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Are fusion peptides really “sided” insertional helices?
Cell, Vol. 70, 531432,
August 21, 1992, Copyright
0 1992 by Cell Press
Letter to the Editor
Are Fusion Peptides Really “Sided” Insertional Helices? Membrane fusion is a widespread and essential biochemical process critical for endocytosis, exocytosis/secretion, entry of enveloped viruses into susceptible cells, and polykaryocytosis occurring during myogenesis and virusinduced pathogenesis. Specifically, virus-induced fusion has been localized to hydrophobic sequences within transmembrane fusion proteins termed “fusion peptides” (White, 1990). The mechanism of action of such fusion peptides has long been of intense interest. While current evidence is strong that fusion peptides contact the lipid environment of target membranes (Harter et al., 1988, 1989; Stegmann et al., 1991) to produce hexagonal lipid structures of negative curvature (Ellens et al., 1989) the mechanism remains unknown. In a recent description of a possible fusion-inducing protein from sperm, Blobel et al. (1992) alluded to the most commonly advanced hypothesis that fusion peptides act as “sided” insertional helices. Such “sided” helices with an asymmetric concentration of bulky, hydrophobic residues, as found in the fusion peptides of a paramyxovirus and of human immunodeficiency virus (HIV), have a favorable free energy for insertion of the more hydrophobic side at a shallow angle into a lipid bilayer (Brasseur et al., 1988). The hypothesis has been experimentally supported: the periodicity of labeling of the influenza virus fusion peptide with a photoactivatable, lipid-embedded reagent has been purported to follow the 3-4 residue repeat for a-helical turns (Harter et al., 1989). More indirectly, mutations affecting the theoretical angle of insertion for the HIV fusion peptide inhibit fusion (Horth et al., 1991). We agree that many viral fusion peptides can be drawn as a “sided” helix. However, in examining the expanding data base of fusion peptides, we have observed a number of properties discordant with this hypothesis. First, several putative retroviral fusion peptides present a completely hydrophobic face over only three helical turns, and bulky hydrophobic amino acids are also on the less hydrophobic side. Second, the most highly conserved amino acids do not uniformly fall on the more hydrophobicside and, particularly among the alphaviruses, are preponderantly glycine and proline. Finally, the frequent presence of the strong helix-breaking residues, glycine, proline, and serine, even in pairs, makes it difficult to project some fusion peptides (such as that of an alphavirus [Levy-Mintz and Kielian, 19911 or the sperm fusion protein PH-30a [Blobel et al., 1992)) as helices by algorithms of protein structure. These discordant notes caused us to reexamine the labeling data of Hatter et al. (1989), which are the sole direct experimental findings supporting this hypothesis. In Figure lA, the data are plotted on a helical wheel, in which amino acids in an a helix rotate 100° per residue, generating a residue per 20° increment in projection down the
helical axis. While there is concentration of label in the direction of the helical moment (O”) about the hemicylinder of 290°-70°, there is also label on the other side (150°1 70°) and gaps in labeling on the more hydrophobic side, particularly at 31 O ” and 330°.
120,
(10
100 . 80
88
3
6: . 0 -4 I;,
60 40
2
20 I
i
d
lb amino
i0 acid position
3b
C
0
s cpm x 10
8
10
-2
Figure 1. Labeling of Influenza A Fusion Peptide as a Function Helical Wheel Position and Side Chain Volume
of
(A) The relative distribution of radioactive photolabel from 3-(trifluoromethyl)-3-([‘251]-iodophenyl) diazirine to amino acids of the aminoterminus of influenza A virus hemagglutinin 2, as reported by Harter et al. (1969) is plotted as a function of position on a helical wheel. Numbers indicate position in the sequence; the amino acid at each position is denoted by its single-letter abbreviation. Arrow, direction of helical moment (0.26/residue). (B) The distribution of label over the 22 aa sequence (closed symbols) co-plotted with the residue volume of each amino acid (open symbols). Residue volume was determined by measuring the aqueous displacement of space-filling models of tripeptides Gly-X-Gly, where X is the residue in question and then subtracting the volume of the dipeptide Gly-Gly. (C) A scatter plot of label as a function of residue volume.
An alternate explanation may lie in reactivity to the phospholipid-embedded reagent. In this and other studies using carbene-generating photolabeling, glycine and alanine have labeled very poorly (Hoppe et al., 1984; Brunner at al., 1985; Meister et al., 1985). This is in accord with a carbene philicity scale for such photolabeling, to which it has been noted these data generally conform (Sigrist et al., 1990). We have explored thevolume occupied by each residue as a variable in reactivity. When label and amino acid residue volume are plotted by the linear position of each residue in the peptide sequence (Figure 1 B) or in a scatter plot of label versus residue volume (Figure lC), the degree of labeling is directly proportional to residue volume. A more cogent explanation of the findings of Harter et al. (1989) would be that the entire sequence is in contact with lipid, rather than existing as a sided structure. We conclude that, while these data demonstrate insertion of the 22 residue amino-terminal fusion peptide into the hydrophobic lipid environment, they cannot be taken in support of the “sided” helical hypothesis. Also contravening the hypothesis, circular dichroism of the measles fusion peptide immersed in a lipid environment shows 73% 8 structure (Epand et al., 1992). We therefore feel that there is insufficient information to rely so heavily on the “sided” insertional helix hypothesis as a mechanism for induction of membrane fusion. Indeed, given the wide sequence diversity among fusion peptides, perhaps multiple structures and molecular mechanisms may have been adopted by cells or viral agents to achieve the same end of membrane fusion. It is essential to maintain an open mind and field of experimentation. W. R. Gallaher,*t J. P. Segrest,* and E. Huntert *Department of Microbiology, Immunology, and Parasitology Louisiana State University Medical Center New Orleans, Louisiana 70112 fDepartment of Microbiology *Department of Medicine University of Alabama at Birmingham Medical Center Birmingham, Alabama 35294 References Blobel, C. P., Wolfsberg, T. G., Turck, C. W., Myles, D.G., Primakoff, P., and White, J. M. (1992). Nature 356, 248-252. Brasseur, R., Cornet, B., Burny, A., Vandenbranden, Ruysschaert, J. M. (1988). AIDS Res. Hum. Retroviruses Brunner, J., Franzusoff, G. (1985). Biochemistry
M., and 4, 83-90.
A. J., Luscher, B., Zugliani, C., and Semenza, 24, 5422-5430.
Ellens, H., Siegel, D. P., Alford, D., Yeagle, P. L., Boni, L., Lis, L. J., Quin, P. J., and Bent& J. (1989). Biochemistry 28, 3892-3703. Epand, R. M., Cheetham, J. J., Epand, P. F., Yeagle, P. L., Richardson, C. D., and Degrado, W. F. (1992). Biopolymers 32, 309-314. Harter, C., Bachi, T., Semenza, G., and Brunner, J. (1988). Biochemistry 27, 1856-1864. Harter, C., James, P., Bachi, T., Semenza, G., and Brunner, J. (1989). J. Biol. Chem. 264, 64596464. Hoppe, J., Brunner, 5610-5816. Horth, M., Lambrecht,
J., and Jorgensen,
B. (1984). Biochemistry
23,
B., Khim, M. C. L., Bex, F., Thiriat, C., Ruys-
schaert, J.-M., Burny, A., and Brasseur, 2755. Levy-Mintz,
R. (1991). EMBO J. 70,2747-
P., and Kielian, M. (1991). J. Virol. 65, 4292-4300.
Meister, H., Bachofen, R., Semenza, Biol. Chem. 260, 18326-16331. Sigrist, H., Muehlemann, Photobiol. B. 7, 277-287.
G., and Brunner,
J. (1985). J.
M., and Dolder, M. (1990). J. Photochem.
Stegmann, T., Delfino, J. M., Richards, J. Biol. Chem. 266, 18404-18410.
F. M., and Helenius, A. (1991).
White, J. M. (1990). Annu. Rev. Physiol. 52, 675-897.
August 21, 1992, Copyright
0 1992 by Cell Press
Letter to the Editor
Are Fusion Peptides Really “Sided” Insertional Helices? Membrane fusion is a widespread and essential biochemical process critical for endocytosis, exocytosis/secretion, entry of enveloped viruses into susceptible cells, and polykaryocytosis occurring during myogenesis and virusinduced pathogenesis. Specifically, virus-induced fusion has been localized to hydrophobic sequences within transmembrane fusion proteins termed “fusion peptides” (White, 1990). The mechanism of action of such fusion peptides has long been of intense interest. While current evidence is strong that fusion peptides contact the lipid environment of target membranes (Harter et al., 1988, 1989; Stegmann et al., 1991) to produce hexagonal lipid structures of negative curvature (Ellens et al., 1989) the mechanism remains unknown. In a recent description of a possible fusion-inducing protein from sperm, Blobel et al. (1992) alluded to the most commonly advanced hypothesis that fusion peptides act as “sided” insertional helices. Such “sided” helices with an asymmetric concentration of bulky, hydrophobic residues, as found in the fusion peptides of a paramyxovirus and of human immunodeficiency virus (HIV), have a favorable free energy for insertion of the more hydrophobic side at a shallow angle into a lipid bilayer (Brasseur et al., 1988). The hypothesis has been experimentally supported: the periodicity of labeling of the influenza virus fusion peptide with a photoactivatable, lipid-embedded reagent has been purported to follow the 3-4 residue repeat for a-helical turns (Harter et al., 1989). More indirectly, mutations affecting the theoretical angle of insertion for the HIV fusion peptide inhibit fusion (Horth et al., 1991). We agree that many viral fusion peptides can be drawn as a “sided” helix. However, in examining the expanding data base of fusion peptides, we have observed a number of properties discordant with this hypothesis. First, several putative retroviral fusion peptides present a completely hydrophobic face over only three helical turns, and bulky hydrophobic amino acids are also on the less hydrophobic side. Second, the most highly conserved amino acids do not uniformly fall on the more hydrophobicside and, particularly among the alphaviruses, are preponderantly glycine and proline. Finally, the frequent presence of the strong helix-breaking residues, glycine, proline, and serine, even in pairs, makes it difficult to project some fusion peptides (such as that of an alphavirus [Levy-Mintz and Kielian, 19911 or the sperm fusion protein PH-30a [Blobel et al., 1992)) as helices by algorithms of protein structure. These discordant notes caused us to reexamine the labeling data of Hatter et al. (1989), which are the sole direct experimental findings supporting this hypothesis. In Figure lA, the data are plotted on a helical wheel, in which amino acids in an a helix rotate 100° per residue, generating a residue per 20° increment in projection down the
helical axis. While there is concentration of label in the direction of the helical moment (O”) about the hemicylinder of 290°-70°, there is also label on the other side (150°1 70°) and gaps in labeling on the more hydrophobic side, particularly at 31 O ” and 330°.
120,
(10
100 . 80
88
3
6: . 0 -4 I;,
60 40
2
20 I
i
d
lb amino
i0 acid position
3b
C
0
s cpm x 10
8
10
-2
Figure 1. Labeling of Influenza A Fusion Peptide as a Function Helical Wheel Position and Side Chain Volume
of
(A) The relative distribution of radioactive photolabel from 3-(trifluoromethyl)-3-([‘251]-iodophenyl) diazirine to amino acids of the aminoterminus of influenza A virus hemagglutinin 2, as reported by Harter et al. (1969) is plotted as a function of position on a helical wheel. Numbers indicate position in the sequence; the amino acid at each position is denoted by its single-letter abbreviation. Arrow, direction of helical moment (0.26/residue). (B) The distribution of label over the 22 aa sequence (closed symbols) co-plotted with the residue volume of each amino acid (open symbols). Residue volume was determined by measuring the aqueous displacement of space-filling models of tripeptides Gly-X-Gly, where X is the residue in question and then subtracting the volume of the dipeptide Gly-Gly. (C) A scatter plot of label as a function of residue volume.
An alternate explanation may lie in reactivity to the phospholipid-embedded reagent. In this and other studies using carbene-generating photolabeling, glycine and alanine have labeled very poorly (Hoppe et al., 1984; Brunner at al., 1985; Meister et al., 1985). This is in accord with a carbene philicity scale for such photolabeling, to which it has been noted these data generally conform (Sigrist et al., 1990). We have explored thevolume occupied by each residue as a variable in reactivity. When label and amino acid residue volume are plotted by the linear position of each residue in the peptide sequence (Figure 1 B) or in a scatter plot of label versus residue volume (Figure lC), the degree of labeling is directly proportional to residue volume. A more cogent explanation of the findings of Harter et al. (1989) would be that the entire sequence is in contact with lipid, rather than existing as a sided structure. We conclude that, while these data demonstrate insertion of the 22 residue amino-terminal fusion peptide into the hydrophobic lipid environment, they cannot be taken in support of the “sided” helical hypothesis. Also contravening the hypothesis, circular dichroism of the measles fusion peptide immersed in a lipid environment shows 73% 8 structure (Epand et al., 1992). We therefore feel that there is insufficient information to rely so heavily on the “sided” insertional helix hypothesis as a mechanism for induction of membrane fusion. Indeed, given the wide sequence diversity among fusion peptides, perhaps multiple structures and molecular mechanisms may have been adopted by cells or viral agents to achieve the same end of membrane fusion. It is essential to maintain an open mind and field of experimentation. W. R. Gallaher,*t J. P. Segrest,* and E. Huntert *Department of Microbiology, Immunology, and Parasitology Louisiana State University Medical Center New Orleans, Louisiana 70112 fDepartment of Microbiology *Department of Medicine University of Alabama at Birmingham Medical Center Birmingham, Alabama 35294 References Blobel, C. P., Wolfsberg, T. G., Turck, C. W., Myles, D.G., Primakoff, P., and White, J. M. (1992). Nature 356, 248-252. Brasseur, R., Cornet, B., Burny, A., Vandenbranden, Ruysschaert, J. M. (1988). AIDS Res. Hum. Retroviruses Brunner, J., Franzusoff, G. (1985). Biochemistry
M., and 4, 83-90.
A. J., Luscher, B., Zugliani, C., and Semenza, 24, 5422-5430.
Ellens, H., Siegel, D. P., Alford, D., Yeagle, P. L., Boni, L., Lis, L. J., Quin, P. J., and Bent& J. (1989). Biochemistry 28, 3892-3703. Epand, R. M., Cheetham, J. J., Epand, P. F., Yeagle, P. L., Richardson, C. D., and Degrado, W. F. (1992). Biopolymers 32, 309-314. Harter, C., Bachi, T., Semenza, G., and Brunner, J. (1988). Biochemistry 27, 1856-1864. Harter, C., James, P., Bachi, T., Semenza, G., and Brunner, J. (1989). J. Biol. Chem. 264, 64596464. Hoppe, J., Brunner, 5610-5816. Horth, M., Lambrecht,
J., and Jorgensen,
B. (1984). Biochemistry
23,
B., Khim, M. C. L., Bex, F., Thiriat, C., Ruys-
schaert, J.-M., Burny, A., and Brasseur, 2755. Levy-Mintz,
R. (1991). EMBO J. 70,2747-
P., and Kielian, M. (1991). J. Virol. 65, 4292-4300.
Meister, H., Bachofen, R., Semenza, Biol. Chem. 260, 18326-16331. Sigrist, H., Muehlemann, Photobiol. B. 7, 277-287.
G., and Brunner,
J. (1985). J.
M., and Dolder, M. (1990). J. Photochem.
Stegmann, T., Delfino, J. M., Richards, J. Biol. Chem. 266, 18404-18410.
F. M., and Helenius, A. (1991).
White, J. M. (1990). Annu. Rev. Physiol. 52, 675-897.