letters to nature .............................................................. step process. It requires coordinated functions of many protein complexes that are conserved from yeast to humans. Screens in Structure of the ESCRT-II endosomal Saccharomyces cerevisiae have identified numerous genes required trafficking complex for the sorting and trafficking of proteins into the MVB. Mutations in these genes result in defects in the sorting of transmembrane Aitor Hierro1, Ji Sun2, Alexander S. Rusnak2, Jaewon Kim1, Gali Prag1, proteins, such as the G-protein-coupled receptor, Ste2p, from the Scott D. Emr2 & James H. Hurley1 plasma membrane to the vacuolar lumen by means of the MVB pathway. Thus far, more than 20 genes referred to as class E VPS 11,12 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive (vesicular protein sorting) genes have been identified . and Kidney Diseases, National Institutes of Health, US Department of Health and During MVB sorting, Hrs–Vps27 is first recruited to the early Human Services, Bethesda, Maryland 20892-0580, USA endosome by virtue of its FYVE domain interaction with PI(3)P 2Department of Cellular and Molecular Medicine and Department of Chemistry (refs 13–15) and its UIM (ubiquitin interacting motif) interaction and Biochemistry and Howard Hughes Medical Institute, University of California with ubiquitinated cargo. It then recruits the ESCRT-I complex at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0668, USA (composed of Vps23, 28, 37) to the membrane16. ESCRT-I recruits ............................................................................................................................................................................. the downstream ESCRT-II and ESCRT-III complexes9. After the The multivesicular-body (MVB) pathway delivers transmem- ESCRTs have been recruited to the endosome membrane, the brane proteins and lipids to the lumen of the endosome. The AAA-type ATPase Vps4 binds ESCRT-III and following MVB vesicle multivesicular-body sorting pathway has crucial roles in growth- formation catalyses the dissociation of the ESCRT protein com- factor-receptor downregulation1, developmental signalling2–4, 5 plexes in an ATP-dependent manner for further rounds of protein regulation of the immune response and the budding of certain sorting17. The MVB sorting process is topologically equivalent to the envelope viruses such as human immunodeficiency virus6. Ubi- 7,8 budding process for envelope viruses such as human immunode- quitination is a signal for sorting into the MVB pathway , which ficiency virus (HIV), equine infectious anaemia virus (EIAV) and also requires the functions of three protein complexes, termed Ebola, suggesting a potential role for the ESCRTs in this event. In ESCRT-I, -II and -III (endosomal sorting complex required for 7,9,10 fact, it has been shown that TSG101–Vps23p binds to the p6 transport) . Here we report the crystal structure of the core of domain of HIV-1 Gag protein and inhibition of this binding the yeast ESCRT-II complex, which contains one molecule of the inhibits viral budding6. The same inhibitory effect is seen with Vps protein Vps22, the carboxy-terminal domain of Vps36 and dominant negative SKD1, the mammalian Vps4 orthologue6. two molecules of Vps25, and has the shape of a capital letter ‘Y’. Here we report the 3.6 A˚ resolution structure of the quaternary The amino-terminal coiled coil of Vps22 and the flexible linker ESCRT-II complex containing Vps22, Vps36 and two Vps25 mol- leading to the ubiquitin-binding NZF domain of Vps36 both ecules. Apart from the two NZF domains of Vps36, nothing is protrude from the tip of one branch of the ‘Y’. Vps22 and Vps36 known about the structures of any of the ESCRTsubunits alone or in form nearly equivalent interactions with the two Vps25 mol- the complex. These structures, in conjunction with mutational ecules at the centre of the ‘Y’. The structure suggests how analyses, provide a framework for an initial understanding of the ubiquitinated cargo could be passed between ESCRT components mechanisms for ESCRT assembly and function. of the MVB pathway through the sequential transfer of ubiqui- The 3.6 A˚ crystal structure of the ESCRT-II core complex (Sup- tinated cargo from one complex to the next. plementary Table 1 and Fig. 1a) contains two molecules of Vps25, The MVB pathway delivers transmembrane proteins and lipids one molecule of Vps22 and one copy of the C-terminal 171 amino into small vesicles that invaginate into the lumen of the endosome. acid residues of Vps36. The observed stoichiometry of 1 Vps22: 2 MVBs then fuse with the vacuole/lysosome and release the vesicles Vps25: 1 Vps36 leads to a calculated molecular weight of 138 kDa into the lumen where they are degraded by the hydrolases contained for the intact complex. This is consistent with the 155 kDa estimated 1 in the vacuole/lysosome . Proteins that remain in the limiting on the basis of size exclusion chromatography of ESCRT-II purified membrane of the MVB are either delivered to the lysosomal/ from yeast9. The complex adopts the shape of a capital letter ‘Y’,with vacuolar limiting membrane or they are recycled to the Golgi overall dimensions of 120 £ 85 £ 52 A˚ (Fig. 1b). The base of the ‘Y’ complex or the plasma membrane. Therefore, the MVB sorting consists of one of the two molecules of Vps25; one of the branches of pathway plays a critical role in the trafficking of numerous cargo the ‘Y’ consists of the second molecule of Vps25, and the other proteins within the endosomal membrane system. branch is formed by a subcomplex consisting of Vps22 and the Sorting of proteins into the MVB pathway is a complex, multi- C-terminal domain of Vps36. The N terminus of Vps22 is a single a-helix that protrudes away from the tip of the ‘Y’ shape. Figure 1 Structure of the ESCRT-II complex. a, Electron density from solvent-flattened Figure 2 Winged helix folds in ESCRT-II. a, WH folds are highlighted in light and dark blue SIRAS map contoured at 0.7 j in the vicinity of the Vps25–Vps36 interface showing the (Vps22 WH1 and WH2), light and dark green (Vps25 WH1 and WH2), and orange and red refined structure in a stick model for Vps36 Tyr 555, Tyr 556 and Tyr 557. b, Overall (Vps36 WH1 and WH2); the rest of the structure is coloured grey. b, Superposition of WH2 structure of the complex: Vps22 is blue, Vps36 red and Vps25 green. of Vps22 and WH2 of Vps36. Colours are as in Fig. 1. NATURE | doi:10.1038/nature02914 | www.nature.com/nature 1 letters to nature Vps22 consists of a single N-terminal coiled coil followed by two C-terminal extensions and a short loop linking the two domains. winged-helix (WH) domains (Supplementary Fig. 1a). All but the Crystallization trials were initiated with a mixture of intact Vps22, first 19 residues are ordered. The N-terminal domain consists of Vps25 and Vps36. Upon microsequencing of redissolved crystals, it helices H2–H4, the C-terminal half of H1 and the N-terminal half of was determined that the crystallized complex contained a fragment H5. Helix H5 is shared between the helical domain and the first WH of Vps36 comprising residues 396–566. The cleavage is attributed to domain. WH domains take the form HSHHSWSW, where H trace amounts of protease contamination in the crystallized denotes a helix, S a b-strand and W a “wing”18, a loop inserted material. The proteolytic stability of the C-terminal portion of between or after the latter two of the three b-strands. Thus WH1 of Vps36 is consistent with the structural finding that it Vps22 consists of H5–H7 and S1–S3. Strands 1–3 form an anti- makes extensive interactions within the core of the complex. The parallel b-sheet with strand order S1-S3-S2. A one-turn helix N-terminal portion of Vps36 contains two copies of a NZF zinc- follows WH2 and immediately precedes the C terminus. finger motif. The second NZF finger of Vps36 binds ubiquitin and is The first helix projects 45 A˚ away from the core and forms a thought to be the main site of interaction between ESCRT-II and dimeric coiled coil with a symmetry-related Vps22 molecule. The ubiquitinated cargo proteins19. The proteolytic susceptibility of the presence of a coiled coil in this region was anticipated on the basis of region connecting the second NZF motif at residue 203 and the start sequence analysis9. We do not observe dimerization of ESCRT-II of the C-terminal domain at 396 suggests that a flexible linker complexes isolated from yeast9 or in solution (not shown), and connects these two domains. ESCRT-II dimerization is therefore unlikely to be the normal None of the three ESCRT-II subunits has discernable homology function of the Vps22 coiled coil. All of the ESCRT-III subunits between each other at the amino acid sequence level. However all contain predicted coiled coils. On the basis of predictions from three have in common a structure based on two copies of the WH sequence analysis, it was proposed that an interaction occurs fold (Fig. 2a). For example, the WH2 of Vps36 superimposes on the between a Vps22 coiled coil and coiled coils in the ESCRT-III WH2 of Vps22 with a root mean square deviation (r.m.s.d.) of 1.8 A˚ subunits9. over 59 Ca positions (Fig. 2b). The internal WH folds also super- Vps25 is the most flexible of the three subunits. Flexible internal impose well on each other. The WH1 and WH2 folds of Vps22 can loops between residues 56–70, 110–115, 146–149 and 162–164, as be superimposed with a r.m.s.d. of 1.9 A˚ for 51 Ca positions. In well as the C-terminal ten residues, could not be visualized in contrast to the similarity in the individual WH folds, the relative electron density.