Portal de Periódicos da CAPES

Alternative intermolecular contacts underlie the rotavirus VP5* two- to three-fold rearrangement

2006; Springer Nature; Volume: 25; Issue: 7 Linguagem: Inglês

10.1038/sj.emboj.7601034

1460-2075

Joshua D. Yoder, Philip R. Dormitzer,

Virus-based gene therapy research

Article2 March 2006free access Alternative intermolecular contacts underlie the rotavirus VP5* two- to three-fold rearrangement Joshua D Yoder Joshua D Yoder Search for more papers by this author Philip R Dormitzer Corresponding Author Philip R Dormitzer Program in Virology, Laboratory of Molecular Medicine, Harvard Medical School, Children's Hospital, Boston, MA, USA Search for more papers by this author Joshua D Yoder Joshua D Yoder Search for more papers by this author Philip R Dormitzer Corresponding Author Philip R Dormitzer Program in Virology, Laboratory of Molecular Medicine, Harvard Medical School, Children's Hospital, Boston, MA, USA Search for more papers by this author Author Information Joshua D Yoder and Philip R Dormitzer 1 1Program in Virology, Laboratory of Molecular Medicine, Harvard Medical School, Children's Hospital, Boston, MA, USA *Corresponding author. Laboratory of Molecular Medicine, Harvard Medical School, Children's Hospital, Enders 673, 320 Longwood Avenue, Boston, MA 02115, USA. Tel.: +1 617 355 3026; Fax: +1 617 730 1967; E-mail: [email protected] The EMBO Journal (2006)25:1559-1568https://doi.org/10.1038/sj.emboj.7601034 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The spike protein VP4 is a key component of the membrane penetration apparatus of rotavirus, a nonenveloped virus that causes childhood gastroenteritis. Trypsin cleavage of VP4 produces a fragment, VP5*, with a potential membrane interaction region, and primes rotavirus for cell entry. During entry, the part of VP5* that protrudes from the virus folds back on itself and reorganizes from a local dimer to a trimer. Here, we report that a globular domain of VP5*, the VP5* antigen domain, is an autonomously folding unit that alternatively forms well-ordered dimers and trimers. Because the domain contains heterotypic neutralizing epitopes and is soluble when expressed directly, it is a promising potential subunit vaccine component. X-ray crystal structures show that the dimer resembles the spike body on trypsin-primed virions, and the trimer resembles the folded-back form of the spike. The same structural elements pack differently to form key intermolecular contacts in both oligomers. The intrinsic molecular property of alternatively forming dimers and trimers facilitates the VP5* reorganization, which is thought to mediate membrane penetration during cell entry. Introduction Cell entry by rotavirus is mediated by a series of molecular rearrangements and interactions that translocate a 710 Å diameter subviral particle across a cellular membrane and into the cytoplasm. The two outermost proteins of the nonenveloped virion, VP7 and VP4, are the moving parts of the translocation apparatus. VP7, a glycoprotein, forms calcium-dependent trimers, which pack together in the smooth outer shell of the virion (Yeager et al, 1990; Dormitzer and Greenberg, 1992; Prasad and Chiu, 1994). The dissociation of VP7 trimers in low calcium may underlie the controlled disassembly of the outer capsid during entry. VP4 forms spikes (Figure 1A), which protrude from the VP7 shell in electron cryomicroscopy image reconstructions of trypsin-primed virions (Shaw et al, 1993; Yeager et al, 1994). Trypsin primes rotavirus for efficient infectivity by cleaving VP4 into two fragments: VP8* and VP5* (Estes et al, 1981). VP8* forms the 'heads' of the spikes (Dormitzer et al, 2002) and binds sialic acid in some rotavirus strains (Ciarlet et al, 2002). Together with the N-terminus of VP8*, VP5* forms the spike 'body' (Dormitzer et al, 2004). The body is linked by an asymmetric stalk to a 'foot,' which is buried beneath the VP7 shell (Shaw et al, 1993; Yeager et al, 1994). Figure 1.Models of two VP4 conformations. (A) The primed state. Two rigid subunits form the spike visible in electron cryomicroscopy image reconstructions of trypsin-primed virions. A third subunit is flexible. VP8* is gray, with an N-terminal tether and a globular head creased by the sialoside-binding site. The VP5* antigen domain is green bean-shape, with a red membrane interaction region and a yellow GH loop. An additional β-strand C-terminal to the antigen domain is also yellow. The spike body includes the VP5* antigen domain, part of the VP8* tether, and the GH loop. The foot is blue, as is a protruding region that rearranges into the coiled-coil. (B) The putative post-membrane penetration state. VP8* has dissociated; the yellow parts of each subunit have joined in a β-annulus; the α-helical triple coiled-coil has zipped up; and the VP5* antigen domain has folded back. The models were produced by Digizyme, Inc. Download figure Download PowerPoint VP4 performs a series of molecular gymnastics during viral entry. Prior to trypsin cleavage, it is flexible (Crawford et al, 2001). The priming trypsin cleavage triggers its rearrangement into rigid spikes with approximate two-fold symmetry of their protruding parts (Shaw et al, 1993; Yeager et al, 1994; Figure 1A). After an unknown second triggering event, cleaved VP4 undergoes another rearrangement, in which two VP5* subunits fold back on themselves and join a third subunit to form a tightly associated trimer, shaped like a folded umbrella (Dormitzer et al, 2004; Figure 1b). VP8* probably dissociates from VP5* before or during the fold-back. The reorganization translocates the body domain's hydrophobic apex, which is a potential membrane interaction region, by at least 55 Å towards the molecule's base. The fold-back resembles those of enveloped virus membrane fusion proteins and may disrupt a cellular membrane. The primary evidence for the trimeric, folded-back state of VP5* is a 3.2 Å resolution X-ray crystal structure of VP5CT (Figure 2A), a protease cleavage fragment of rhesus rotavirus (RRV) VP4 that consists of residues A248 to L525 or F528 (Dormitzer et al, 2004). Key structural elements holding the trimer together include a C-terminal triple α-helical coiled-coil, a nine-stranded β-annulus, and a 'cap' of hydrophobic residues. Some electron cryomicroscopy evidence suggests that three VP4 subunits clustered at each peripentonal channel join to form the trimers. Although the molecular envelope reconstructed for the protruding part of the primed spike has approximate two-fold symmetry, the foot has apparent three-fold (or pseudo-hexameric) symmetry (Yeager et al, 1994). In addition, high pH treatment causes VP4 to coalesce into three foreshortened protrusions at each peripentonal channel (Pesavento et al, 2005). Therefore, the primed conformation of VP4 may include not only the rigid, two-fold clustered protruding spike but also a third protruding subunit, which remains flexible and, therefore, absent from averaged image reconstructions (Figure 1A). Figure 2.VP5CT and the VP5* antigen domain. (A) Ribbon diagram of the VP5CT trimer, colored to match Figure 1. VP5CT does not include the foot region. (B) Ribbon diagram of a single VP5CT subunit. The part that forms the VP5* antigen domain is green, yellow, and red; the remainder is drawn in outline. Download figure Download PowerPoint Each subunit of the VP5CT trimer contains a globular domain between residues R247 and D479 (Figure 2B). This globular domain does not include the coiled-coil and is missing one of the β-strands that each subunit contributes to the β-annulus. It does include the potential membrane interaction region and all known antibody neutralization escape mutations, including mutations selected by heterotypically neutralizing antibodies, on VP5* (summarized in Dormitzer et al, 2004). This domain also includes the minimal in vitro-translated fragment immunoprecipitated by neutralizing monoclonal antibodies recognizing VP5* (Mackow et al, 1990). Therefore, we refer to this region as the 'VP5* antigen domain.' Because VP5CT is stable, soluble, and presents heterotypic neutralizing epitopes, it might be considered a promising antigen for use in a recombinant vaccine against rotavirus, a pathogen that kills approximately 440 000 children annually (Parashar et al, 2003). However, this fragment is produced very inefficiently by proteolysis from VP4 (Dormitzer et al, 2001). Direct expression of a fragment equivalent to VP5CT yields insoluble protein (unpublished data). Examination of the VP5CT structure suggests that its N-terminal and C-terminal parts, which make trimer contacts, may only fold properly on an intact VP4 precursor, accounting for the insolubility of the directly expressed protein. We hypothesized that an engineered domain that lacks the residues that form the coiled-coil and β-annulus might be expressed directly and efficiently as a soluble protein. We have expressed the VP5* antigen domain in insect cells and report biochemical and X-ray crystallographic analyses of the purified protein. These analyses demonstrate that this autonomously folding domain self-associates in both dimers and trimers, with many of the same structural elements making key intersubunit contacts in both states. This intrinsic molecular property, together with the 'zipping-up' of a coiled-coil, underlies the molecular gymnastics of VP4 during cell entry. Results and discussion Expression and biochemical characterization We expressed the RRV VP5* antigen domain with an N-terminal histidine tag in bacteria and insect cells. When expressed in Escherichia coli, the domain is insoluble (data not shown). When expressed in Sf9 insect cells from a recombinant baculovirus vector, the domain is soluble and readily purified by nickel affinity chromatography and size exclusion chromatography, with anion exchange chromatography interposed when higher purity is needed (Supplementary Figure 1A and B). This procedure yields up to 4 mg of purified protein from each liter of insect cell culture. The purified domain is soluble to 4 mg/ml. SDS–PAGE, mass spectrometry, and N-terminal sequencing reveal some heterogeneity due to inconsistent cleavage of the histidine tag by adventitious proteases (Supplementary Figure 1B). The solubility of the directly expressed VP5* antigen domain suggests that further structure-based engineering may yield an optimized version of the domain that can be produced efficiently enough to be a practical immunogen for inclusion in a subunit rotavirus vaccine. Equilibrium analytical ultracentrifugation demonstrates self-association of the domain. The domain's hydrodynamic behavior can be modeled as a dynamic equilibrium between monomers and trimers with an association constant of between 18.7 and 62.5 (Supplementary Figure 1C). The domain's equilibrium distribution is similar at 4 and 22°C and at pH 5.6 and 8.0 (Supplementary Table). The hydrodynamic data cannot rule out the possibility that a small proportion of oligomers other than trimers are also present in solution, but the simple monomer–trimer equilibrium model is sufficient to fit the observed distributions closely. Crystal structure of the VP5* antigen domain dimer At 25°C and pH 5.6, the VP5* antigen domain crystallizes by the hanging drop method, using 2-methyl-2,4-pentanediol (MPD) as a precipitant. When frozen, these crystals diffract X-rays coherently to 1.5 Å interplanar spacing (although anisotropy limits the resolution of useable data to 1.6 Å). We determined the structure of these crystals by molecular replacement, using VP5CT as an initial phasing model (See Materials and methods and Table I). Table 1. X-ray diffraction data collection and refinement statistics Dimer Trimer Data collection statistics Resolution limit (Å) 1.6 2.0 Unique reflections 73654 55552 Redundancya 4.70 (4.36) 4.71 (4.56) Completenessa (%) 93.6 (69.9) 94.9 (97.0) I/σa 28.36 (4.54) 26.85 (4.25) Rsyma,b (%) 4.0 (29.4) 4.6 (40.8) Refinement statistics Polypeptide chains 2 3 Protein atoms 3535 5542 Water molecules 418 488 MPD molecules 4 0 Tris molecules 1 0 Residues in allowed regions of Ramachandran plot (%) 100 100 Residues in most favored regions of Ramachandran plot (%) 91.3 89 RMSD bond lengths (Å) 0.012 0.0078 RMSD bond angles (deg) 1.61 1.50 Mean B value (Å2) 32.0 40.1 RMSD main chain B (Å2) 2.20 1.52 Resolution range (Å) 45.5–1.6 50–2.0 R-factorc 20.3 22.2 Free R-factorc 22.5 25.2 aValues for last shell given in parentheses. bRsym=∑(I−〈I〉)/I. 〈I〉 is the average intensity over symmetry equivalent reflections. cR-factor=(∑∣∣Fobs∣−∣Fcalc∣∣)/∑∣Fobs∣, where the summation is over the working set of reflections. For the free R-factor, the summation is over the test set of reflections (5% of the total reflections). This VP5* antigen domain crystal structure reveals a dimer. Figure 3A shows the dimer in the orientation of the spike on a trypsin-primed virion. In this orientation, the heads would be located above the diagram, and the foot would be buried under the VP7 shell below the diagram. The dimer has maximal dimensions of 78 Å (height), 45 Å (width), and 30 Å (depth), as measured on a Cα trace. The N- and C-termini of each subunit are at the bottom, and three loops (B′C′, D′E′, and F″G′) with hydrophobic tips are at the top. In the dimer, the F″G′ loop, which resembles the Semliki Forest virus fusion loop in primary sequence (Mackow et al, 1988), is distal to the approximate two-fold axis, and the B′C′ and D′E′ loops are adjacent to this axis. Like the protruding VP4 dyad on virions (Shaw et al, 1993; Yeager et al, 1994), the crystallized antigen domain dimer is slightly asymmetrical. A highly ordered molecule of MPD (not shown) is bound between the sheets of the β-sandwich of the green subunit in Figure 3A, but is absent from the blue subunit. In solution at pH 5.6 and 22°C, the antigen domain is in a dynamic equilibrium, with monomers and trimers as the main species (Supplementary Table). The change in solvent characteristics caused by MPD and glycerol in the crystallization mother liquor or the asymmetry introduced by the insertion of an ordered MPD molecule into some molecules may alter the equilibrium in favor of dimer formation. Figure 3.Crystal structures of the VP5* antigen domain dimer and trimer. (A) Ribbon diagram of the dimer. The orientation matches the model of the rigid spikes on the virion in Figure 1A. The red, blue, and black boxes show the regions detailed in panels (C), (E), and (F), respectively. The termini of the green subunit are indicated. Secondary structural elements, including the three hydrophobic loops of the blue subunit are labeled. (B) Ribbon diagram of the trimer. The orientation matches the model of the rearranged trimer in Figure 1B. The red, blue, and black boxes show the regions detailed in panels (D), (G), and (H), respectively. The termini of the blue subunit are indicated. (C, E, F) Atomic details of the key intersubunit contacts of the dimer. Black ovals indicate the approximate two-fold axis. In panel (F), the L261 side chain is below the W262 side chain. (D, G, H) Atomic details of the key intersubunit contacts of the trimer. Black triangles indicate the three-fold axis. In panel (H), the W262 rings are seen on-edge with the five-atom pyrrole ring closest to the viewer. Panels (C)–(H) are drawn as if looking down from the tops of the ribbon diagrams in panels (A) and (B). Panels (C) and (D), panels (E) and (G), and panels (F) and (H) are pairs, showing alternative packing of the same residues. The depicted side chains are discussed in the text. Dashed black lines indicate hydrogen bonds. Download figure Download PowerPoint The contacts between subunits at the top of the dimer are insubstantial—salt bridges between E293 of one subunit and R341 of the other subunit and a hydrogen bond between the hydroxyl of S293 and its counterpart across the approximate two-fold axis (not shown). Instead, the dimer is primarily held together by extensive two-fold interactions near the bottom of the structure. These contacts include an intersubunit β-sheet (red box in Figure 3A and C) and a hydrophobic core (blue and black boxes in Figure 3A, E, and F). The central strands of the intersubunit β-sheet (strand G from each subunit) share eight backbone amide-to-backbone carbonyl hydrogen bonds and an additional hydrogen bond between the S412 side chain and its symmetry mate (Figure 3C). This interaction creates a continuous 10-stranded β-sheet (CDEHGGHEDC), which forms a saddle across the dimer interface (Figure 3A). The hydrophobic core on the two-fold axis is below the new β-sheet. One dimer contact is made by the aromatic ring of Y367 stacking against its symmetry mate (Figure 3E). Below this, two W262 aromatic rings pack tangentially against each other, separated at their distal ends by a sandwiched pair of L473 side chains (Figure 3F). At the bottom of the structure, the L261 side chain contacts its symmetry mate (Figure 3F). An additional intersubunit contact, formed by the N-terminus of one subunit (blue in Figure 3A) crossing over to pack as strand A against the other subunit, is discussed below under 'Comparison of VP5* subunit structures.' The dimer contacts bury 1846 Å2 (15.2%) of the surface of each subunit. Excluding the crossed-over N-terminus, 1371 Å2 (11.9%) are buried. Fit of the dimer to the body of the primed VP4 spike A 12 Å resolution electron cryomicroscopy image reconstruction of a trypsin-primed rotavirus virion provides a molecular envelope of the dimeric, protruding region of cleaved VP4 (Figure 4). Previously, we described the 'in silico' trimming and reorientation of two subunits of the VP5CT trimer to model a potential fit of a hypothesized dimeric arrangement of the subunits to this molecular envelope (Dormitzer et al, 2004). In that fit, the trimmed subunits were arranged as a parallel dyad (although they did not contact each other), with the hydrophobic apices occupying the 'shoulders' of the spike body and each F″G′ loop located distal to the approximate two-fold axis (Supplementary Figure 2). The dimeric crystal structure demonstrates that the VP5* antigen domain actually forms a dimer with parallel subunits in which the F″G′ loop of each subunit is distal to the two-fold axis (Figure 3A). The VP5* antigen domain dimer fits the electron cryomicroscopy envelope in the orientation reported for the models carved from the VP5CT structure (Figure 4). The gap between the subunits of the dimer in the crystal structure corresponds to the hole in the spike body, the hydrophobic apices correspond to the 'shoulders,' and the C-termini extend into the stalk to connect with the buried foot domain. Figure 4.The VP5* antigen domain dimer fit to the molecular envelope of the primed spike. The molecular envelope of an approximately 12 Å resolution electron cryomicroscopy image reconstruction of a VP4 spike on a trypsin-primed SA11-4F rotavirus virion is contoured at 0.5 σ. (A) Depicted from the perspective of Figures 1A and 3A. The Cα trace includes residues T259-N477 of one subunit (on the left) and residues T259-S476 of the second subunit (on the right). The termini and the F″G′ loop of one subunit are indicated. (B) Rotated 90° about a vertical axis from panel (A). Download figure Download PowerPoint Despite the good overall match of the dimeric crystal structure to the electron cryomicroscopy envelope, the fit is imperfect, with 25.6% of the atoms protruding beyond an envelope contoured at 0.5σ (Figure 4). In the previously reported fit of trimmed VP5CT subunits (Supplementary Figure 2, Dormitzer et al, 2004), which were not constrained in their orientation relative to each other by dimer contacts, 18.9% of the atoms protruded. Relative to the equivalent parts of the trimmed VP5CT subunits, the hydrophobic apices of the VP5* antigen domain dimer extend further beyond the spike shoulders. The dimer subunits are closer to the approximate dyad axis of the molecular envelope, leaving the lateral parts unfilled. Although not apparent from the perspectives in Figure 4, the flexible tip (with high thermal parameters) of each GH hairpin of the dimer protrudes from the molecular envelope. A shift in the GH hairpin (described under 'Comparison of VP5* subunit structures') from its position in VP5CT to form the central strands of the intersubunit β-sheet in the dimer fills in some of the envelope at the base of the body on the approximate dyad axis. The fit of the dimeric crystal structure to the molecular envelope could be improved if the dimer were extended about the 'joint' formed by its proximal two-fold contact so that the apices separated from each other. This distortion would produce a better match to the previously reported independent fit of each electronically trimmed subunit from VP5CT (Supplementary Figure 2). The imperfect match between the dimeric crystal structure and the molecular envelope of the primed spike probably reflects the conformational effects of interactions of the VP5* antigen domain with VP8* and with the stalk and foot regions of VP5* on the virion. In the primed spike, approximately 60 N-terminal residues of each VP8* fragment tether each head to the body (Figure 1A; Dormitzer et al, 2004). Many of these residues probably insert between the two hydrophobic VP5* apices to form the distal dyad contact of the spike body. The alternative possibility, that the VP8* N-termini fill the lateral part of the electron cryomicroscopy envelope, is less likely, as antibody neutralization escape mutations are broadly distributed over the surface of the VP5* antigen domain, indicating surface exposure on the virion (Dormitzer et al, 2004). Insertion of the N-terminal residues of VP8* into the insubstantial distal two-fold contact of the dimeric crystal structure would force the VP5* apices apart, producing the extension about the proximal two-fold contact that improves the fit to the envelope. Because the VP8* N-terminus forms the distal two-fold contact and tethers the heads to the body, VP8* probably either dissociates from the folded back, trimeric form of VP5* or has a substantially altered association. Early dissociation of the heads could allow the VP5* antigen domains to flex about their proximal two-fold contact and achieve the conformation in the dimeric crystal structure prior to trimerizing and folding back. Crystal structure of the VP5* antigen trimer During storage at 8°C at pH 8.0, the VP5* antigen domain crystallizes in batch without added precipitant. These crystals, when frozen, diffract X-rays coherently to 2 Å interplanar spacing. We determined the structure of these crystals by molecular replacement, using VP5CT as an initial phasing model (see Materials and methods and Table I). The structure reveals a VP5* antigen domain trimer (Figure 3B). Like the VP5CT trimer (Figure 2A), the VP5* antigen domain trimer is shaped like an umbrella. Unlike the VP5CT trimer, the VP5* antigen domain trimer lacks the coiled-coil or β-annulus that form the umbrella's 'post' due to the C-terminal truncation of the recombinant construct. In the VP5* antigen domain trimer, 1286 Å2 (11.0%) of the surface of each subunit are buried. The additional three-fold contacts of VP5CT raise its buried surface area to 3956 Å2 (25.8%). The tips of the globular 'shades' of the VP5* antigen domain umbrella approach each other more closely than those of VP5CT because they are not separated by a coiled-coil 'post.' Except for the position of the GH loop (discussed under 'Comparison of VP5* subunit structures'), the globular regions of the VP5* antigen domain and VP5CT trimers are essentially identical (RMSD of 0.76 Å for Cα of residues Y267-T410 and S423-L470). The fold-back rearrangement of VP5* (Figure 1) inverts the antigen domain of the trimer relative to the dimer. Correspondingly, in Figure 3, the dimer and the trimer are both depicted with the probable position of the foot at the bottom, but the subunits of the trimer are inverted relative to those of the dimer. In Figure 3B, the trimer is presented with each subunit's termini at the top and hydrophobic apex at the bottom. The maximal dimensions of the trimer are 74 Å (height) and 33 Å (radius), as measured on a Cα trace. The view of the blue subunit in Figure 3B displays features that are apparent at the 1.6 and 2.0 Å resolutions of the VP5* antigen domain structures, but not at the 3.2 Å resolution of the VP5CT structure (Dormitzer et al, 2004). Specifically, adjacent pairs of glycines interrupt the β-structure of the F′G loop, so that strand F′ from the VP5CT structure is divided into strands F′ and F″ (by G382 and G383) in the antigen domain structures, and strand G is divided into strands G′ and G (by G399 and G400). The more radial limb of the shortest loop at the bottom of the trimer structure (the loop that includes strand H′) had weak electron density in the VP5CT maps and was modeled as a coil (Figure 2A). In the new maps, these residues have strong density and form β-strand I (Figure 3B). Accordingly, VP5CT strand I becomes strand J, and VP5CT strand J becomes strand K in the antigen domain structures. The antigen domain trimer is held together by a hydrophobic core centered on the three-fold axis near the top of the structure. The packing of this hydrophobic core is the same in the VP5* antigen domain trimer and in VP5CT. Three main levels of hydrophobic interactions are apparent (red, blue, and black boxes in Figure 3B). At the bottom level (red box in Figure 3A and D), the F415 aromatic ring of each subunit packs about the three-fold axis and forms the base of a large solvent filled cavity (volume 390 Å3; Supplementary Figure 3). This cavity is also present in VP5CT and communicates with the molecule's exterior through narrow channels. During entry, it could allow room for movements associated with molecular rearrangements. The ceiling of the cavity is formed by Y367 aromatic rings (blue box in Figure 3B and G), which pack about the three-fold axis, reinforced by interdigitating V366 side chains. Above this (black box in Figure 3B and H), a tight 'propeller' of three W262 aromatic side chains is held together by interactions between the hydrogen bound to each pyrrole nitrogen and the π electrons of the adjacent indole ring. The propeller is reinforced by interdigitating L473 side chains. The packing of each L261 side chain against the L473 side chain of the adjacent subunit caps the hydrophobic core (Figure 3H). The structure of the VP5* antigen domain trimer has several implications for VP4 rearrangements. First, near the bottom of the structure, the subunits do not interact. In VP5CT, the interactions between the lower portions of each domain and the coiled-coil are polar. Thus, the lack of a central coiled-coil in the VP5* antigen domain trimer does not expose hydrophobic patches. The overall hydrophilicity of the sides (but not the apex) of the globular domain allows for its free rotation through solvent during the fold-back translocation. Second, alternative packing of the same residues in the dimer and the trimer allows the formation of two well-ordered oligomers. Residues L261, W262, Y367, and L473 make key hydrophobic contacts about both the two-fold axis of the dimer (Figure 3E and F) and the three-fold axis of the trimer (Figure 3G and H). Other residues, such as V366 (Figure 3E and G) and F415 (Figure 3C and D), only make intersubunit contacts in the trimer. Comparison of VP5* subunit structures Superposition of a subunit from the VP5* antigen domain dimer on a subunit from the VP5CT trimer (Figure 5) demonstrates that most of the globular domain remains rigid during the two- to three-fold reorganization (RMSD 0.85 Å for Cα of residues D270-Q360, A372-T410, and S423-L470). However, the GH loop rotates by approximately 61° relative to the rest of the domain, displacing its tip (residue D417) by 18.4 Å (two-headed arrow in Figure 5). In the dimer, the GH loop forms the central strands of the intersubunit β-sheet (Figure 3C); in the VP5CT trimer, this loop forms six of nine strands of the intersubunit β-annulus (Figure 2A). The shift of the loop disrupts the intersubunit β-sheet of the dimer and allows the F415 aromatic rings to pack around the three-fold axis of the trimer (Figure 3D). In VP5CT, hydrogen bonds in the β-annulus between strand G and an additional β-strand formed by residues just N-terminal to the coiled-coil substitute for the disrupted bonds in the intersubunit β-sheet and clamp the subunits in the folded-back conformation (Dormitzer et al, 2004). The shift in the GH loop is accompanied by the partial uncoiling of an adjacent, short α-helix (labeled 'α' in the green subunits of Figure 3A and B) between β-strands E and F. The GH loop of the VP5* antigen domain trimer does not form part of an intersubunit β-sheet or β-annulus, and its position may not model a conformation attained during the rearrangements that lead to cell entry. Figure 5.Superposition of the VP5* antigen domain and VP5CT. Residues S260-S476 of a VP5* antigen domain subunit in the dimer conformation are drawn as a green Cα trace. Residues I254-L522 of a VP5CT subunit are drawn as a blue Cα trace. The black arrow indicates the movement of the GH loop between the two states. Download figure Download PowerPoint Comparison of the VP5* antigen domain dimer and VP5CT trimer subunits also reveals significant rearrangements of the domains' termini (Figure 5). The C-termini of the dimer and trimer point in opposite directions, reflecting the fold-back relative to the foot domain. The N-terminus of one subunit of the dimer (the blue subunit in Figure 3A) crosses over to form intersubunit β-strand A, which is hydrogen bonded to strand B of the other subunit. The equivalent residues of the