REPORTS
`
`S5A). Hence, p53 phosphorylation in Ser9 and
`Ser6 serves as integration node in the cross-talk
`between Ras/MAPK and TGF-b.
`This prompted us to consider the possibil-
`ity that, although p53 is a ubiquitous protein,
`FGF might spatially pattern p53’s activity. In
`Xenopus, expression of different FGFs (eFGF,
`FGF3, and FGF8) is enriched in the marginal
`zone of the embryo, from which the mesoderm
`emerges, whereas lower FGF activity is present
`in the animal pole (10) (Fig. 3A). Using phos-
`phospecific antibodies, we found that kinase
`activities targeting Ser9 and Ser6 are localized in
`the marginal zone; in contrast, phosphorylation
`in other residues appears constitutive (Fig. 3B).
`To determine whether endogenous FGF sig-
`naling is responsible for this graded p53 phos-
`phorylation along the animal-vegetal axis,
`embryos were treated with the FGF-receptor in-
`hibitor SU5402 or injected with DN-Raf mRNA.
`Blockade of FGF signaling causes specific
`down-regulation of P-Ser9 and P-Ser6 (Fig. 3C).
`Conversely, ectopic FGF expression in animal
`cap cells specifically raises P-Ser6 and P-Ser9
`levels (Fig. 3D). Similarly, at the biochemical
`level, FGF is required for p53/Smad2 interaction
`because the formation of this complex is inhib-
`ited by SU5402 (fig. S6). However, introduction
`of Ser to Glu phosphomimicking substitutions
`in Ser6 and Ser9 (p53S6,9E), renders p53 able
`to complex with Smad2 in an FGF-independent
`manner (fig. S6). Together, the results indicate
`that FGF patterns the phosphorylation status of
`p53 in the embryo, restricting its cooperation
`with TGF-b to the prospective mesoderm.
`Next, we wished to gain insight into the ki-
`nase responsible for inducing p53 phosphoryl-
`ation in response to FGF/Ras/MAPK signaling.
`Both Ser6 and Ser9 conform to a CK1 consen-
`sus: There are seven mammalian CK1 genes,
`but p53 has been shown to associate specifically
`with CK1e and CK1d (11). In Xenopus em-
`bryos, inhibition of these kinases with dominant-
`negative CK1e (DN-CK1e) (12, 13) antagonizes
`FGF-mediated Ser6 and Ser9 phosphoryla-
`tion (fig. S7). Biologically, increasing levels of
`CK1e promote mesoderm induction in a p53-
`dependent manner (Fig. 3E and fig. S8); con-
`loss-of-CK1e by microinjection of
`versely,
`DN-CK1e or CK1e morpholino inhibits endog-
`enous and p53-mediated mesodermal gene ex-
`pression (Fig. 3, F and G, and fig. S9). Thus,
`CK1e lies downstream of FGF to promote p53
`phosphorylation and Smad cooperation in Xeno-
`pus mesoderm development.
`We next investigated the relevance of CK1e/d-
`mediated p53 phosphorylation on the activa-
`tion of the TGF-b cytostatic program in human
`cells. To this end, p53-reconstituted H1299
`cells were transfected with siRNAs to deplete
`endogenous CK1e and CK1d. CK1e/d knock-
`down leads to down-regulation of P-Ser6 and
`P-Ser9 levels (Fig. 3H) and to loss of TGF-b–
`mediated p21Waf1 induction (Fig. 3I, compare
`lanes 3 and 4 with lanes 7 and 8). By contrast, a
`
`phosphomimicking substitution of Ser9 with
`Glu (p53S9E) renders p53 able to sustain TGF-
`b–mediated p21Waf1 induction even in the ab-
`sence of CK1e/d (Fig. 3I, compare lane 4 with
`lane 8 and lane 6 with lane 10). Hence, p53S9E
`acts epistatically to CK1e/d. This indicates the
`key role of p53 N-terminal phosphorylation as
`mediator of the positive effect of CK1e/d in
`supporting TGF-b cytostatic responses.
`We have established a role for p53 as signal-
`ing integrator, outside of its widely investi-
`gated response to genotoxic stress (8). We provide
`evidence that p53 activity, rather than stability,
`can be qualitatively patterned by RTK/Ras-
`induced phosphorylation through CK1e/d. This
`phosphorylation step enables a robust biochemical
`interaction of p53 with TGF-b–activated Smads,
`leading to mesoderm induction in embryos and,
`in human cells, to the deployment of the TGF-b
`cytostatic program.
`These data establish a mechanistic link be-
`tween three key regulators of cell proliferation
`that are dysregulated in human cancers: Ras,
`p53, and TGF-b. This could provide an expla-
`nation for the p53-dependent tumor-suppressive
`function of Ras/MAPK reported in primary cells
`(14, 15). Activated Ras may well have general
`growth-promoting effects but, in the presence of
`wild-type p53, this would be balanced by the
`positive role played on p53/Smad cooperation
`that would sustain TGF-b growth control and
`thus limit neoplastic transformation.
`
`References and Notes
`1. L. Attisano, J. L. Wrana, Science 296, 1646 (2002).
`2. J. Schlessinger, Cell 103, 211 (2000).
`3. C. LaBonne, M. Whitman, Development 120, 463 (1994).
`4. R. A. Cornell, D. Kimelman, Development 120, 453 (1994).
`5. P. P. Hu et al., J. Biol. Chem. 274, 35381 (1999).
`6. R. T. Bottcher, C. Niehrs, Endocr. Rev. 26, 63 (2005).
`7. M. Cordenonsi et al., Cell 113, 301 (2003).
`8. A. M. Bode, Z. Dong, Nat. Rev. Cancer 4, 793 (2004).
`9. T. Mitsudomi et al., Oncogene 7, 171 (1992).
`10. C. LaBonne, B. Burke, M. Whitman, Development 121,
`1475 (1995).
`11. U. Knippschild et al., Oncogene 15, 1727 (1997).
`12. X. Zeng et al., Nature 438, 873 (2005).
`13. G. Davidson et al., Nature 438, 867 (2005).
`14. S. W. Lowe, E. Cepero, G. Evan, Nature 432, 307 (2004).
`15. E. Roper, W. Weinberg, F. M. Watt, H. Land, EMBO Rep.
`2, 145 (2001).
`16. We thank G. Bressan and D. Volpin for discussion. We also
`thank G. Del Sal, M. Mechali, D. Lane, C. Niehrs, J. Graff,
`D. Morrison, and K. Vousden for gifts of plasmids or
`antibodies, G. Blandino for the gift of p53-reconstituted
`cells, and O. Wessely for comments. This work is supported
`by grants to S.P. from Associazione Italiana per la Ricerca
`Sul Cancro, TELETHON-Italy GGP04030, MIUR (CoFin, FIRB),
`Agenzia Spaziale Italiana, Istituto Superiore di Sanita, and
`Swissbridge. A.M. is recipient of a European Union Marie
`Curie Research Training Network fellowship (Epiplast
`Carcinoma). M.C. was supported by a FIRC fellowship.
`Supporting Online Material
`www.sciencemag.org/cgi/content/full/1135961/DC1
`Materials and Methods
`Figs. S1 to S9
`References
`
`5 October 2006; accepted 22 December 2006
`Published online 18 January 2007;
`10.1126/science.1135961
`Include this information when citing this paper.
`
`Structure of the Prefusion Form
`of the Vesicular Stomatitis Virus
`Glycoprotein G
`Stéphane Roche, Félix A. Rey,* Yves Gaudin,† Stéphane Bressanelli
`Glycoprotein G of the vesicular stomatitis virus triggers membrane fusion via a low pH–induced
`structural rearrangement. Despite the equilibrium between the pre- and postfusion states, the
`structure of the prefusion form, determined to 3.0 angstrom resolution, shows that the fusogenic
`transition entails an extensive structural reorganization of G. Comparison with the structure of the
`postfusion form suggests a pathway for the conformational change. In the prefusion form, G has
`the shape of a tripod with the fusion loops exposed, which point toward the viral membrane,
`and with the antigenic sites located at the distal end of the molecule. A large number of G
`glycoproteins, perhaps organized as in the crystals, act cooperatively to induce membrane merging.
`
`The Rhabdoviridae are enveloped bullet-
`
`shaped viruses that are widespread among
`a great variety of organisms, including
`plants, insects, fishes, mammals, reptiles, and
`
`CNRS, Unité Mixte de Recherche (UMR) 2472, Institut Na-
`tional de la Recherche Agronomique (INRA), UMR 1157,
`Institut Fédératif de Recherche 115, Laboratoire de Virologie
`Moléculaire et Structurale, 91198, Gif sur Yvette, France.
`*Present address: Département de Virologie, Institut Pasteur,
`25 rue du Docteur Roux, 75724 Paris cedex 15, France.
`†To whom correspondence should be addressed. E-mail:
`gaudin@vms.cnrs-gif.fr
`
`crustaceans (1). This family includes vesicular
`stomatitis virus (VSV) as well as notable hu-
`man pathogens, such as rabies virus (RV) and
`Chandipura virus (2).
`The rhabdoviruses enter the cell via the
`endocytic pathway and subsequently fuse with a
`cellular membrane within the acidic environ-
`ment of the endosome (3). Both receptor recog-
`nition and membrane fusion are mediated by a
`single transmembrane (TM) viral glycoprotein
`(G) that is trimeric and forms the spikes that
`protrude from the viral surface. The large ecto-
`
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`REPORTS
`
`domain of G (446 out of 495 amino acids for
`the VSV Indiana strain) is also the target of
`neutralizing antibodies, and the antigenic sites
`of G in both VSV and RV have been described
`in detail (4–6).
`Similar to other viral fusion proteins, G un-
`dergoes a fusogenic structural transition during
`cell entry (7, 8). As for influenza virus hemag-
`glutinin (HA), flavivirus E protein, and Semliki
`Forest virus E1 protein,
`the conformational
`change is triggered at low pH (9). G can adopt
`at least three conformational states (7, 8, 10–14):
`the native prefusion state detected at the viral
`surface above pH 7; the activated hydrophobic
`state, which interacts with the membrane as a
`first step of the fusion process (11); and the
`fusion-inactive postfusion conformation that
`is antigenically distinct from both the native
`and activated states. There is a pH-dependent
`equilibrium between the different states of G
`that is shifted toward the postfusion confor-
`mation at low pH (15). Thus, unlike fusogenic
`proteins from other viral families, the native pre-
`fusion conformation is not metastable (9). In-
`deed, the reversibility of the low pH–induced
`conformational change is essential to allow G to
`be transported through the acidic compartments
`of the Golgi apparatus and to recover its native
`functional state at the viral surface (16).
`We have recently determined the low-pH
`postfusion three-dimensional structure of the
`VSV G ectodomain (residues 1 to 422), gener-
`ated by limited proteolysis of the virions with
`thermolysin (Gth) (17). In spite of having an
`unrecognized fold distinct from those of other
`fusion proteins previously described, the post-
`
`fusion conformation of G displays the classic
`hairpin conformation of other viral fusogenic
`proteins [i.e., an elongated structure with the
`fusion domain and the TM domain at the same
`end of the molecule (18)]. As in class I fusion
`proteins (19–21), the postfusion trimer displays
`a six-helix bundle with the fusion domains at
`the N terminus of the central helices and the TM
`domains at the C terminus of the antiparallel
`outer helices. Each fusion domain bears two fu-
`sion loops located at the tip of an elongated b
`sheet, which is a marked convergence with class II
`fusion proteins (22–24). Unexpectedly, G turned
`out to be homologous to glycoprotein gB of
`herpesviruses, the atomic structure of which was
`published at the same time (25). Because the low
`pH–induced conformational change of rhabdo-
`viral G is reversible, it remained unclear to what
`extent the pre- and postfusion conformations
`differed for this class of fusion proteins.
`Among the different crystal forms obtained
`with Gth (17) (see also the materials and meth-
`ods in the supporting online material), one of
`
`them, which was grown at pH 8.7, appeared to
`be particularly notable, because the asymmetric
`unit could not accommodate the postfusion form
`(125 Å in length) but was consistent with the
`presence of one protomer of the prefusion form
`[8.5 nm in length as measured for the RV G
`ectodomain by electron microscopy (EM) (26)].
`This crystal structure of Gth was determined to
`3.0 Å resolution by molecular replacement with
`the use of domains I, III, and IV (Table 1) of the
`low-pH form as search models. Data collection
`and refinement statistics are given in table S1.
`The structure of Gth is depicted in Fig. 1. Its
`length (88 Å), the location of the antigenic sites,
`and the comparison with the low-pH structure
`indicate that this Gth structure corresponds to the
`prefusion conformation of the molecule. The chain
`can be traced up to residue 413 (see the electron
`density for the final model in fig. S1). Clear
`density is also present for the first residues of
`both oligosaccharide chains (on N163 and N320)
`(27), which were disordered in the structure of
`the low-pH form.
`
`Table 1. Domain nomenclature used in the text. Root mean square deviation (RMSD) is between
`the pre- and postfusion structures. The number of alpha carbons (Ca) used in superposing the
`domains is indicated in parentheses.
`Domain
`Domain name
`Color
`DI
`Lateral domain
`Red
`DII
`Trimerization domain
`Blue
`DIII
`PH domain
`Orange
`DIV
`Fusion domain
`Yellow
`Cter
`C-terminal part
`Magenta
`RbI-II
`Rigid block
`-
`
`Residues
`1 to 17 and 310 to 382
`18 to 35, 259 to 309, and 383 to 405
`36 to 46 and 181 to 258
`53 to 172
`406 to 413
`1 to 25 and 273 to 382
`
`RMSD
`0.42 Å (80 Ca)
`-
`0.40 Å (82 Ca)
`0.77 Å (94 Ca)
`-
`0.56 Å (122 Ca)
`
`Fig. 1. Overall Gth
`structure in pre- and
`postfusion conforma-
`tions. (A) View of the
`G protomers superim-
`posed on their fusion
`domains (DIV) and col-
`ored by domain (as de-
`fined in Table 1) with
`the fusion loops in
`green. The two glycosyl-
`ated asparagines [N163
`(labeled “1”) and N320
`(labeled “2”)] are dis-
`played as dark green
`spheres. (B) View of the
`G trimers, colored by
`domains as in (A). The
`trimers were superim-
`posed on the rigid blocks
`made of DI and the
`invariant part of DII
`(RbI-II, defined in Table
`1 and highlighted in the
`boxed inset for one pro-
`tomer of each conformation). Helix E is indicated on both trimers. (C) Domain
`architecture of VSV G plotted on a linear diagram, color-coded according
`to Table 1 with domain boundaries numbered. The unobserved C-terminal
`
`segment is in gray, with a checkerboard pattern for the TM domain. The
`regions that refold in the transition are hatched. All structural figures were
`generated with PyMOL (38).
`
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`REPORTS
`
`The overall architecture of Gth in its prefusion
`state resembles a tripod (Fig. 1B). Each leg is
`composed of a fusion domain with the fusion
`loops pointing toward the viral membrane. The
`last residues that we can see (including the
`conserved H407 and P408) pack against the side
`of the fusion domain. This organization, which
`is reminiscent of the low-resolution structure of
`retroviruses’ envelope spikes that was recently
`determined by EM (28, 29), suggests that the
`TM segments are separate in the membrane.
`Nevertheless, we cannot exclude the possibility
`that the missing C-terminal segments of the ec-
`todomain (residues 414 to 446) that lead to the
`TM segments come together toward the three-
`fold axis.
`In the tripod arrangement, the fusion do-
`mains are set wide apart, keeping the fusion
`loops separate (Fig. 1B, left). In contrast to class
`I and class II fusion proteins, the fusion loops
`of G are not buried at an oligomeric interface
`in the prefusion conformation. The hydropho-
`bic residues Y116, A117, W72, and Y73 are ex-
`posed (Fig. 1, A and B), even though they
`cluster near crystal contacts (fig. S3D). The
`tips of the fusion domains are the most flexible
`part of the structure (fig. S4) and thus are the
`least well defined in the electron density maps.
`
`The conformational change involves a dra-
`matic reorganization of the G molecule. Figure
`S2 shows a comparison of the secondary struc-
`ture elements of the two conformations with their
`nomenclature. The pre- and postfusion states are
`related by flipping both the fusion domain and a
`C-terminal segment (composed of residues 383
`to 413) relative to a rigid block (RbI-II) made
`by the lateral domain and residues of the tri-
`merization domain that include helix F2 of the
`prefusion form (Table 1 and Fig. 1B, inset).
`During the structural transition, both the fusion
`loops and the TM domain move ~160 Å from
`one end of the molecule to the other. Thus, the
`observed conformational change, although re-
`versible, appears to be similar to that of para-
`myxovirus F glycoprotein (30). It also suggests
`that similar intermediates are formed during the
`fusion-associated refolding of G, HA, and para-
`myxovirus F glycoprotein (19, 30). In one of
`these intermediates (Fig. 2C and movie S1), the
`fusion domain is projected at the top of the spike,
`allowing the initial interaction with the target
`membrane.
`In spite of large rearrangements in their rela-
`tive orientation (Fig. 2, A and B), domains I, III,
`and IV retain their folded structure (Table 1 and
`Figs. 1A and 2). In this and the following para-
`
`graphs, we describe the conformational change of
`a protomer by considering RbI-II as invariant
`(Fig. 1B, inset). The flippings of both the fusion
`domain and the TM segment relative to RbI-II
`occur through a concerted rearrangement of dis-
`tinct regions of the molecule. Although we have
`only snapshots of the initial and final states,
`analysis of the two structures (see the descrip-
`tion of movie S1 in the supporting online ma-
`terial) suggests a plausible sequence of events
`leading from pre- to postfusion conformations.
`The fusion domain is projected toward the
`target membrane through the combination of
`two movements (Fig. 2C): a 94° rotation around
`the hinge between the fusion and pleckstrin ho-
`mology (PH) domains (Fig. 2A) and the reposi-
`tioning of the latter domain at the top of the
`trimerization domain (Fig. 2B). The rotation
`involves the reorganization of two segments
`(residues 47 to 52 and 173 to 180) of the poly-
`peptide chain. In the former segment, helix A0
`unfolds whereas, in the latter segment, helix C
`forms (Fig. 2A). Mutations M44 → V or I in
`RV G, which kinetically stabilize the native con-
`formation (31), map to this region. Their location
`suggests that they impede the slight distortion of
`strands b and j of the PH domain that accom-
`panies the movement.
`
`Fig. 2. Structural changes in the
`protomer between the pre- and
`postfusion conformations and rel-
`ative movements of domains. In
`(A) and (B), fragments of the pre-
`and postfusion conformations are
`displayed to the left and right, re-
`spectively. Secondary structure
`elements of the prefusion form
`that refold are named and num-
`bered according to fig. S2. (A)
`Relative movement of PH (DIII,
`orange) and fusion (DIV, yellow)
`domains. The protomers are super-
`imposed on DIII. Hinge residues 47
`to 52 (prefusion helix A0) and 173
`to 180 (postfusion helix C) are
`colored in cyan and gray-blue, re-
`spectively. (B) Domain II refolding.
`DI and DIII are omitted in the top
`panels for clarity but are shown in
`the bottom panels to provide the
`relative orientations in the two
`forms. The protomers are super-
`imposed on the invariant part of
`DII, which is indicated in dark
`blue, whereas the three segments
`that refold and/or relocate are
`indicated in shades of green. In
`the prefusion form, strands a1 and
`y1 form an interchain b sheet. The
`DIII-DIV hinge (bottom panels) is
`displayed and colored as in (A), with the two segments connected by a yellow
`bar to mark the location of the fusion domain. (C) Cartoon representation of
`the relative organization of domains with respect to the viral membrane
`during the conformational change. The one-sided black arrows indicate the
`relative movements of domains. The N- to C-terminal orientations of helices F2
`
`(blue; left), F (blue; middle and right), and H (dark blue; right) are indicated
`with white arrows. Pre- (left) and postfusion (right) conformations are shown.
`The trimer axes are indicated. The middle cartoon shows how the fusion loops
`(in green) would be projected after the refolding of both the DIII-DIV hinge and
`the DII-DIII connection and before the C-terminal refolding of helix H.
`
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`

`REPORTS
`
`The trimerization domain undergoes a major
`refolding event during the transition between the
`pre- and postfusion structures (Fig. 2B). This
`refolding drives the repositioning of the PH do-
`main and the flipping of the C-terminal part and
`involves all three segments of the trimerization
`domain (Fig. 1C).
`As a first step, central helix F2 (residues 276
`to 294) is lengthened by the recruitment of a
`segment (made up of residues 263 to 275) to
`form the long helix F. The second segment that
`refolds is composed of residues 26 to 35, which,
`in the prefusion conformation, is buried in a
`groove of RbI-II that is closed by residues 263 to
`275. A sharp bend is introduced right after the
`conserved motif C24P25: The peptide bond be-
`tween P25 and S26 flips, which redirects the poly-
`peptide chain at an 80° angle, and short helix A
`(residues 24 to 29) is formed. The conformation
`of short strand a1 (residues 22 to 24), involved in
`the interchain b-sheet a1y1 in the prefusion
`conformation, is unchanged, although it is not
`paired to strand y1 of the adjacent protomer in the
`postfusion conformation (Fig. 2B).
`The small b-sheet q1y2 of the native form is
`then broken, although the individual strands q1
`and y2 retain their b conformation in the post-
`fusion form, and residues 384 to 400 (including
`helices H1 and H2 and strand y1) refold into
`helix H. This helix then positions itself in the
`grooves of the central core in an antiparallel man-
`ner to form the six-helix bundle. This move-
`ment repositions the TM domains at the same
`end of the molecule as the fusion domains
`(Figs. 1B and 2B). Finally, residues 259 to 261
`and 403 to 405, which are distant by ~30 Å in
`the prefusion conformation, form sheet qz that
`zips together helices F and H in the postfusion
`state (Fig. 2B). Strands q and z are already in an
`extended b structure in the native conformation,
`primed to form sheet qz in the postfusion form.
`The buried interface between two subunits in
`the trimer is 1600 Å2 per protomer, as calculated
`by the Protein Interfaces, Surfaces, and Assem-
`blies server (32). This value is less than half of
`that of the buried interface in the low-pH form.
`This explains the increased stability of the
`oligomeric structure of G at low pH (8). The
`interactions between protomers are all located
`in domain II (fig. S5) but are different from
`those observed in the postfusion form (Fig. 3,
`A and B). Not only is prefusion helix F2 shorter
`than postfusion helix F, it is also tilted and its
`C-terminal end is kept away from the trimer
`axis (Fig. 3A). This results from repulsive
`forces between the carboxylates of the three
`E286 amino acids (Fig. 3C). In contrast to the
`postfusion form,
`the main contribution to
`trimer stability is not due to the central helix
`bundle but appears to come from interchain
`b-sheet a1y1 [which must break during the
`fusogenic transition, before the formation of
`helix H (Fig. 2B)] and its environment,
`burying 1250 Å2 per protomer (Fig. 3D). The
`conformational change occurs at
`the viral
`
`surface even in absence of the target mem-
`brane. This seems to be topologically im-
`possible without transient dissociation of the
`trimer. This hypothesis is in agreement with
`the large differences in the trimeric interfaces
`between the native and the postfusion con-
`formations of G.
`A number of the few conserved residues
`(fig. S2) are involved in key networks of in-
`teractions that are different in the two forms
`(Fig. 4). This set of residues includes amino
`acids D137, Y139, H407, and P408 that cluster to-
`gether in the postfusion conformation to stabi-
`
`lize b-sheet qz of the trimerization domain
`(Fig. 4B). In the prefusion conformation, the
`qz sheet does not exist: D137 and Y139 remain
`associated with the segment corresponding to
`the q strand and contribute to a network of
`hydrogen bonds that also involves conserved
`W236 of the PH domain (Fig. 4A, top). This
`network is disorganized during the rotation of
`the fusion domain relative to the PH domain
`(Fig. 2A). Conserved histidines—H407 [involved
`in a salt bridge with D137 in the low-pH structure
`(Fig. 4B)], H162 [previously shown to be in-
`volved in the interactions between fusion do-
`
`Fig. 3. The trimeric interface of the prefusion conformation [(A), (C), and (D)] as compared to that of
`the postfusion conformation (B). For clarity, only DII [the only domain involved in the interface in the
`prefusion conformation (fig. S5)] is represented, and the three protomers are colored in three shades of
`blue. Secondary structure elements that refold and/or relocate are labeled. (A) Top view (orientation as
`in fig. S5, looking down toward the viral membrane) of the trimeric interface of the prefusion
`conformation. The arrow indicates the viewpoint used in (D). (B) Trimeric interface of the postfusion
`conformation, superimposed on the invariant parts of DII in (A). The view therefore would now be from
`the membrane. (C) Zoom of image in (A) showing only the three helices F2 and the side chains involved
`in their interactions, which are colored by atom type (oxygen, red; nitrogen, blue; sulfur, yellow; carbon:
`green, magenta, or dark blue, depending on the protomer) and labeled. As in the postfusion state, V275
`and L279 contribute to hydrophobic stabilizing interactions at the center of the molecule, but L283 now
`makes a lateral interaction with I278. The three E286 amino acids in the center are 4 Å apart in native
`crystals. In theYbCl3-derivative crystal used for refinement of the model (table S1), they chelate an
`ytterbium ion (not shown), bringing their side-chain oxygen atoms within 3.5 Å. (D) Close-up view of
`the outer region of the prefusion trimeric interface seen from the side. Contact residues are colored as
`in (C), with main-chain atoms included only when they participate in the contacts. Besides the
`canonical hydrogen bonds of the b sheet, the interface is stabilized through extended van der Waals
`contacts and a hydrogen bond between the imidazole ring and the carboxyl group of T265 of the
`neighboring protomer. Finally, carboxyl groups of L384 and I387 make two hydrogen bonds with the
`guanidium group of R277 of the other chain. These three hydrogen bonds are displayed as magenta
`dashed lines.
`
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`mains in the low-pH conformation (17)], and
`H60—cluster together (H60 is absent in RV G,
`but H86, which corresponds to S84 in VSV G,
`replaces it) (Fig. 4A, bottom). Protonation of
`these residues at low pH is likely to destabilize
`the interaction between the C-terminal segment
`of Gth and the fusion domain in the prefusion
`
`conformation, priming the initial movement of
`the fusion domain toward the target membrane.
`Conversely, the acidic amino acids that were
`either buried at the trimer interface (D268) or
`brought close together (D274 with D395 and E276
`with D393) in the postfusion acidic conformation
`(17) are solvent-exposed in the prefusion state
`
`Fig. 4. Alternative net-
`works of conserved res-
`idues in the pre- (A)
`and postfusion (B) con-
`formations. The orien-
`tation is as that in Fig.
`2A. Conserved residues
`are displayed in stick rep-
`resentation (main-chain
`atoms are not shown un-
`less they participate in
`interdomain contacts).
`Hydrogen bonds are
`displayed as magenta
`dashed lines.
`[(A) and
`(B), top] Close-up views
`of the DIII-DIV connec-
`tion are shown. The pre-
`fusion hydrogen bonds
`of Y139 to the main
`chain of W236 are relo-
`cated to the postfusion
`qz sheet, whereas D137
`switches from making a
`bidentate hydrogen bond
`to the main chain to engaging in a salt bridge with H407. [(A), bottom] A close-up view of the prefusion
`DIV-Cter interface that has to be disrupted for DIV to move is shown. Note the cluster of conserved
`histidines, including H407.
`
`Fig. 5. Antigenic sites of Rhabdo-
`viridae mapped onto the surface of
`the pre- (A) and postfusion (B) VSV
`G trimers. Sites are colored on both
`forms and labeled on the form(s) in
`which they are recognized. VSV
`sites are labeled in bold, and RV
`sites are labeled in italics within
`parentheses. VSV sites A1 (residues
`37 to 38, corresponding to RV an-
`tigenic site II located on segments
`composed of residues 34 to 42 and
`198 to 200) and A2 (located at the
`surface of helix E indicated in Fig.
`1) are indicated in shades of red.
`The RV G site recognized by anti-
`body 17D2 (between residues 255
`and 270) is in orange. NS (extend-
`ing from amino acid 10 to 15) is in
`dark blue. VSV site B (extending
`from amino acid 341 to 347), cor-
`responding to RV G minor antigen-
`ic site a (amino acid 340 to 342),
`is in magenta.
`In the prefusion
`conformation, the cleft between DI
`and DIII
`is colored black.
`It
`is
`flanked by residues 331 and 334,
`in gray, whose counterparts in RV
`affect virulence.
`
`REPORTS
`
`(not shown). Thus, the histidines in the prefusion
`form and the acidic residues in the postfusion
`form appear to constitute two pH-sensitive mo-
`lecular switches.
`The major antigenic sites of rhabdoviruses
`are located in the lateral and PH domains (4–6)
`(Fig. 5). The accessibility of antigenic sites to
`antibodies has been studied in detail for RV G.
`Antibodies directed against RV G site II are un-
`able to recognize the protein in its low-pH con-
`formation (7, 15). Indeed, during the structural
`transition, this site moves from the top of the
`molecule to a less accessible location at the sur-
`face of the virus. Conversely, the N-terminal
`epitope of RV G (NS) is only accessible in the
`low-pH conformation at the viral surface (31).
`Finally, RV G minor site a is recognized in both
`conformations (7). As for monoclonal antibody
`17D2 (33) that binds only the prefusion confor-
`mation (34), its epitope is located in the segment
`of helix F that is unfolded in the native structure.
`The cellular receptor of VSV G has not been
`identified. Nevertheless, a canyon located be-
`tween the lateral and PH domains is exposed at
`the top of the molecule and could be involved in
`ligand binding (Fig. 5A). In support of this,
`residues 330 and 333 of RV G, which are
`involved in the recognition of the putative viral
`receptor p75 (low-affinity nerve growth factor
`receptor) (35) and which affect viral pathogenesis,
`align with residues 331 and 334 of VSV G, which
`are located at either end of the canyon.
`In a previous study, we estimated the mini-
`mal number of trimeric spikes involved in the
`formation of a RV fusion complex as about 15
`(15). At the viral surface, a local organization of
`the spikes resembling the P6 lattice found in the
`crystal (in which all
`the spikes are oriented
`identically, with the major antigenic sites ex-
`posed at their tops) (fig. S3) might organize the
`glycoproteins in an optimal manner for a con-
`certed conformational change. It might also fa-
`cilitate the formation of the initial intermediates
`on the fusion pathway. Indeed, the initial lipidic
`deformations leading to the formation of the
`stalk and the initial fusion pore (36) can form
`inside the inner rim of such a hexagon. Re-
`inforcing the idea that the P6 organization may
`reflect the structure of a fusion relevant complex,
`a local hexagonal lattice of spikes of similar
`dimensions has been observed at low temperature
`under mildly acidic conditions at the surface of
`some RV G mutants that were affected in the
`kinetics of their low pH–induced structural
`transition (31).
`It is often considered that fusogenic proteins
`drive membrane fusion by coupling irreversible
`protein refolding to membrane deformation (37).
`At least for rhabdoviral G, this is not the case.
`Rather, it appears that a concerted cooperative
`change of a large number of glycoproteins
`(perhaps organized in a hexagonal lattice, like
`the one present in the crystals) is used to over-
`come the high energetic barrier encountered dur-
`ing fusion.
`
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`REPORTS
`References and Notes
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`2. B. L. Rao et al., Lancet 364, 869 (2004).
`3. I. Le Blanc et al., Nat. Cell Biol. 7, 653 (2005).
`4. L. H. Luo, Y. Li, R. M. Snyder, R. R. Wagner, Virology
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`5. A. Benmansour et al., J. Virol. 65, 4198 (1991).
`6. S. B. Vandepol, L. Lefrancois, J. J. Holland, Virology 148,
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`7. Y. Gaudin, C. Tuffereau, D. Segretain, M. Knossow,
`A. Flamand, J. Virol. 65, 4853 (1991).
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`J. Virol. 67, 1365 (1993).
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`J. Biol. Chem. 270, 17575 (1995).
`12. B. L. Fredericksen, M. A. Whitt, Virology 217, 49 (1996).
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