Figure 1: Model of paramyxovirus-mediated membrane fusion. A: Previous studies in the Lamb lab have shown the paramyxovirus fusion protein (F) adopts a series of conformations while mediating membrane fusion: the native structure (which is in a metastable conformation), a temperature-arrested intermediate (which forms after hemagglutinin-neuraminidase [HN] binds its receptor at nonfusion-permissive temperatures and is susceptible to N1 binding), a prehairpin intermediate (which adheres to target cells independent of HN and is susceptible to C1 binding), and the fusogenic form of F (which couples 6HB formation to membrane merger). The present work is consistent with residues 447 and 449 having distinct interaction sites on the F protein, one in the native structure and the other in the cavity formed by HRA trimers in the 6HB of the fusogenic form. The strong correlation between 6HB stability and C-peptide inhibitory potency in the present study and the coincidence of C1 inhibition and F protein binding in a previous study show that C1 inhibits membrane fusion by binding to transiently exposed HRA triple-stranded coiled coils in the prehairpin intermediate.
B: Biophysical studies on the N1/C-peptide mutants show that the aliphatic (al) and aromatic (ar) mutations at L447 and I449 decrease the amount of energy released by mutant 6HB formation; whereas, functional studies on the F mutants are consistent with the aromatic mutations decreasing and the aliphatic mutations increasing the activation energy of the native state. The hyperactive fusion phenotype of the aromatic mutants is consistent with the facile activation (and subsequent inactivation in the absence of a fusion target) of these F mutants overcompensating for the decrease in energy released by mutant 6HB formation.
From Russell, C.J., Kantor, K.L., Jardetzky, T.S., and Lamb, R.A. 2003. ;Journal of Cell Biology 163:363–374. © 2003 The Rockefeller University Press.
Figure 2: The structure of prefusion PIV5 (SV5) F protein. A: Schematic diagram of the F-GCNt domains. Important domains are colored, and their corresponding residue ranges are indicated.
B: Ribbon diagram of the F trimer, with each chain colored by residue number in a gradient from blue (N terminus) to red (C terminus). The head and stalk regions are indicated. HRB linker residues 429–432 could not be modeled in one subunit and had high-temperature factors in the other two.
C: Ribbon diagram of one subunit of the F trimer colored by domain. The domains are labeled, and the colors correspond to those used in Figure 1A. The cleavage/activation site is indicated with an arrow.
D: Top view of the trimer, colored as in A. Cleavage/activation sites are indicated by arrows.
E: Surface representation of the F trimer colored by subunit. Blue, the exposed surface of the fusion peptide.
F: Close-up view of the fusion peptide (residues 103–128). The peptide, which is folded back on itself with a small hydrophobic core, contains a mixture of extended chain, one ;b-strand, and a C-terminal ;a ;helix. The fusion peptide is sandwiched between two subunits of the trimer, between DII and DIII domains.
From Yin, H.S., Wen, X., Paterson, R.G., Lamb, R.A., and Jardetzky, T.S. 2006. Nature 439:38–44. © 2006 by Nature Publishing Group.
Figure 3: Structure of postfusion uncleaved soluble human parainfluenza virus 3 (hPIV3) F protein (solF0). A: Schematic of the domain structure of the hPIV3 solF0 protein. Domain regions are indicated with hPIV3 sequence numbers shown below and with colors corresponding to those used in 1B, D, and E.
B: Ribbon diagram of the hPIV3 solF0 trimer. The three chains are colored similarly from blue (amino terminus) to red (carboxyl terminus). Residues 95–135 are disordered in all chains. Residue 94 is labeled in one chain and residues 136–140 at the base of the stalk are ordered in one chain because of the crystal packing interactions.
C: Surface representation of the solF0 trimer. Each chain is a different color, and domains I–III and heptad repeat HRB for one chain (yellow) are indicated by labels. One radial channel is readily apparent below domains I and II of the yellow chain and above domain III of the red chain.
D: Ribbon diagram of the solF0 protein monomer colored by domain. The direct distance within one monomer between residue 94 at the end of HRC and residue 142 at the base of the stalk region is 122 Å.
E: Ribbon diagram of the monomer rotated by 90°, indicating the width and height of the solF0 monomer. An arrow at the carboxyl terminus of the HRB segment points toward the likely position of the transmembrane anchor domain that would be present in the full-length protein.
From Yin, H.S., Paterson, R.G., Wen, X., Lamb, R.A., and Jardetzky, T.S. 2005. ;Proceedings of the National Academy of Sciences USA ;102:9288–9293. © 2005 by the National Academy of Sciences.
Figure 4: Structural changes between the pre- and postfusion F protein conformations. A: Ribbon diagram of the SV5 F-GCNt trimer. DI, yellow; DII, red; DIII, magenta; HRB, blue; GCNt, gray.
B: Ribbon diagram of the hPIV3 (postfusion) trimer, colored as in A.
C: Ribbon diagram of a single subunit of the SV5 F-GCNt trimer, colored as in A, except for HRA residues (green).
D: Ribbon diagram of a single subunit of the hPIV3 F trimer, colored as in C.
From Yin, H.S., Wen, X., Paterson, R.G., Lamb, R.A., and Jardetzky, T.S. 2006. Nature 439:38–44. © 2006 Nature Publishing Group.
Figure 5: The role of DIII in HRA folding and transformation. A: HRA refolds from 11 distinct segments (h1, h2, b1, b2, h3, h4, and the intervening residues) in the prefusion conformation into a single ~120-Å-long helix in the postfusion form.
B: The HRA helices wrap around the domain III core in the prefusion conformation. The heptad repeat residues (magenta) do not form any coiled-coil interactions in the prefusion conformation. Breakpoints in the HRA helix (N133, T147, T158, and a stutter observed in the postfusion coiled coil) are labeled.
C: Secondary structure diagram for DIII in the prefusion (SV5) conformation. The DIII core includes three antiparallel strands: HRC, a helical bundle (HB), and h4 of HRA. HRA segments are colored as in A and the cleavage site (//) and fusion peptide (Fpep) are indicated. The DIII core sheet is extended by the b1 and b2 strands from HRA.
D: Secondary structure diagram for DIII in the postfusion (hPIV3) conformation, colored as in C. The DIII core sheet is extended by one strand from HRB linker from a neighboring subunit (dark violet).
E: Ribbon diagram of DIII in the prefusion conformation, colored as in C.
F: Ribbon diagram of DIII in the postfusion conformation colored as in D.
From Yin, H.S., Wen, X., Paterson, R.G., Lamb, R.A., and Jardetzky, T.S. 2006. Nature 439:38–44. © 2006 Nature Publishing Group.
Figure 6: Electron microscopy of F-GCNt and rosette formation. The majority of uncleaved F-GCNt trimers resemble a "ball and stem" (A). These F molecules are ~13 nm in length, and their shape resembles that of the atomic structure of F-GCNt . After cleavage with trypsin, the shape of the trimers was not altered noticeably (B).
When uncleaved F-GCNt was heated to 50°C, the trimers converted to a crutch-like shape (C), ~16 nm in length. Their shape resembled that of the atomic structure of postfusion hPIV3 F. The data indicate that, as for hPIV3 Fsol, PIV5 F-GCNt can refold into its postfusion conformation in the absence of cleavage.
When F-GCNt was treated with trypsin and then heated to 50°C (D) or when F-GCNt was heated to 50°C, cooled to 4°C, and then treated with trypsin (E), the trimers not only adopted a crutch-like shape but also organized into rosettes with the wide ends of the crutches oriented on the outside. These data suggest that the cleaved postfusion conformation F molecules aggregate through interactions of the fusion peptides. The data also indicate that order of performing heating and cleavage does not prevent prefusion F from adopting a postfusion protein conformation with release of the fusion peptide. For comparison, the morphology soluble hPIV3 F was also examined by electron microscopy.
Uncleaved hPIV3 F exhibited a crutch-like shape (F) similar to the PIV5 F-GCNt postfusion form (C). When uncleaved hPIV3 F was treated with trypsin to cleave F, rosettes formed with the head of the crutch outward (G). The shape of the hPIV3 F and the PIV5 F-GCNt rosettes was indistinguishable (E and G).
From Connolly, S.A., Leser, G.P., Yin, H.-S., Jardetzky, T.S., and Lamb, R.A. 2006. ;Proceedings of the National Academy of Sciences USA ;103:17903–17908. © 2006 National Academy of Sciences, U.S.A.
Figure 7: A model for F-mediated membrane fusion. A: Structure of the prefusion conformation. HRB, blue; HRA, green; domains I, II, and III are, respectively, yellow, red, and magenta.
B: An "open stalk" conformation, in which the HRB stalk melts and separates from the prefusion head region. HRB is shown as three extended chains because the individual segments are unlikely to be helical. This conformation is consistent with a low-temperature intermediate that is inhibited by HRA peptides, but not HRB peptides. Mutations of the switch peptide residues 443, 447, and 449 would influence the formation of this intermediate, by affecting stabilizing interactions between the prefusion stalk and head domains.
C: A prehairpin intermediate can form by refolding of DIII, allowing the formation of the HRA coiled coil and insertion of the fusion peptide into the target cell membrane. This intermediate can be inhibited by peptides derived from both HRA and HRB regions.
D: Prior to forming the final 6HB, the close approach of viral and cellular membranes may be trapped by folding of the HRB linker onto the newly exposed DIII core, with the formation of twob ;strands.
E: The formation of the postfusion 6HB is tightly linked to membrane fusion and pore formation, juxtaposing the membrane interacting fusion peptide and transmembrane domains.
From Yin, H.S., Wen, X., Paterson, R.G., Lamb, R.A., and Jardetzky, T.S. 2006. Nature 439:38–44. © 2006 Nature Publishing Group.
Figure 8: Atomic structure of the SV5 HN protein. Schematic cartoon diagrams show the top and side views of an SV5 HN monomer. Cylinders, helices; belted arrows, ;b ;strands. Blue, the N terminus; red, the C terminus.
From Yuan, P., Thompson, T.B., Wurzburg, B.A., Paterson, R.G., Lamb, R.A., and Jardetzky, T.S. 2005. Structure ;13:803–815. © 2005 with permission from Elsevier.
Figure 9: SV5 HN tetramers. Active sites are marked by space-filling representations of the ligand sialyllactose. The four subunits are shown in different colors. A: Top view. B: Side view.
From Yuan, P., Thompson, T.B., Wurzburg, B.A., Paterson, R.G., Lamb, R.A., and Jardetzky, T.S. 2005.Structure ;13:803–815. © 2005, with permission from Elsevier.
Figure 10: A model for HN tetramer rearrangement upon cell-surface receptor binding. The HN tetramer is primarily stabilized by the amino-terminal stalk region and can interact with F protein. Sialic acid receptors are displayed at the cell surface, where binding of the individual HN head domains could perturb the NA tetramer arrangement, consistent with the weak interactions between NA domains. Changes in the HN head domain tetramer could affect F interactions and stimulate membrane fusion.
From Yuan, P., Thompson, T.B., Wurzburg, B.A., Paterson, R.G., Lamb, R.A., and Jardetzky, T.S. 2005.Structure ;13:803–815. © 2005, with permission from Elsevier.
Figure 11: Model for activation of the M2 ion channel, showing only the transmembrane domain from residues 24 to 44 (green); the selectivity filter His37 (red) and the gate Trp41 (blue) are highlighted. For clarity these residues are shown for only two of the four subunits; the residues facing the viewer and the subunit closest to the viewer are omitted.
Upper panel, scheme for the closed wild-type M2 channel. The channel is closed when pHoutis high because His37 is not charged and Trp41 obstructs the pore near its cytoplasmic end.
Middle panel, the wild-type M2 channel in the open state. With low pHout, His37 is charged, allowing rotation of Trp41 to a conformation parallel to the pore's axis, permitting H+ ;to flow.
Lower panel, the M2-W41F mutant protein. In this case the smaller side chain of the Phe mutant permits passage through the pore regardless of pHout.
From Tang, Y., Zaitseva, F., Lamb, R.A., and Pinto, L.H. 2002. ;Journal of Biological Chemistry ;277:39880–39886. © 2002 American Society for Biochemistry and Molecular Biology.
Figure 12: Immunofluorescent staining of the influenza B virus BM2 integral membrane.
Cover image, ;Virology, February 1, 2003. © 2003, with permission from Elsevier. See also Paterson, R.G., Takeda, M., Ohigashi, Y., Pinto, L.H., and Lamb, R.A. 2003. ;Virology306:7–17.
Figure 13: Schematic diagram indicating the conservation of the influenza B virus BM2 transmembrane (TM) domain His and Trp residues as compared to residues in the influenza A virus M2 proton-selective ion channel protein. Histidine residues are colored red, tryptophan residues are colored green, and in BM2 the serine residues are colored magenta.
From Paterson, R.G., Takeda, M., Ohigashi, Y., Pinto, L.H., and Lamb, R.A. 2003. ;Virology ;306:7–17. © 2003, with permission from Elsevier.
