of the large ATPase domains in the different dimers (Fig. 2b). Each protomer-protomer interface can be split in two interacting surfaces, the conserved hydrophobic (H) interface between the small ATPase domain and the large ATPase domain from the neighboring protomer (Fig. 2c) and the variable (V) interface between two large ATPase domains from neighboring protomers (Fig. 2d). The H interface is hydrophobic and most of the interactions are conserved in all six interfaces (Supplementary Table 1). The V interface is dominated by polar interactions which vary substantially between the three different interprotomer interfaces (Supplementary Table 1). However, key interacting residues within the V and H interfaces are conserved between archaeal and human Vps4 (Supplementary Fig. 1). Superpositioning of the hexamer on itself by stepwise 60° rotations reveals the extensive conformational space that the pseudohexamer can adopt via its V and H interface in the absence of nucleotide (Supplementary Movie 1).
Table 1.
Data collection | MsVps4ΔMIT | ADP/ MsVps4ΔL- MIT |
ADP/ MsVps4ΔMIT |
---|---|---|---|
Space group | P212121 | P65 | P65 |
Cell dimensions | |||
a, b, c (Å) | 99.70, 127.39, 191.23 | 65.27, 65.27, 112.5 | 151.19, 151.19, 98.59 |
α, β, γ (°) | 90, 90, 90 | 90, 90, 120 | 90, 90, 120 |
Resolution (Å) | 48–3.6 (3.8–3.6)* | 2.4 (2.49–2.40)* | 3.2 (3.4–3.2)* |
Rmerge | 9.1 (61.6) | 5.8 (63.1) | 14.4 (51) |
I / σI | 8.31 (1.76) | 16.7 (2.3) | 9.9 (3.1) |
Completeness (%) | 99.1 (99.6) | 99.7 (99.7) | 99.6 (99.8) |
Redundancy | 5.3 (5.5) | 4.1 (4.2) | 4.6 (4.7) |
Refinement | |||
Resolution (Å) | 3.6 | 2.4 | 3.2 |
No. reflections | 27368 | 10642 | 21217 |
Rwork / Rfree | 26.4/31.8 | 21.2/26.4 | 22.9/26.8 |
No. atoms | |||
Protein | 12875 | 2133 | 6528 |
Ligand/ion | 0 | 27 | 81 |
Water | 0 | 7 | 12 |
B-factors | |||
Protein | 142.61 | 55.8 | 53.5 |
Ligand/ion | 43.1 | 43.1 | |
Water | 41.7 | 21.2 | |
R.m.s deviations | |||
Bond lengths (Å) | 0.01 | 0.013 | 0.011 |
Bond angles (°) | 1.716 | 1.59 | 1.54 |
1 crystal was used for each structure.*Highest resolution shell is shown in parenthesis.
In order to probe the conformation upon nucleotide binding, hexameric MsVps4ΔMIT and a shorter MsVps4 construct lacking the linker region (MsVps4ΔL-MIT, residues 88–369) were crystallized in the presence of ADP and Mg2+. Both constructs crystallized with P65 helical symmetry (Table 1) with similar H interfaces as observed in the hexamer structure and a V interface close the to A–B interface. ADP is coordinated between the large and small ATPase domains (Fig. 3a; Supplementary Fig. 3b and c) and P-loop residues Gly141, Cys142, Gly143, Lys144 and Thr145 (located on the large ATPase domain) bind the α- and β-phosphate groups of ADP while Tyr282, Arg310 and Val103 (located on the small ATPase domain) make hydrogen bonds with the sugar and the base. The base is stacked between the small and large ATPase domain in a pocket composed of residues Met146, Leu278 and Ala306 (Fig. 3a). Comparison of the nucleotide-free apo structure present in the ring structure showed that ADP binding induced a 23° rotation of the small ATPase domain compared to the large one (Fig. 3b and Supplementary Movie 2). Aligning the large ATPase domain of MsVps4ΔMIT with those of yeast Vps4 bound to ATPγS and ADP and mouse SKD1/VPS4B in complex with ATP and ADP19,24,25 showed significant differences in the position of the small ATPase domain including α6. ADP bound to MsVps4 occupies a similar position as ATPγS bound to yeast Vps4p while ADP bound to yeast Vps4p penetrates less into the binding pocket (Fig. 3c). Substantial differences are also observed between all known Vps4 apo structures (Fig.3d)19,20,25,29,35, representing the overall conformational flexibility of the nucleotide-free structure.
The nucleotide binding cleft is open and accessible to nucleotides in all protomers of the apo structure. This is consistent with ITC experiments that demonstrated a 1:1 nucleotide binding ratio when ATP binding to MsVps4ΔMIT was determined at the MsVps4ΔMIT protein concentration that ensures nucleotide independent hexamer formation in vitro. The overall KD of ATP interaction is 3.3 µM (Fig. 3e). Measurement of ADP binding to MsVps4ΔMIT showed that the MsVps4ΔMIT hexamer binds 5 ADP with low affinity (KD 5.0 µM) and 1 ADP with higher affinity (KD 0.4 µM) (Fig. 3f; 0.3 mM MsVps4ΔMIT). Together, these data indicate that MsVps4 can bind six ATP with low affinity and six ADP in a cooperative way in vitro.
Interface residues important for Vps4 assembly and ATPase activity
In order to validate the interactions present in the MsVps4ΔMIT hexamer we mutated residues conserved between archaeal and eukaryotic Vps4 homologues. Because removal of the His-tag from wild type MsVps4 was rather inefficient, the experiments with the mutants were carried out with N-terminally His-tagged MsVps4. The oligomeric state of interface mutants was first tested by SEC. This revealed four main peaks corresponding to dodecamers, hexamers, dimers and monomers for wild-type MsVps4 (Figure 4a). Notably, His-tagged MsVps4 has an approximately 6 times higher ATPase activity compared to untagged MsVps4, indicating that the presence of the tag seems to facilitate activation. First, we changed Phe126 and Met318 from the conserved H interface to alanine (Fig. 2c). SEC analyses of MsVps4 Phe126Ala showed a single peak corresponding to a monomer (Figure 4a), which was confirmed by SEC-MALLS (Supplementary Fig. 4). This indicates that the Phe side chain is crucial for oligomer formation and/or stability. Consistent with the defect in assembly, the Phe126Ala mutant has no ATPase activity (Table 2). In contrast, the Met318Ala mutation in close proximity to Phe126 (Fig. 2c) produces hexamers, dimers and monomers upon SEC analysis, but dodecamer formation is impaired (Figure 4a). Hexamer formation was confirmed by SAXS analysis (Supplementary Fig. 5). MsVps4 Met318Ala revealed an Rg of 4.64 nM and a calculated Dmax of 17.2 nM. Because the Rg and Dmax are smaller for the MsVps4ΔMIT structure (4.29 nm and 13.65 nm, respectively), we conclude that the MIT domain is flexibly linked and detached from the ring in this construct. Its ATPase activity is also enhanced by a factor of 1.5 compared to wild type MsVps4 (Table 2) corroborating the proposal that dodecamer formation is not required for ATPase activity29.
Table 2.
MsVps4 and hVPS4 constructs | Domain | Elution volumes (ml) | Complex1 | ATPase2 | ESCRT-III3 | HIV-14 | |||
---|---|---|---|---|---|---|---|---|---|
MsVps4_wild-type | 11.6 | 13.4 | 15.8 | 17.1 | dodecamer | 100% | - | - | |
hVPS4B_E235Q +ATP | 14.1 | 17.8 | hexamer | - | - | - | |||
hVPS4B_wild-type +ATP/Mg | - | - | - | - | - | 100% | 100% | ||
hVPS4A_wild type | - | - | - | - | - | - | - | 100% | |
MsVps4_Phe126Ala | H | 16.4 | monomer | 0.0 ± 0.1% | - | - | |||
hVPS4B_E235Q_Phe160Ala + ATP | 14.7 | 16.7 | 17.9 | monomer | - | - | - | ||
hVPS4B_Phe160Ala + ATP/Mg | - | - | - | - | - | 0.0 ± 0.1% | Inhib. | - | |
hVPS4A_Phe153Ala | - | - | - | - | - | - | - | Inhib. | |
MsVps4_Met318Ala | H | 13.1 | 14.6 | 17.2 | hexamer | 154 ± 2,5% | - | - | |
hVPS4B_E235Q_Met350Ala + ATP | 14.3 | 16.6 | monomer | - | - | ||||
hVPS4B_Met350Ala + ATP/Mg | - | - | - | - | - | 0.0 ± 0.1% | ~20% | - | |
MsVps4_Ile89Glu | AAA | 12.6 | 15.8 | 17.5 | dodecamer | 85.6 ± 16.4% | - | - | |
hVPS4B_E235Q_Ile124Glu + ATP | 13.6 | 18 | hexamer | - | - | - | |||
hVPS4B_Ile124Glu + ATP/Mg | - | - | - | - | - | 8.10 ± 1.95% | Inhib. | - | |
hVPS4A_Val117Glu | - | - | - | - | - | - | - | 100% | |
MsVps4_Glu174Ala_Glu176Ala | V | 10.5 | 13.7 | 16 | 17.6 | dodecamer | 0.14 ± 0.1% | - | - |
hVPS4A_Glu204Ala_Glu206Ala | - | - | - | - | - | - | - | Inhib. | |
MsVps4_Arg259Ala_Arg260Ala | V | 13.7 | 15.4 | 17.3 | hexamer | 0.1 ± 0.02% | - | ||
MsVps4_Glu214Tyr | pore loop 2 | 10.7 | 13.5 | 15.9 | 17.6 | dodecamer | 7.9 ± 1.5% | - | - |
hVPS4A_Glu240Tyr | - | - | - | - | - | - | - | Inhib. | |
MsVps4_Ser159Ala | V | 11.9 | 14.6 | - | 17.4 | dodecamer | 14.5 ± 3.2% | - | - |
hVPS4B_Arg253Ala + ATP/Mg | V | - | - | - | - | - | 69.5 ± 5% | ~50% | - |
Main complex formed in vitro
ATPase activity ; reported ATPase values correspond to the mean ± SD of at least 3 distinct experiments carried out on 2 or more batches of MsVps4.
Disassembly of ESCRT-III CHMP2A-CHMP3 tubular structures
Inhibition (Inhib.) of HIV-1 budding
Within the variable V interface the conserved Glu176 hydrogen bonds to Ser169 only in the C–D dimer, but not in the other dimer interfaces (A–B and D–E) (Fig. 2d, Supplementary Table 1). A double mutation of Glu176Ala and Glu174Ala within this variable V interface produced dodecamers, hexamers, dimers and monomers by SEC (Fig. 4a), however, its ATPase activity was almost completely abolished (Table 2). Vice versa, a Ser169Ala mutant formed dodecamers and hexamers as analyzed by SEC (Fig. 4a), but still showed ATPase activity, suggesting that Glu174 is more important than Glu176 and its hydrogen bond to Ser169. Mutation of the two conserved Arg fingers, Arg259Ala and Arg260Ala, located within the V interface, abrogates dodecamer formation. However, the hexamers (and dimers and monomers) formed in vitro (Fig. 4a) have no ATPase activity (Table 2).
The N-terminal region comprising residues 88 to 95 is disordered in the ring structure, but form a short β’-strand and α-helix in the ADP-bound monomer structure, thus possibly acting as a flexible arm helping to reorient the MIT domain and its bound substrate during the ATP cycle. Mutation of Ile89 to Glu located in the β’-strand produced hexamers, but no dodecamers by SEC indicating a linker contribution in higher order assembly (Fig.4a). Although, the elution volume indicates a slightly larger hexameric structure, which is likely due to the displacement of the MIT domain thereby increasing the hydrodynamic radius of the complex. However, its ATPase activity was only slightly reduced (Table 2). Because substrate has been suggested to pass through the central pore of the ring structure, we mutated the pore loop 2 residue Glu214 to Tyr. SEC analysis revealed the same elution profile (dodedecamers, hexamers, dimers and monomers) as observed for wild-type MsVps4 (Fig. 4a), however, the ATPase activity was reduced to 8% of the wild type activity (Table 2). In summary, our mutagenesis data confirm the role of conserved key residues for MsVps4 assembly and demonstrate that ring formation is not sufficient for ATPase activity, which is coupled to pore loop 2 and V interface residues.
Because the above described key residues are conserved between archaeal and human VPS4 (A and B) (Supplementary Fig. 1, Supplementary Table 1), we mutated VPS4B homologous positions of three key residues that affect MsVps4 assembly and its ATPase activity. In order to test assembly of VPS4B in vitro in the presence of ATP, ATP hydrolysis must be inhibited. Therefore, the Phe160 (MsVps4 Phe126), Met350 (MsVps4 Met318) and Ile124 (MsVps4 Ile89) to alanine mutations were tested by SEC with a VPS4B Glu235Gln active site mutant. This showed that VPS4B Glu235Gln elutes as a monomer from a SEC column in the absence of ATP and as a hexamer in the presence of ATP (Fig. 4b). The Phe160 mutant is mostly monomeric, but still forms some higher order oligomers (Fig. 4b), which are absent in case of MsVps4. Met350, which is juxtaposed to Phe160 of the neighboring protomer in MsVps4 (Fig. 2c) and thus occupying the same hydrophobic pocket generated by hexamer assembly does no longer oligomerize (Figure 4b). Consistent with no significant oligomerization in the presence of ATP, both the Phe160 and Met350 mutant of wild type VPS4B have no ATPase activity (Table 2). Together, the data on the Phe160 and Met350 mutants indicate that these hydrophobic interactions are essential for VPS4B assembly and thus ATPase activity. The mutant Ile124Glu (MsVps4 Ile89) within the N-terminal β’-strand still assembled into hexamers, which elute slightly larger from a SEC column than the wild type (Fig.4b). However, in contrast to MsVps4 Ile89Glu, which still had ~ 85% ATPase activity, its ATPase activity is reduced to ~ 10% (Table 2).
Residues important for HIV-1 budding and ESCRT-III disassembly
To further confirm the physiological relevance of the two interfaces, we generated hVPS4A mutants and compared their effect on HIV-1 budding to the dominant negative effect of catalytic inactive VPS4A (GFP-hVPS4A-Glu228Gln) as described previously38. Over expression of the mutants that show no ATPase activity in the context of MsVps4 in vitro, Phe153Ala (MsVps4 Phe126), the double mutant Glu204Ala and Glu206Ala (MsVps4 Glu174 and 176) and the pore loop mutant Glu240Tyr (MsVps4 Glu214) in HIV-1 infected cells exerted a strong dominant negative effect on HIV-1 budding comparable to catalytic inactive hVPS4A-Glu228Gln. No or very small amounts of Gag are detected in the cell culture supernatant and Gag processing is affected as evident from the presence of CAp2 and CA cleavage products detected in cells (Fig. 5a and Supplementary Fig. 6). In contrast, the Val117Glu mutation had no effect on virus budding despite the detection of CAp2 in cells (Fig. 5a and Supplementary Fig. 6). Expression levels of GFP-hVPS4A wild type and mutants were comparable in these experiments (Fig. 5a and Supplementary Fig. 6). We conclude that the key residues identified in affecting ATPase activity in vitro are also important for hVPS4A function in HIV-1 budding. In addition, the mutation of Val117 to Glu that disrupted dodecamer but not hexamer assembly had little effect on its ATPase activity in vitro (Table 2, MsVps4Ile89Glu) and no influence on HIV-1 budding, further indicating that dodecamer assembly is not essential for its physiological function.
Because the dominant negative effect on HIV-1 budding observed with the above described mutants might be due to a defect in ESCRT-III disassembly, we tested a set of VPS4B mutants for their capacity to disassemble ESCRT-III CHMP2A-CHMP3 helical tubular structures in vitro as described before39. Fluorescein-labeled CHMP2A-CHMP3 tubes treated with wild type VPS4B and AMP-PNP (adenylyl-imidodiphosphate) and Mg2+ showed no changes in fluorescence emission intensity as a function of time (Fig. 5b). Dequenching, indicating disassembly of ESCRT-III CHMP2A-CHMP3, was measured as an increase in fluorescence emission when wild type VPS4B was added together with ATP and Mg2+ (Fig. 5b). As expected the catalytic mutant Glu235Gln did not disassemble the tubes, similar to the Ile124Glu and Phe160Ala mutants. The Met350Ala mutant had some disassembly activity and the Arg253Ala mutation within the V interface revealed an intermediate phenotype with ~50% disassembly activity over 20 minutes (Fig. 5b). We conclude that VPS4 catalyzed ESCRT-III disassembly requires conserved H and V interface residues.
Discussion
Here we report the crystal structure of the hexameric AAA-type ATPase Vps4 from M. sedula. Although several structures of Vps4 have been reported19,20,24,25,29,35, none presented the native oligomeric conformation that is required for enzymatic activity26. The structure reveals an asymmetric pseudohexamer with three sets of opposing protomers making three different dimer-dimer interactions with neighboring protomers. The same principles of asymmetry have been described for nucleotide-free and nucleotide-bound bacterial AAA-type ClpX ring structures with nucleotide-loaded and free protomers facing each other (Supplementary Fig.7)40,41. Although the MsVps4 structure is nucleotide-free, the conserved asymmetry indicates that opposing protomers may act together as reported for ClpX40,41.
AAA-type ATPases bind nucleotide at the interface between the large and small ATPase domain of one protomer and the large ATPase domain from an adjacent protomer, generating six binding sites26. ADP binding to MsVps4 induces rotation of the small ATPase domain with respect to the large domain as reported for other Vps4 structures19,24,25 and ClpX40,41. While this rotation is facilitated by a single coil linking the large and small ATPase domains of ClpX, the two MsVps4 domains are connected by the corresponding linker and the C-terminal helix 10, which might render the monomer more rigid. Notably, comparison of MsVps4 and ClpX demonstrates that nucleotide binding induces a much larger rotation of the small ATPase domain of ClpX than of MsVps4 (Supplementary Fig. 7). ClpX hexamers have been reported to bind four nucleotides in solution42, although all six sites can also be occupied41. The crystal structure of MsVps4 shows that all nucleotide binding sites are accessible and ITC analyses demonstrate that MsVps4 can bind six ATP molecules with low affinity at room temperature with no apparent cooperativity, similar to the p97-VCP D1 ring that also binds six ATP per hexamer43. ADP bound in a cooperative way to MsVps4 with at least one high affinity and five low affinity binding sites, at a MsVps4ΔMIT protein concentration that allows hexamer formation independent of nucleotide in vitro. Nevertheless, we cannot exclude the possibility that some hexamer assembly may have contributed to the ITC signal. MsVps4 nucleotide binding in vitro differs from data on ClpX, which was reported to have two high affinity ATP binding sites and two low affinity sites at the nucleotide loading step41. Because the structures of ClpX adopt asymmetric conformations with 4-loaded and 2-unloaded states, it was suggested that the reaction cycle might pass via a 4-loaded 2-unloaded state or alternatively via a 5-loaded 1-unloaded state40,41,44.
Vps4 ring assembly requires nucleotides20,29, although archaeal Vps4 forms ring structures under non-physiological protein concentration and temperature conditions29,34. However, MsVps4 ring assembly is also induced by nucleotides under physiological conditions, because we measure significant ATPase activity at 0.25 µM concentration when MsVps4 is monomeric. Because MsVps4 rings bind six nucleotides in vitro, we propose that binding of six nucleotides leads to assembly of the ring structure and might thus present the starting or an early intermediate conformation at the onset of the nucleotide reaction cycle. In case of eukaryotic VPS4, assembly is further regulated by Ist1 and Vta1 upon ESCRT-III interaction, two factors that are absent in Crenarchaea28,45.
Both full-length MsVps4 and MsVps4ΔMIT exert ATPase activity in vitro, which is substantial higher in the presence of the MIT domain, in contrast to data reporting increased ATPase activity in linker and MIT domain deletion mutants46. However, the linker region of eukaryotic Vps4 was also reported to be important for ring assembly and activity18. Our crystal structures reveal that the β’-strand at the N-terminus is ordered in the ADP-bound conformation and disordered in the nucleotide-free conformation, suggesting that this conformational flexibility might be important for MIT domain orientation. A mutation within the β’-strand abrogates MsVps4 dodecamer formation, but not hexamer formation and its ATPase activity is reduced. The corresponding VPS4B mutant also still forms hexamers, but its ATPase activity is greatly reduced consistent with no ESCRT-III disassembly activity. The MIT domain is necessary to assemble MsVps4 dodecamers as its deletion produces only MsVps4 hexamers, which are enzymatically active consistent with the proposal that the physiological unit of Vps4 is hexameric29. This is further validated by two MsVps4 mutants (Met318Ala and Ile89Glu) that are hexameric and have wild-type like or slightly reduced ATPase activities. SAXS analysis of the MsVps4 Met318Ala mutant suggests hexameric ring structures with the MIT domains detached from the ring by the flexible linker. Because the MIT domain auto-inhibits ESCRT-III engagement47 a conformation with the MIT domains assembled on top of the ring as observed by electron microscopy35–37 might constitute an intermediate state during the activation cycle. The question remains whether there is a physiological role for dodecameric Vps4 such as recruiting multiple Vps4 hexamers to the site of ESCRT-III function thereby accelerating ESCRT-III disassembly? Notably double ring structures of ClpA unfold proteins faster than single ring ClpX structures48.
The potential physiological relevance of the observed MsVps4 interfaces was confirmed by mutating V and H interface residues conserved between MsVps4, yeast Vps4 and human VPS4A and B. The effects of the mutations are twofold. They either influence VPS4 assembly leading to no or low ATPase activity or they have no affect on hexamer assembly, but are nevertheless physiologically inactive because the mutations disturb the ATPase cycle thereby inhibiting HIV-1 budding and ESCRT-III disassembly in vitro. The H interface mutant Phe126Ala (MsVps4) and its corresponding VPS4A and B mutants show defects in oligomerization, no ATPase activity, inhibit HIV-1 budding and ESCRT-III disassembly in vitro. The second H interface mutant Met318 (MsVps4), which form together with Phe126 a hydrophobic pocket is still hexameric and exerts ATPase activity, while its human VPS4B counterpart is defective in hexamer assembly and thus exerts no ATPase activity. It shows some residual ESCRT-III disassembly activity in vitro, which is consistent with the proposal that ESCRT-III polymers stimulate VPS4 assembly28. Reduced ATPase activity and hexamer formation in vitro were also reported for H interface mutants of SsoVps4 (Tyr121Asp and Phe328Ala)29. Notably, the effect of mutations within the V interface is similar in MsVps4 and human VPS4. A double mutation in the V interface (Glu174Ala, Glu176Ala) impaired the ATPase activity and blocked HIV-1 budding. Because the glutamate residues are ~ 22 Å distant from the active site glutamate 203, their substitution by alanine might inhibit the propagation of signals from one protomer to the next during the ATPase cycle. The arginine fingers (Arg259 and 260) have been implicated in nucleotide binding and ATP hydrolysis49. The structure shows that Arg259 makes different polar contacts depending on the dimer, while Arg260 has no clear stabilizing role in the unliganded MsVps4 structure. Nevertheless, the double mutation prevents dodecamer, but not hexamer formation. However, the hexamers lack ATPase activity in agreement with a previous report on SsoVps429. Pore loop 1 and 2 mutants have been implicated in exerting a dominant negative effect on HIV-1 budding20,25 and ESCRT-III disassembly46. Mutation of Glu214 (MsVps4) to Tyr had no effect on dodecamer and hexamer assembly, but reduced the ATPase activity to less than 10% and its corresponding mutation in VPS4A (Glu240Tyr) blocked HIV-1 budding almost completely, similar to the reported Glu240Ala mutation25. Recent work showed that this conserved glutamate residue affects substrate binding within the pore47. This is also consistent with the importance of pore loop residues for ESCRT-III unfolding when passing through the central pore50. The defect induced by the alanine and tyrosine mutations indicates that the negative charge of the glutamate is critical for the ATPase cycle and thus for ESCRT-III unfolding.
In summary, we identified several key residues that are located at the conserved V and H interfaces, far from the active site, but important for MsVps4 and human VPS4 function. Based on these observations we propose the following mode of action for Vps4. First, Vps4 assembles in the presence of ATP molecules into a stable asymmetric ring structure that might resemble our nucleotide-free structure. ATP hydrolysis then leads to different intermediate states including potentially one when the ring has one high affinity binding site and five low affinity binding sites for ADP (Figure 6). The different potential conformations adopted by the three opposing dimers coupled to the movement of the small ATPase domain upon nucleotide binding and release indicates how these conformational motions might be propagated throughout the ring structure. Key interface residues such as Glu174 and Glu176 (located on α3), the arginine finger and the pore loop residue Glu214 are important for transferring the ATPase activity from two opposing protomers to their neighboring set. This may generate flexing motions of the whole ring as suggested for ClpX40 whereby the nucleotide reaction cycle, free state, ATP binding, hydrolysis and ADP-bound states is converted into mechanical work41. The asymmetric structure of MsVps4 provides a snapshot of the conformational flexibility of a hexameric Vps4 AAA-type ATPase, which will help to understand its reaction cycle that is most likely similar to other asymmetric AAA-type ATPases functioning as proteases, helicases and unfoldases51–53.
Methods
Cloning, expression and purification of MsVps4 and hVPS4B
The gene encoding the full length MsVps4 or truncated constructs were amplified by PCR from genomic DNA and using High-Fidelity PCR master mix (Phusion, Finnzymes) and gene-specific primers encoding a Tobacco Etch Virus (TEV) site. Mutations were introduced by overlapping PCR. The resulting products were cloned into the pDEST-17 vector using the Gateway cloning system from Invitrogen and following the manufacturer’s instructions. The gene encoding the full length human VPS4B (hVPS4B) was cloned into the pProExHT vector. Mutations were introduced by overlapping PCR.The sequences of all constructs were confirmed by DNA sequencing.
Purification of MsVps4 constructs
The E. coli C41(DE3) strain was used as the host for protein expression. The cells were grown in LB medium supplemented with ampicillin (100µg/ml) to an absorbance of approximately 0.5 and expression was induced for 1 hour at 37 °C by addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). To obtain selenomethionine (Se-Met) incorporated MsVps4ΔMIT, cells were cultured in M9 medium supplemented with Se-Met (60 mg/l) and other natural amino acids. Induction conditions were the same as for the native protein. The cells were pelleted by centrifugation (5,000 × g, 20 min), resuspended in lysis buffer (50 mM Tris pH 8.8, 50 mM NaCl, 10 mM imidazole, 1% CHAPS and Complete Protease Inhibitor® (Roche Diagnostics)), then disrupted by sonication. After centrifugation (20,000 rpm, 30 min, 4°C) the bacterial lysate was applied onto a Ni2+-NTA column (Qiagen) for affinity purification. After extensive washings (50 mM Tris pH 8.8, 50 mM NaCl 50, 20 mM imidazole) the protein was eluted in presence of 300 mM imidazole. The fractions containing MsVps4ΔMIT were pooled and incubated overnight at room temperature with (Tobacco Etch Virus) TEV (0.2 mg /10 mg of protein) and dialyzed against 50 mM Tris pH 8.8, 50 mM NaCl. The TEV protease and uncleaved protein were removed by passing the solution through Ni2+-NTA column (Qiagen) and collecting the unbound protein. Cleaved MsVps4ΔMIT was then concentrated to 150–200 µM using an Amicon 400 concentrator with 30-kDa cutoff membrane and purified by anion-exchange chromatography (monoQ) and Superose 6 size exclusion chromatography (SEC) on an ÄKTA FPLC™ system (GE Healthcare, UK). The peak fractions were analyzed on 12% SDS-PAGE and Commassie blue staining and concentrated to 10 mg/ml for crystallization. Incorporation of Se-met was confirmed by mass spectrometry. Extensive crystallization trials that included 576 conditions in 96-well sitting-drop vapor diffusion format were performed on various constructs at the high-throughput crystallization facility of the EMBL Grenoble outstation. Following optimization, crystals used for diffraction studies were grown at 20°C in hanging drop vapour diffusion plates. Wild type and mutant MsVps4 were purified by the same protocol.
Purification of hVPS4B constructs
The E. coli C41(DE3) strain was used as the host for protein expression. The cells were grown in LB medium supplemented with ampicillin (100µg/ml) to an absorbance of approximately 0.8 and expression was induced over night at 20 °C by addition of 0.1 mM IPTG. The cells were pelleted by centrifugation (5,000 × g, 20 min), resuspended in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM imidazole and Complete Protease Inhibitor® (Roche Diagnostics)), then disrupted by sonication. After centrifugation (20,000 rpm, 30 min, 4°C) the bacterial lysate was applied onto a Ni2+−NTA column (Qiagen) for affinity purification. After extensive washing (50 mM Tris pH 8.0, 150 mM NaCl 50, 20 mM imidazole) the protein was eluted in presence of 300 mM imidazole. The fractions containing MsVps4ΔMIT were pooled and incubated overnight at room temperature with (Tobacco Etch Virus) TEV (0.2 mg /10 mg of protein) and dialyzed against 25 mM Hepes, 150 mM NaCl, 10 mM Imidazole. The TEV protease and uncleaved protein were removed by passing the solution through Ni2+-NTA column (Qiagen) and collecting the unbound protein. Cleaved MsVps4ΔMIT was concentrated to 80–150 µM using an Amicon 400 concentrator with 10-kDa cutoff membrane and purified by Superose 6 size exclusion chromatography (SEC) on an ÄKTA FPLC™ system (GE Healthcare, UK) in a buffer containing 25 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM, DTT.
Chemical cross-linking
Untagged MsVps4ΔMIT was purified by SEC (Superose 6) in 50 mM Hepes pH 7.5 and the fractions corresponding to oligomers were cross-linked for 20 minutes at room temperature with 0.05% glutaraldehyde, as reported for the yeast Vps4p complexes. Following addition of an equal volume of 1M glycine pH7 to quench the reaction, the sample was re-injected on the superose 6 and peak fractions were collected for mass spectrometry.
Mass spectrometry
Cross-linked samples were desalted and buffer exchanged using ultrafiltration (Vivaspin 500, Sartorius) with a 50 kDa cutoff, then diluted to a concentration of 5–8 µM in 250 mM NH4Ac at pH7. Protein complex ions were generated using a nanoflow electrospray (nano-ESI) source. Mass spectrometry analyses were carried out on a quadrupole time-of-flight mass spectrometer (Q-TOF Ultima, Waters Corporation, Manchester, U.K.). The instrument was modified for the detection of high masses. The following instrument parameters were used: capillary voltage up to 1.2–1.3 kV, cone potential = 40 V, RF lens-1 potential = 40 V, RF lens-2 potential = 1 V, aperture-1 potential = 0 V, collision energy = 20–40 V, and microchannel plate (MCP) = 1900 V. All mass spectra were calibrated externally using a solution of cesium iodide (6 mg/mL in 50% isopropanol) and were processed with the Masslynx 4.0 software (Waters Corporation, Manchester, U.K.).
ATPase activity
The ATPase activity of the different constructs was carried out at 60°C and according to the manufacturer's instructions using the QuantichromeTM ATPase/GTPase Assay Kit (BioAssay Systems). Solutions containing 0.1 µM – 10 µM MsVps4, 25 mM Hepes pH 7.5, 50 mM NaCl, 2 mM MgCl2 and 1mM ATP were prepared on ice. They were then incubated at 60°C for various times (1 to 30 min). For hVPS4B, solutions containing 10 µM hVPSB, 25 mM Hepes pH 7.5, 150 mM NaCl were prepared on ice, 10 mM MgCl2 and 1mM ATP were added at the start of the experiment. They were then incubated at 37°C for various times (10 to 30 min). The reaction was stopped on ice and reagent was added and incubated 30 min at room temperature. The complex formed by the reagent and free phosphate was detected by absorbance at 620 nm using a plate reader. A sodium phosphate standard curve was used to estimate the amount of phosphate released during ATP hydrolysis.
Isothermal titration calorimetry
The binding constants of ATP to MsVps4ΔMIT were measured by ITC (MicroCal Inc.). All samples used in the ITC experiments were dialyzed against 25mM Hepes pH 7.5, 50 mM NaCl and 2 mM MgCl2. The ITC measurements were performed at 15°, 20° and 25°C by making 28 injections (10 µl) of ATP or ADP to 1.4 ml of MsVps4ΔMIT. The concentrations of the MsVps4ΔMIT and ATP were 100 µM or 300 µM and 1 mM, respectively. Curve fitting was performed with the MicroCal Origin software. The experiments were done in triplicate.
ESCRT-III CHMP2A-CHMP3 polymer disassembly
CHMP2AΔC/CHMP3 polymers were assembled as described previously39. For amine specific labelling of the co-polymer, NHS ester (or succinimidyl ester) of Alexa Fluor® 488 (Life Technologies) was dissolved in anhydrous DMSO and added to the co-polymer solution in HBS. The reaction was carried out at 4°C over night at a final concentration of 2 mg/ml of protein and 300 µM of the fluorophore. The reaction mixture was centrifuged and the pellet was extensively washed in order to remove free fluorophore and resuspended in HBS. The efficiency of the labelling was estimated by measuring the absorbance of the protein at 280 nm (taking in account the absorbance of the dye at 280 nm) and the fluorescence of Alexa Fluor 488 at 495 nm and the estimated value was 1.5 dye per CHMP2AΔC/CHMP3 dimer. The disassembly of the polymer was monitored on a BMG FLUOstar OPTIMA Microplate Reader. The different constructs of hVPS4B (10 µM) were mixed with the CHMP2AΔC/CHMP3 polymers (3µM) in reaction volumes of 100 µl HBS. After about 150 seconds, ATP/Mg (1mM/10mM) was added to the mixtures containing hVPS4B wt, hVPS4B E235Q, hVPS4B I124E, hVPS4B M350A, hVPS4B F160A, and hVPS4B R253A. After about 150 seconds, AMPPNP/Mg (1mM/10mM) was added to the mixture containing hVPS4B wt. Emission intensity was measured at 520 nm as a function of time by exciting the Alexa 488 at 490 nm.
Mammalian expression plasmids and HIV-1 budding assay
The GFP-VPS4A mutations were generated via overlapping PCR and the correctness of the sequence was confirmed by DNA sequencing. To examine the effects of wild-type and mutated GFP-VPS4A on HIV-1 release, 293T (ATCC CRL-3216) cells (1.2 × 106) were seeded into T25 flasks and transfected 24 hr later using a calcium phosphate precipitation technique. The cultures were transfected with 1 µg Vpu-negative proviral DNA (HIV-1HXB2) together with expression vectors for wild-type or mutated GFP-VPS4A. The total amount of transfected DNA was brought to 8 µg with carrier DNA (pTZ18U). Twenty-four hours post-transfection, the cells were lysed in radioimmunoprecipitation assay buffer (140 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 1% NP-40, 0.5% sodium deoxycholate, 0.05% SDS), and the culture supernatants were clarified by low-speed centrifugation and passed through 0.45 µm filters. Virions released into the medium were pelleted through 20% sucrose cushions by ultracentrifugation for 2 hr at 27,000 rpm and 4°C in a Beckman SW41 rotor. Pelletable material and the cell lysates were analyzed by SDS-PAGE and western blotting, using the anti-HIV CA antibody 183-H12-5C to detect Gag proteins. GFP-VPS4A was detected using an anti-GFP antibody (Molecular Probes/Life Technologies).
SAXS analysis of MsVps4
Small Angle X-ray Scattering Experiment (SAXS) Data Collection and Analysis. MsVPS4 M318A was purified as described above (his tag was cleaved) and used for the SAXS experiments. SAXS experiments were performed at the BM29 BioSAXS beamline at the ESRF (Grenoble, France). Data were collected using an online size-exclusion chromatography (SEC). 500 µl of MsVPS4 M318A at 220 µM was injected on a superose 6 10/300 GL, equilibrated in 50 mM Tris pH 8.8 50 mM NaCl. Data corresponding to the elution peek of MsVPS4 M318A was analysed; the data was normalized to the intensity of the incident beam, the scattering of the buffer was subtracted and the resulting intensities were scaled for concentration. Data processing and analysis were performed using the ATSAS package54 and molecular weights were estimated55. The final merged scattering data were further evaluated using PRIMUS56. The isotropic scattering intensity I(q) was transformed to the distance distribution function P(r) using the program GNOM, which was also used to calculate the maximum dimensions of the particle, the Dmax57.
Size exclusion chromatography and multi-angle laser light scattering
SEC was performed with a superose 6. The column was equilibrated in 50 mM Tris pH 8.8 50 mM NaCl and MsVps4 Phe126Ala was injected at 200 µM. The experiment was performed with multi-angle laser light scattering (MALLS) using a DAWN-EOS detector (Wyatt Technology Corp., Santa Barbara, CA) and refractive index measurement using a RI2000 detector (Schambeck SFD). Light scattering intensities were measured at different angles relative to the incident beam, and analysis of the data was performed with the ASTRA software (Wyatt Technology Corp., Santa Barbara, CA).
Crystallization, data collection and structure determination
MsVps4ΔMIT was crystallized in 20 mM Tris, 50 mM NaCl, 0.2 M sodium malonate pH 7 and19% PEG 3350 by mixing 1 µl of the reservoir to 1 µl of protein at 10.8 mg/ml. The crystals were cryoprotected in the same conditions containing 25% ethylene glycol and flash cooled in liquid N2 before data collection. Se-Met derivatized crystals of MsVps4ΔMIT grew under the same conditions. A data set from a Se-Met crystal was collected at 0.97 Å wavelength to 3.6 Å resolution on the ID14-EH4 beam line at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Data were processed with the program XDS58 and the crystals belong to space group P212121 (Table 1).
The structure was solved by molecular replacement using the program Phaser59 and a Vps4 model generated with the Phenix Ensembler program60 using human Vps4 (PDB code 1XWI), yeast Vps4 (PDB code 2RKO), yeast Vps4 (PDB code 2QP9) and yeast (PDB code 3EIH) as input models. The molecular replacement model was confirmed by the single anomalous dispersion method employing the data set collected at the peak wavelength (0.9791 Å). The phases were improved and initial model building was performed with the program Crank261 using both the anomalous signal and the molecular replacement solution described previously. Further model building was completed manually with Coot62 and the model was refined with REFMAC63 to an Rfactor of 26.4% and Rfree of 31.8%. The model contains chain A residues 97 to 369, chain B residues 98 to 367, chain C residues 96 to 367, chain D residues 97 to 369, chain E residues 96 to 367 and chain F residues 96 to369. The N-terminal residues 75 to 95 were disordered in all protomers and 82.92/8.35 % of the residues are within the most favored and allowed regions of a Ramachandran plot64.
Crystallization and structure solution of MsVps4 in complex with ADP
Two constructs of MsVps4 were crystallized with ADP: MsVps4ΔMIT (residues 75 to 369) and MsVps4ΔL-MIT (residues 88 to 369). Final ADP/ MsVps4ΔL-MIT cocrystals were grown by mixing 1 µl of 8 mg/ml MsVps4ΔL-MIT, 5 mM ADP, 10 mM MgCl2, and 1 µl of the reservoir solution containing 40% (w/v) polyethylene glycol 200 (PEG200) and 100 mM Tris-HCl buffer (pH 8.8). Crystals were directly flash cooled in liquid N2 at 100 K. Data were collected on beamline BM14 at the ESRF at a wavelength of 0.9737 Å. Final ADP/ MsVps4ΔMIT cocrystals were grown by mixing 1 µl of 10 mg/ml MsVps4ΔMIT, 5 mM ADP, 5 mM MgCl2, and 1 µl of the reservoir solution containing 1,4 M sodium/potassium phosphate pH 5.6 (1.26 M sodium dihydrogen phosphate monohydrate and 0.14 M di-potassium hydrogen phosphate) and 7.5% (v/v) glycerol. Crystals were soaked in the reservoir solution supplemented with 25 % (v/v) glycerol and flash cooled in liquid N2 at 100 K. Data were collected on beam line ID23-2 at the ESRF.
Data were processed with the program XDS58. Both crystals belong to space group P65 but with different cell dimensions (Table 1). The structures were solved by molecular replacement using the program Phaser59 and the apo MsVps4ΔMIT model. The model was rebuilt using COOT62 and refined using REFMAC63, Phenix65 and PDB_REDO web server66. Statistics for data reduction and structure refinement are presented in Table 1. The models were evaluated by using COOT and Phenix. In both structures the nucleotides gave clear electronic density and were present in all MsVps4 subunits. For the ADP/ MsVps4ΔL-MIT complex, one molecule of ADP and one molecule of MsVps4ΔL-MIT containing residues 97 to 363 are present in the asymmetric unit. The structure was refined against 2.4 Å data to an R/Rfree of 20.6/26.6% and 98.1 /99.6 % of the residues are within the most favored and allowed regions of a Ramachandran plot. For the ADP/ MsVps4ΔMIT complex, three molecules of ADP and three molecules of MsVps4ΔMIT are present in the asymmetric unit. MsVps4ΔL-MIT chain A contains residues 97 to 366, chain B contains residues 88 to 366 and chain C contains residues 90 to 366. The structure was refined against 3.2 Å data to an R/Rfree of 22.9/26.8% and 96.9/99.9 % of the residues are within the most favored and allowed regions of a Ramachandran plot.
Structure analysis
Molecular graphics figures were generated with PyMOL (W. Delano; http://www.pymol.org). Further details are described in the supplementary information.
Supplementary Material
Acknowledgments
This work was initiated by the ANR (ANR-08-BLAN-0271) and supported by the ANR (ANR-12-BSV8-0004), by the NIH/NIAID grant AI029873 (H.G) and the Institut Universitaire de France (W.W.). G.E. was supported by a post-doctoral fellowship from the ANRS. We acknowledge the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) supported by the French Infrastructure for Integrated Structural Biology Initiative FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We further thank the ESRF-EMBL Joint Structural Biology Group for access and support at the ESRF beam lines, J. Marquez (EMBL) from the crystallization platform, M. Jamin (UVHCI) for SEC-MALLS analysis, L. Signor (IBS, Grenoble) and P. A. Klein (IBS, Grenoble) for mass spectrometry, N. Pannu (Leiden University) for help with Crank2 and J. Radzimanowski for help with SAXS data analysis. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 monoclonal antibody (183-H12-5C) from Dr. Bruce Chesebro and Kathy Wehrly.
Footnotes
Accession codes. Co-ordinates and structure factures have been deposited in the Protein Data Bank with accession IDs 4D80, 4D81, 4D82.
Author Contribution
P.R., H.G. and W.W. designed the experiments; P.R. carried out cloning; C.C. and P.R. performed protein purification and protein crystallization; X-ray diffraction data were collected by P.M., C.C. and W.W. Structure determination was performed by P.M., A. McC. and C.C.; C.C. and P.R. performed the ITC and ATPase assays. E.D. performed mass spectrometry analysis and G.E. protein quality control by electron microscopy. Y.W. performed the HIV-1 budding assay and C.C the ESCRT-III disassembly assay. W.W. wrote the manuscript with help from P.R.
Competing financial interests. The authors declare no competing financial interests.
References
- 1.Henne WM, Buchkovich NJ, Emr SD. The ESCRT pathway. Dev Cell. 2011;21:77–91. doi: 10.1016/j.devcel.2011.05.015. [DOI] [PubMed] [Google Scholar]
- 2.Babst M, Davies BA, Katzmann DJ. Regulation of Vps4 during MVB sorting and cytokinesis. Traffic. 2011;12:1298–1305. doi: 10.1111/j.1600-0854.2011.01230.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Agromayor M, Martin-Serrano J. Knowing when to cut and run: mechanisms that control cytokinetic abscission. Trends Cell Biol. 2013;23:433–441. doi: 10.1016/j.tcb.2013.04.006. [DOI] [PubMed] [Google Scholar]
- 4.McCullough J, Colf LA, Sundquist WI. Membrane fission reactions of the mammalian ESCRT pathway. Annu Rev Biochem. 2013;82:663–692. doi: 10.1146/annurev-biochem-072909-101058. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Votteler J, Sundquist WI. Virus budding and the ESCRT pathway. Cell Host Microbe. 2013;14:232–241. doi: 10.1016/j.chom.2013.08.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Martin-Serrano J, Neil SJ. Host factors involved in retroviral budding and release. Nat Rev Microbiol. 2011;9:519–531. doi: 10.1038/nrmicro2596. [DOI] [PubMed] [Google Scholar]
- 7.Usami Y, et al. The ESCRT pathway and HIV-1 budding. Biochem Soc Trans. 2009;37:181–184. doi: 10.1042/BST0370181. [DOI] [PubMed] [Google Scholar]
- 8.Fabrikant G, et al. Computational model of membrane fission catalyzed by ESCRT-III. PLoS Comput Biol. 2009;5:e1000575. doi: 10.1371/journal.pcbi.1000575. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Bajorek M, et al. Structural basis for ESCRT-III protein autoinhibition. Nat Struct Mol Biol. 2009;16:754–762. doi: 10.1038/nsmb.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Guizetti J, et al. Cortical Constriction During Abscission Involves Helices of ESCRT-III-Dependent Filaments. Science. 2011;331(6024):1616–1620. doi: 10.1126/science.1201847. [DOI] [PubMed] [Google Scholar]
- 11.Frickey T, Lupas AN. Phylogenetic analysis of AAA proteins. J Struct Biol. 2004;146:2–10. doi: 10.1016/j.jsb.2003.11.020. [DOI] [PubMed] [Google Scholar]
- 12.Babst M, Wendland B, Estepa EJ, Emr SD. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 1998;17:2982–2993. doi: 10.1093/emboj/17.11.2982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Weissenhorn W, Poudevigne E, Effantin G, Bassereau P. How to get out: ssRNA enveloped viruses and membrane fission. Current Opinion in Virology. 2013;3:159–167. doi: 10.1016/j.coviro.2013.03.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Lindas AC, Karlsson EA, Lindgren MT, Ettema TJ, Bernander R. A unique cell division machinery in the Archaea. Proc Natl Acad Sci U S A. 2008;105:18942–18946. doi: 10.1073/pnas.0809467105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Samson RY, Obita T, Freund SM, Williams RL, Bell SD. A role for the ESCRT system in cell division in archaea. Science. 2008;322:1710–1713. doi: 10.1126/science.1165322. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Snyder JC, Samson RY, Brumfield SK, Bell SD, Young MJ. Functional interplay between a virus and the ESCRT machinery in archaea. Proc Natl Acad Sci U S A. 2013;110:10783–10787. doi: 10.1073/pnas.1301605110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Ellen AF, et al. Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components. Extremophiles. 2009;13:67–79. doi: 10.1007/s00792-008-0199-x. [DOI] [PubMed] [Google Scholar]
- 18.Shestakova A, Curtiss M, Davies BA, Katzmann DJ, Babst M. The linker region plays a regulatory role in assembly and activity of the Vps4 AAA ATPase. J Biol Chem. 2013;288:26810–26819. doi: 10.1074/jbc.M113.497032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Inoue M, et al. Nucleotide-Dependent Conformational Changes and Assembly of the AAA ATPase SKD1/VPS4B. Traffic. 2008;9:2180–2189. doi: 10.1111/j.1600-0854.2008.00831.x. [DOI] [PubMed] [Google Scholar]
- 20.Scott A, et al. Structural and mechanistic studies of VPS4 proteins. EMBO J. 2005;24:3658–3669. doi: 10.1038/sj.emboj.7600818. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Azmi IF, et al. ESCRT-III family members stimulate Vps4 ATPase activity directly or via Vta1. Dev Cell. 2008;14:50–61. doi: 10.1016/j.devcel.2007.10.021. [DOI] [PubMed] [Google Scholar]
- 22.Zhang X, et al. Structure of the AAA ATPase p97. Mol Cell. 2000;6:1473–1484. doi: 10.1016/s1097-2765(00)00143-x. [DOI] [PubMed] [Google Scholar]
- 23.Roll-Mecak A, Vale RD. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature. 2008;451:363–367. doi: 10.1038/nature06482. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Xiao J, Xia H, Yoshino-Koh K, Zhou J, Xu Z. Structural characterization of the ATPase reaction cycle of endosomal AAA protein Vps4. J Mol Biol. 2007;374:655–670. doi: 10.1016/j.jmb.2007.09.067. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Gonciarz MD, et al. Biochemical and Structural Studies of Yeast Vps4 Oligomerization. J Mol Biol. 2008;384:878–895. doi: 10.1016/j.jmb.2008.09.066. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Hill CP, Babst M. Structure and function of the membrane deformation AAA ATPase Vps4. Biochim Biophys Acta. 2012;1823:172–181. doi: 10.1016/j.bbamcr.2011.08.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Lottridge JM, Flannery AR, Vincelli JL, Stevens TH. Vta1p and Vps46p regulate the membrane association and ATPase activity of Vps4p at the yeast multivesicular body. Proc Natl Acad Sci U S A. 2006;103:6202–6207. doi: 10.1073/pnas.0601712103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Shestakova A, et al. Assembly of the AAA ATPase Vps4 on ESCRT-III. Mol Biol Cell. 2010;21:1059–1071. doi: 10.1091/mbc.E09-07-0572. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Monroe N, et al. The oligomeric state of the active Vps4 AAA ATPase. J Mol Biol. 2014;426:510–525. doi: 10.1016/j.jmb.2013.09.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Vild CJ, Li Y, Guo EZ, Liu Y, Xu Z. A novel mechanism of regulating the ATPase VPS4 by its cofactor LIP5 and the endosomal sorting complex required for transport (ESCRT)-III protein CHMP5. J Biol Chem. 2015;290:7291–7303. doi: 10.1074/jbc.M114.616730. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Hobel CF, Albers SV, Driessen AJ, Lupas AN. The Sulfolobus solfataricus AAA protein Sso0909, a homologue of the eukaryotic ESCRT Vps4 ATPase. Biochem Soc Trans. 2008;36:94–98. doi: 10.1042/BST0360094. [DOI] [PubMed] [Google Scholar]
- 32.Scheuring S, et al. Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking. J Mol Biol. 2001;312:469–480. doi: 10.1006/jmbi.2001.4917. [DOI] [PubMed] [Google Scholar]
- 33.Lupas AN, Martin J. AAA proteins. Curr Opin Struct Biol. 2002;12:746–753. doi: 10.1016/s0959-440x(02)00388-3. [DOI] [PubMed] [Google Scholar]
- 34.Moriscot C, et al. Crenarchaeal CdvA forms double-helical filaments containing DNA and interacts with ESCRT-III-like CdvB. PLoS One. 2011;6:e21921. doi: 10.1371/journal.pone.0021921. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Hartmann C, et al. Vacuolar protein sorting: two different functional states of the AAA-ATPase Vps4p. J Mol Biol. 2008;377:352–363. doi: 10.1016/j.jmb.2008.01.010. [DOI] [PubMed] [Google Scholar]
- 36.Landsberg MJ, Vajjhala PR, Rothnagel R, Munn AL, Hankamer B. Three-dimensional structure of AAA ATPase Vps4: advancing structural insights into the mechanisms of endosomal sorting and enveloped virus budding. Structure. 2009;17:427–437. doi: 10.1016/j.str.2008.12.020. [DOI] [PubMed] [Google Scholar]
- 37.Yu Z, Gonciarz MD, Sundquist WI, Hill CP, Jensen GJ. Cryo-EM structure of dodecameric Vps4p and its 2:1 complex with Vta1p. J Mol Biol. 2008;377:364–377. doi: 10.1016/j.jmb.2008.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Garrus J, et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell. 2001;107:55–65. doi: 10.1016/s0092-8674(01)00506-2. [DOI] [PubMed] [Google Scholar]
- 39.Lata S, et al. Helical structures of ESCRT-III are disassembled by VPS4. Science. 2008;321:1354–1357. doi: 10.1126/science.1161070. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Glynn SE, Martin A, Nager AR, Baker TA, Sauer RT. Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine. Cell. 2009;139:744–756. doi: 10.1016/j.cell.2009.09.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Stinson BM, et al. Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine. Cell. 2013;153:628–639. doi: 10.1016/j.cell.2013.03.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Hersch GL, Burton RE, Bolon DN, Baker TA, Sauer RT. Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine. Cell. 2005;121:1017–1027. doi: 10.1016/j.cell.2005.05.024. [DOI] [PubMed] [Google Scholar]
- 43.Briggs LC, et al. Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase. J Biol Chem. 2008;283:13745–13752. doi: 10.1074/jbc.M709632200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Stinson BM, Baytshtok V, Schmitz KR, Baker TA, Sauer RT. Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX. Nat Struct Mol Biol. 2015;22:411–416. doi: 10.1038/nsmb.3012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Azmi I, et al. Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1. J Cell Biol. 2006;172:705–717. doi: 10.1083/jcb.200508166. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Merrill SA, Hanson PI. Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition. J Biol Chem. 2010;285:35428–35438. doi: 10.1074/jbc.M110.126318. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Han H, et al. Binding of Substrates to the Central Pore of the Vps4 ATPase is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs) J Biol Chem. 2015;290:13490–13499. doi: 10.1074/jbc.M115.642355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Olivares AO, Nager AR, Iosefson O, Sauer RT, Baker TA. Mechanochemical basis of protein degradation by a double-ring AAA+ machine. Nat Struct Mol Biol. 2014;21:871–875. doi: 10.1038/nsmb.2885. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Wendler P, Ciniawsky S, Kock M, Kube S. Structure and function of the AAA+ nucleotide binding pocket. Biochim Biophys Acta. 2012;1823:2–14. doi: 10.1016/j.bbamcr.2011.06.014. [DOI] [PubMed] [Google Scholar]
- 50.Yang B, Stjepanovic G, Shen Q, Martin A, Hurley JH. Vps4 disassembles an ESCRT-III filament by global unfolding and processive translocation. Nat Struct Mol Biol. 2015;22:492–498. doi: 10.1038/nsmb.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Thomsen ND, Berger JM. Running in reverse: the structural basis for translocation polarity in hexameric helicases. Cell. 2009;139:523–534. doi: 10.1016/j.cell.2009.08.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Forster F, Unverdorben P, Sledz P, Baumeister W. Unveiling the long-held secrets of the 26S proteasome. Structure. 2013;21:1551–1562. doi: 10.1016/j.str.2013.08.010. [DOI] [PubMed] [Google Scholar]
- 53.Zhao M, et al. Mechanistic insights into the recycling machine of the SNARE complex. Nature. 2015;518:61–67. doi: 10.1038/nature14148. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Konarev PV, Petoukhov MV, Volkov VV, Svergun DI. ATSAS 2.1, a Program Package for Small-Angle Scattering Data Analysis. Journal of Applied Crystallography. 2006;39:277–286. doi: 10.1107/S0021889812007662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Putnam CD, Hammel M, Hura GL, Tainer JA. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys. 2007;40:191–285. doi: 10.1017/S0033583507004635. [DOI] [PubMed] [Google Scholar]
- 56.Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. appl. Cryst. 2003;36:1277–1282. [Google Scholar]
- 57.Svergun DI. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 1992;25:495–503. [Google Scholar]
- 58.Kabsch W. Xds. Acta Crystallogr D Biol Crystallogr. 2010;66:125–132. doi: 10.1107/S0907444909047337. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.McCoy AJ, et al. Phaser crystallographic software. J Appl Crystallogr. 2007;40:658–674. doi: 10.1107/S0021889807021206. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60.Wang X, Snoeyink J. Defining and computing optimum RMSD for gapped and weighted multiple-structure alignment. IEEE/ACM Trans Comput Biol Bioinform. 2008;5:525–533. doi: 10.1109/TCBB.2008.92. [DOI] [PubMed] [Google Scholar]
- 61.Skubak P, Pannu NS. Automatic protein structure solution from weak X-ray data. Nat Commun. 2013;4:2777. doi: 10.1038/ncomms3777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486–501. doi: 10.1107/S0907444910007493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63.Murshudov GN, Vagin AA, Dodson EJ. Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Crystallographica Section D. 1997;53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
- 64.CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 1994;50:157–163. doi: 10.1107/S0907444994003112. [DOI] [PubMed] [Google Scholar]
- 65.Adams PD, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66:213–221. doi: 10.1107/S0907444909052925. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 66.Joosten RP, Long F, Murshudov GN, Perrakis A. The PDB_REDO server for macromolecular structure model optimization. IUCrJ. 2014;1:213–220. doi: 10.1107/S2052252514009324. [DOI] [PMC free article] [PubMed] [Google Scholar]
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