Download Hydrophobic-at-Interface Regions in Viral Fusion Protein Ectodomains

Bioscience Reports, Vol. 20, No. 6, 2000
MINI REVIEW
Hydrophobic-at-Interface Regions in Viral Fusion Protein
Ectodomains
José L. Nieva1,2 and Tatiana Suárez1
Receiûed June 20, 2000
In this chapter we shall describe how to apply the hydrophobicity-at-interface scale, as
proposed by Wimley and White [Wimley, W. C. and White, S. H. (1996) Nature Struct.
Biol. 3:842–848], to the detection of amino acid sequences of viral envelope glycoproteins
putatively engaged in interactions with the target membranes. In addition, a new approach
will be briefly introduced to infer the bilayer location at equilibrium of membrane-partitioning sequences. The use of these new procedures may be important in describing the molecular mechanism leading to the formation of a fusion pore by viral glycoproteins.
KEY WORDS: Membrane fusion; viral fusion; HIV-1 gp41; Influenza hemagglutinin;
Sendai virus; Semliki forest virus; vesicular stomatitis virus; fusion peptide; peptide–lipid
interaction; Wimley–White hydrophobicity scale.
INTRODUCTION
Membrane fusion is the basic strategy used by enveloped viruses to introduce their
genomes in the internal milieu of host cells (White, 1992; Hernández et al., 1996;
Zimmerberg et al., 1993). It constitutes a pivotal event during the infection cycle of
important human pathogens such as the influenza, human immunodeficiency, or
Ebola viruses (Wiley and Skehel, 1987; Moore et al., 1993; Feldman et al., 1999).
Consequently, the envelope glycoproteins promoting virus-cell fusion constitute an
important target for the development of antiviral therapies (Luo et al., 1997; Kilby
et al., 1998). These integral membrane type-1 proteins bring about the fusion of the
cell membrane and the viral envelope through a complex mechanistic pathway
involving protein–lipid interactions not fully understood as yet. As a general rule,
under physiological conditions the fusion process is activated by diverse stimuli like
interactions with cell surface receptors or by the pH drop along the endocytic pathway. Thereafter it appears to evolve spontaneously in the absence of additional
energy supply. This implies that the energetic barrier for fusion, imposed by the
formation of the lipid pore necessary to connect the internal compartments of the
virion and the cell (Chernomordik and Zimmerberg, 1995; Siegel and Epand, 1997),
1
Unidad de Biofı́sica (CSIC-UPV兾EHU) and Departamento de Bioquı́mica, Universidad del Paı́s Vasco,
P.O. Box 644, 48080-Bilbao, Spain.
2
To whom correspondence should be addressed. Tel: C34 94 6012615; Fax: C34 94 4648500; E-mail:
[email protected]
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0144-8463兾00兾1200-0519$18.00兾0  2000 Plenum Publishing Corporation
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Nieva and Suárez
must be overcome using free energy supplied mainly by the viral envelope glycoprotein (Siegel, 1993; Chernomordik and Kozlov, 1998; Bentz, 2000). In fact, this
protein complex is synthesized by the infected cell as a thermodynamically metastable intermediate (Doms et al., 1993; Carr et al., 1997).
An important characteristic shared by many of these polypeptides, known for
quite a long time as being crucial for the development of their fusogenic function,
is the presence of a fusion peptide within the ectodomain exposed to the water phase
(Gallaher, 1987; White, 1992). This hydrophobic and conserved sequence is thought
to be involved in driving the initial partitioning of the fusion protein into the target
membrane (Doms and Peiper, 1997; Durell et al., 1998). Hence, at a certain stage
after fusion activation, the fusion peptide is likely to insert into the lipid bilayer of
the target cell, thus making the viral envelope glycoprotein an integral component
of two membranes: that of the virus and that of the target cell. Also, importantly,
such an initial interaction of the fusion peptide with target lipids might lead to the
selection by the protein of a unique spontaneous folding pathway in contact with
the membrane (van der Goot et al., 1992; Engelman, 1996). The inserted envelope
protein trapped in this ‘‘membrane pathway’’ would refold until the newly formed
protein–lipid complex reaches equilibrium. In other words, the target bilayer might
select for characteristic folding intermediates implied in subsequent steps of the
fusion process (van der Goot et al., 1991, 1992; Bañuelos and Muga, 1995; Engelman, 1996; Booth and Curran, 1999; Bogdanov and Dowhan, 1999). From that
point of view, identification in viral envelope glycoproteins of amino acid sequences
showing a tendency to partition into membranes may provide some insight into the
molecular mechanism underlying the fusion process that they catalyze.
In this work a new methodology will be briefly described that can be applied
to the detection of protein sequences showing a tendency to partitioning into membrane interfaces. Since these procedures have been recently developed (Wimley and
White, 1996; White et al., 1998; White and Wimley, 1998, 1999) and even more
recently applied to the detection of membrane-partitioning regions within ectodomains of viral fusion proteins (Pereira et al., 1997; Ruiz-Argüello et al., 1998; Nir
and Nieva, 2000; Nieva et al., 2000; Suárez et al., 2000a; Suárez et al., 2000b), many
of the data and results reported here are still in the process of being published in
the customary research article form.
HYDROPHOBICITY AT THE MEMBRANE INTERFACES
Nonconstitutive membrane proteins show the ability to get stably inserted into
membranes after partitioning from the water phase into the lipid bilayer (Engelman,
1996). Certain amino acid sequences have probably evolved and become fit to carry
out the spontaneous partitioning of the overall water-soluble protein into membranes (Parker et al., 1989; Engelman, 1996; Shatursky et al., 1999). From a thermodynamic point of view this means that the formation of a lipid–protein complex
involving these sequences is energetically more favorable than keeping them as a
constitutive part of the tertiary or quaternary structure of the soluble protein. In the
absence of repulsive or attractive electrostatic interactions, initial partitioning of
peptides between the aqueous and membrane phases is energetically constrained by
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the hydrophobic character of the amino acid side chains, the cost of partitioning the
peptide bonds, and the bilayer effect (White and Wimley, 1999).
Based on the water-to-membrane interface transfer free energies for each amino
acid, Wimley and White have proposed a whole-residue hydrophobicity scale compiling the energetic components that dictate initial partitioning of unfolded peptide
sequences into membranes (Wimley and White, 1996). This ‘‘interfacial hydrophobicity scale’’ was determined for 1-palmitoyl-2-oleoylphosphatidylcholine
(POPC) bilayer interfaces using two types of oligopeptides that allowed the evaluation of both, side-chain and peptide-bond hydrophobicities. As shown by these
authors, the energy cost of the partitioning into the membrane interface of the peptide bond alone is actually comparable to that of the negatively charged Asp residue.
This indicates that transferring the peptide bond to membrane interfaces is a highly
unfavorable process. Peptide bond partitioning is thus underscored by classical
hydropathy indexes, that are usually estimates, based on bulk-phase partitioning, of
side chain hydrophobicity alone (Kyte and Doolittle, 1982). This is one substantial
difference between the Wimley–White scale and other hydrophobicity scales
commonly used to detect membrane interacting sequences in polypeptide chains
(Table 1).
An additional important difference stressed by White and Wimley (1999) is
that, in comparison with bulk-phase hydrophobicity scales, their scale takes into
account the effect of the membrane interface on partitioning. This bilayer region
consists of a complex mixture of water and chemically heterogeneous phospholipid
groups (polar head groups, glyceryl, phosphoryl, carbonyl and methylene groups)
in which significant changes in polarity occur at short range (White and Wimley,
1994, 1998, 1999; Hristova et al., 1999). This originates another important difference
with other scales (Kyte and Doolittle, 1982; Eisenberg et al., 1984; Engelman et al.,
1986); even in the absence of solid physical foundations (Yau et al., 1998; White
and Wimley, 1998) aromatic residues, namely Phe, Tyr and Trp appear to be the
most hydrophobic ones when located at interfaces (Table 1).
Table 1. Relative Contribution (in Percentage) of
Aromatic and Some Apolar Amino Acids to
Maximal Hydrophobicity According to Different
Hydropathy Indexes
Amino acid
a
WW a
b
Trp
Phe
Tyr
100 (1)
81 (2)
76 (3)
Ile
Val
Leu
60 (5)
50 (9)
67 (4)
KD
E
40 (10)
81 (4)
35 (12)
79 (5)
95 (2)
71 (10)
100 (1)
97 (2)
92 (3)
100 (1)
92 (3)
92 (4)
WW, Wimley and White (1996); KD, Kyte and
Doolittle (1982); E, Eisenberg et al. (1984).
b
Numbers in brackets indicate the amino acid position in the scale when going from most hydrophobic to most hydrophilic.
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Finally, in contrast to scales based on normalized hydrophobicity values (Kyte
and Doolittle, 1982; Eisenberg et al., 1984), the interfacial hydrophobicity scale of
Wimley and White is additive and reflects real thermodynamic measurements. Additivity allows computing the energetics of partitioning for arbitrary protein sequences
(White and Wimley, 1999). Consequently, this scale can be used as a theoretical tool
that directly reflects the partitioning capacity of defined sequences within proteins,
thus, it can be used as a suitable hydropathy index (White and Wimley, 1999). The
ectodomains of viral fusion proteins might be considered as beginning nonconstitutive membrane proteins that get inserted into target membranes in the initial step of
the fusion process. In what follows the use of this new hydropathy index will be
described to detect putative regions involved in partitioning within sequences of
several fusogenic viral proteins.
HYDROPATHY PLOTS AND DETECTION OF
MEMBRANE-INTERACTING REGIONS
The plots displayed in Fig. 1 have been constructed using respectively the
Wimley–White and Kyte–Doolittle hydropathy indexes. They illustrate the different
information that might be obtained on the hydrophobicity of the transmembrane
gp41 fusogenic subunit of the human immunodeficiency virus type (HIV-1) envelope
glycoprotein. The plots are not superimposable all along the sequence. The combined use of both plots reveals three interesting regions. At the N-terminus, both
Fig. 1. Hydrophobicity of the sequence spanning residues 500–856 of HIV-1
gp160 precursor, (BH10 isolate). The plots (mean values for a window of 11
amino acids) were made using the Kyte–Doolittle hydropathy index (white)
and Wimley–White interfacial hydrophobicity (black) scale for individual residues. Designated gp41 regions include: FP, fusion peptide; pre-TM, pre-transmembrane segment; TM, transmembrane domain; LLP, Lentivirus Lytic
Peptides.
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plots identified a hydrophobic region FP, corresponding to the fusion peptide. This
implies that this stretch combines the ability of partitioning at the interface with an
overall hydrophobic character arising from the side chain composition of constituent
amino acids. Several findings, including mutational analysis and destabilization of
model membranes by representative synthetic sequences, support the direct involvement of this conserved segment of about 25 amino acids in HIV-1 fusion in ûiûo
(reviewed in: Durell et al., 1997; Nir and Nieva, 2000).
Moving towards the C-terminus, two positive peaks appear at the transmembrane region, of which one, corresponding to the pre-transmembrane region (preTM), is detected by the Wimley–White plot, meaning that this sequence has a
marked tendency to partition into membrane interfaces. It is important to point
out that this stretch could not be previously identified by plots based on classical
hydrophobicity scales. Following the prediction by the Wimley–White algorithm,
the fluorescence spectra displayed in Fig. 2B are consistent with this sequence being
able to partition into POPC bilayers, being most probably located at the interface.
Incubation of the peptide with vesicles results in an increase of the Trp fluorescence
and a blue shift from 346 to 339 nm of the maximum emission wavelength. These
spectral changes in Trp emission indicates that the gp41 sequence proximal to the
transmembrane stretch is able to partition into the apolar milieu of the membrane.
In addition, the preferential bilayer location of this sequence may be deduced from
Fig. 2. (A) Amino acid sequence of gp41 pre-transmembrane
region spanning residues 664–683 of HIV-1 gp160 precursor
(BH10 isolate). Positions of aromatic residues are indicated in bold
characters. (B) Fluorescence emission spectra (excitation wavelength: 280 nm) of a synthetic peptide representing the sequence in
panel A, in buffer (1), and incubated with POPC LUV (2) or
POPC:d-DHPE (9.4:0.6) LUV (3). Spectrum 1 has been corrected
for emission decrease in solution with time. Lipid concentration
was 0.25 mM, and peptide-to-lipid ratio was 1:250.
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the Trp spectrum obtained in the presence of POPC vesicles containing the fluorescent N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero3-phosphoethanolamine (d-DHPE) probe (Ruiz-Argüello et al., 1998). The decrease
in Trp emission concomitant to the increase in d-DHPE emission reflects the existence of resonance energy transfer from the indole group to the dansyl moiety of the
probe. The dansyl moiety in the d-DHPE probe is probably located at the edge of
the lipid–water interface in the vesicles. Given that the R0 distance calculated for the
couple dansyl-Trp is approximately 17 Å (Vaz and Schoellmann, 1976), the observed
strong quenching of Trp fluorescence suggests that these residues remain associated
to the interface and兾or to the acyl chain region close to it.
The pre-TM region is immediately followed by the membrane spanning
sequence TM, that anchors the protein to the membrane envelope (Fig. 1). Interestingly this stretch is detected by Kyte–Doolittle but underscored by Wimley–White.
A plausible explanation for the segregation existing between both regions as detected
by the plots might be that, whereas the pre-TM sequence bears an intrinsic ability
to partition into the interface, the TM stretch, as transferred by the translocation
apparatus (Singer, 1990), only shows the ability to stably reside within the hydrocarbon core of the bilayer. The ability of the gp41 pre-TM sequence to partition
into the membrane interface appears to be based on the presence of several aromatic
residues (Fig. 2A). The unusual concentration of aromatic amino acids at regions
preceding the transmembrane anchors of fusogenic viral proteins belonging to several virus families was first noticed by Gallaher (W. R. Gallaher, personal communication). Moreover, this rather common characteristic might be involved in the
promotion of fusion as recently put forward for the case of the HIV-1 gp41 fusogenic
subunit depicted in Fig. 1 (Salzwedel et al., 1999; Muñoz-Barroso et al., 1999; Nieva
et al., 2000; Suárez et al., 2000a). In a recent report Suárez et al. (2000a) have
demonstrated that the sequence displayed in Fig. 2A is able to induce membrane
fusion and permeabilization, even more efficiently that the N-terminal fusion peptide. Thus, the presence of two membrane-partitioning stretches, FP and pre-TM,
separated by a collapsible intervening sequence might constitute a common structural motif related to the fusogenic function of at least some viral glycoproteins.
At the cytoplasmic tail of the gp41 protein, an additional example of segregation between membrane-partitioning and membrane-residing sequences may be
observed. Three consecutive hydrophobic peaks are detected by the plots. The one
positioned in the first place, LLP-2, and the one in the third position, LLP-1, correspond to Lentivirus Lytic Peptides 2 and 1 respectively (Srinivas et al., 1992). They
are only visible using the Kyte–Doolittle algorithm. These regions have been shown
to interact with and permeabilized cell and model membranes by forming pores of
a defined size (Srinivas et al., 1992; Miller et al., 1993; Chernomordik et al., 1994).
The peak in between those two, only detected by the Wimley–White plot, corresponds to a leucine zipper-like sequence spanning residues 789–815 and representing
the Lentivirus Lytic Peptide-3 (LLP-3) (Kliger and Shai, 1997). The LLP-3 sequence
has been experimentally shown by these authors to interact with and perturb membranes. The relative positions of the three peaks are highly suggestive of this hydrophobic region as consisting of one inside–outside and one outside–inside membranespanning stretches connected through a membrane interface-residing sequence. An
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525
alternative model postulates the interaction of these three sequences exclusively with
the intraviral side of the membrane (Kliger and Shai, 1997). In such a case the
intervening interfacial LLP-3 might be useful to promote the spontaneous insertion
of LLP-1 and LLP-2 into the membrane.
The results displayed in Fig. 1 suggest the existence in gp41 of membranetransferring sequences that precede hydrophobic stretches, the latter being by themselves unable to partition into membranes from the water phase. The possibility
that those ‘‘interfaceCtransmembrane’’ arrangements might represent a common
structural motif is worth exploring, specially among nonconstitutive membrane proteins that need to make use of specialized sequences to get inserted into target membranes. Whether these types of motif also play a role in viral fusion and what their
function could be like in relation to the underlying molecular mechanism remain to
be established (Nieva et al., 2000; Suárez et al., 2000a).
A rather obvious conclusion from the results displayed in Fig. 1, is that the
Wimley–White plot detects previously unidentified membrane-partitioning regions
within the sequence of HIV-1 gp41. This demonstrates a direct application of the
Wimley–White plots: the detection of unidentified membrane interacting regions
in diverse viral fusion proteins, specifically at their ectodomains. In Figs. 3–5 we
present preliminary results on the detection of putative fusion peptides in several
selected instances.
In Fig. 3 we include comparative hydropathy analyses using Wimley–White
and Kyte–Doolittle hydrophobicity scales. The top panel corresponds to the Semliki
Forest virus (SFV) E1 fusion protein. Mutational analysis demonstrated the existence within SFV E1 of a conserved region comprising residues 76YQCKVYT
GVYPFMWGGAYCFC96 that might represent a fusion peptide (Levy-Mintz and
Kielian, 1991). Consistent with this putative role, recent experimental work in our
laboratory has confirmed the ability of synthetic peptides representing this E1
sequence to interact with model membranes (F. B. Pereira et al., unpublished
results). The SFV E1 fusion peptide sequence, indicated as FP1 in the plot, was
barely detectable by the Kyte–Doolittle algorithm. By contrast, the Wimley–White
plot identifies the FP1 sequence as the most prominent positive peak, indicating that
in fact this region might play an important role in the molecular interaction of E1
with the lipids of the target membrane.
In addition, the Wimley–White plot puts forward the presence of a second
sequence in E1 also showing high potentiality to partition into membranes. This
sequence indicated as FP2? in the plot comprises residues 153AVTIGGTQFIF
GPLSSAWTPF173 of the SFV E1 glycoprotein. FP2? might constitute a second
internal fusion peptide as judged by its high interfacial hydrophobicity, the high
degree of conservation among the members of the Togaviridae family, and by the
prediction of a loop as being the secondary structural element bounded by the two
conserved prolines (data not shown). The presence of a predicted loop bounded by
two conserved prolines has been proposed by Delos and coworkers (2000) as a common organization shared by internal fusion peptides. Thus, the information obtained
in this case from the Wimley–White plot points to the fact that more than one
sequence might be involved in the interaction of SFV E1 ectodomain with its target
membrane.
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Fig. 3. Hydrophobicity of SFV E1 and Sendai F1 (residues 100–565 of the Sendai F0 protein
precursor) fusogenic subunits. The stretches plotted include proposed fusion peptide sequences
(FP) and the transmembrane anchor regions (TM). Hydropathy plots (mean values for a window of 11 amino acids) were made using the Kyte–Doolittle hydropathy index (thin lines) and
Wimley–White interfacial hydrophobicity (thick lines) scales for individual residues.
The existence of at least two fusion peptides has been indeed demonstrated for
Sendai F1 glycoprotein (Peisajovich et al., 2000). In addition to the fusion peptide
at the N terminus of the F1 protein (residues 117–140 of the precursor), Peisajovich
et al. (2000) identified an internal segment in the Sendai virus F1 protein (amino
acids 214–226) highly homologous to the fusion peptides of HIV-1 and RSV. A
synthetic peptide, including this region, was found to induce membrane fusion of
vesicles even more effectively than the previously known N-terminal fusion peptide.
As shown by the bottom plot in Fig. 3, the Wimley–White algorithm clearly identifies this F1 region, indicated as FP2, as potentially partitioning into membrane
interfaces. Moreover, in a way consistent with the observed in ûitro behavior of
representative synthetic sequences, the relative intensities of the corresponding peaks
reflect a higher tendency to interact with membranes for internal FP2 than for Nterminal FP1. Peisajovich and coworkers concluded their study suggesting that the
concerted action of two fusion peptides, one N-terminal and the other internal,
might be a key requirement for the actual fusion process in paramyxoviruses.
In summary, the evidence presented in Fig. 3 confirm that the Wimley–White
plots may be used to identify hitherto undetected fusion peptides in viral fusion
proteins. Another aspect of the Wimley–White scale, as applied to the detection of
membrane-partitioning regions in these proteins appears illustrated in Fig. 4. This
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Fig. 4. Effect of protonating Asp and Glu residues on
interfacial hydrophobicity of Influenza HA2 (top) and HIV1 gp41 (bottom) ectodomains. The stretches plotted include
the fusion peptide sequences (FP) and the transmembrane
anchor regions (TM). Thick lines: hydropathy plots using
interfacial hydrophobicity values for protonated side chains
of Asp and Glu residues as reported by Wimley and White
(1996). Thin lines: hydropathy plots using interfacial hydrophobicity values for non-protonated side chains of Asp and
Glu residues.
figure displays the effects of protonation of Asp and Glu side chain carboxylates
on the interfacial hydrophobicity of influenza HA2 and HIV-1 gp41. The fusion
phenomenon mediated by influenza hemagglutinin is activated by the acidic pH of
endosomes (White, J., 1992; Hernández et al., 1996; Zimmerberg et al., 1993). This
low-pH-dependence of fusion is further demonstrated by the fact that isolated
virions can be induced to fuse in ûitro with lipid vesicles by lowering the pH of the
medium (White et al., 1982; Stegmann et al., 1987). In addition, different fragments
of HA2, including the N-terminal fusion peptide, have been shown to interact with
membranes at low pH, as in the physiological process of influenza fusion (Rafalski
et al., 1991; Yu et al., 1994; Kim et al., 1998; Epand et al., 1999). These facts
together suggest that lowering the pH might cause protonation of defined Asp or
Glu residues in HA2, thereby increasing the tendency of certain regions to partitioning into membranes. This is probably not the case for the HIV-1 gp41 transmembrane subunit, since membrane fusion induced by this protein is activated at neutral
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pH through interactions of the envelope glycoprotein complex with the surface
receptors present in the target cell (Doms and Peiper, 1997).
The plots in the top panel of Fig. 4 demonstrate that protonation of the acidic
residues located at the fusion peptide of HA2, would result in an increase of its
interfacial hydrophobicity. In the unprotonated protein this N-terminal region (FP)
appears divided in two segments, each showing a very modest interfacial hydrophobicity. Protonation causes the merging of the two segments into a single region
that shows an overall high tendency to partition into membranes. By contrast, as
evidenced by the superimposable plots shown in the bottom panel of Fig. 4, the
interfacial hydrophobicity of the fusion peptide region in gp41 remains unaltered
upon protonation.
An additional effect induced on HA2 by Glu and Asp protonation, is the emergence of several positive peaks within the sequence intervening the N-terminal fusion
peptide and the C-terminal transmembrane region (top panel in Fig. 4). Several
positive peaks are already present within the corresponding gp41 unprotonated
sequence (bottom panel in Fig. 4, see also Fig. 1). This observation suggests the
existence of a certain tendency of this gp41 sequence to partition into membranes
even at neutral pH. In the case of HA2 such a tendency would only be expressed
upon protonation of certain Glu or Asp residues. It has been argued that regions
other than the fusion peptides may participate in the fusion process through a direct
interaction with the target membrane (Yu et al., 1994; Rabenstein and Shin, 1995;
Santos et al., 1998; Kim et al., 1998; Epand et al., 1999; Ben-Efraim et al., 1999).
The significant interfacial hydrophobicity scattered along the HA2 and gp41
sequences separating the fusion peptide from the transmembrane stretch would be
consistent with such assertion. A thorough hydropathy analysis of these sequences
is under progress in order to further sustain this hypothesis (Nieva, Goñi, and Gallaher, unpublished).
Activation of HA2-mediated fusion at pH well above the pKa values of sidechain carboxylates, implies that Glu and Asp residues whose pKa are increased
within the folded polypeptide would be more prone to participate in the process.
From the plots displayed in Fig. 4 it is impossible to deduce at which positions the
pKa of Asp or Glu lateral side chains have been increased. However, from comparison with the HIV-1 gp41, active at neutral pH, the preferential location of protonable residues at certain HA2 regions might be inferred. Accumulation at
membrane-partitioning regions of fusion-protonable Glu and Asp residues might
contribute toward controlling the process. In conclusion, comparing interfacial
hydrophobicities of low-pH-activated glycoproteins with those activated at neutral
pH may provide an additional tool for detecting regions putatively involved in
fusion.
As a corollary to the results presented in Figs. 3 and 4, we have applied the
former procedures to the case of the vesicular stomatitis virus (VSV) G protein
(Fig. 5). We have selected this particular example because there exists certain controversy as to what the fusion peptide would be in this protein, given the low degree
of conservation existing in the proposed sequence between different rhabdoviruses
(Gaudin et al., 1995). In Fig. 5 we show plots corresponding to residues 1–220 of
the VSV G protein precursor. This sequence includes the proposed fusion peptide
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Fig. 5. Interfacial hydrophobicity of the sequence spanning residues 1–220 of VSV
G glycoprotein precursor (New Jersey serotype). The plots (mean values of free
energies of transfer from membrane interfaces to water for a window of 11 amino
acids) were constructed using the Wimley–White scale for individual residues using
values for unprotonated (A) and protonated (B) Asp and Glu side chains. Designated regions include: SP, signal peptide; FP, fusion peptide.
spanning residues 120–136 (Whitt et al., 1990; Li et al., 1993; Li and Ghosh, 1994;
Fredericksen and Whitt, 1995) and the region about 70 amino acids downstream,
also proposed to participate in the fusion process (Li et al., 1993).
According to the results in Fig. 5, the sequence showing the highest tendency
to partition into membrane interfaces (region 1 in the plot) comprises residues
70VEGFMCHSALWMTTCDFRWYGPKYIT95. Therefore, at least from the
point of view of average interfacial hydrophobicity, this G sequence would constitute a suitable fusion peptide candidate. Centered at position 200, an additional
sharp peak (region 2) that broadens after Asp and Glu protonation can be
detected. The broad peak in the protonated sequence corresponds to residues
191SVCSQLF TLVGGIFFSDSEEITSMGL216. Insertional mutations in this
region have been shown to inhibit low-pH-induced fusion of cells transfected
with the VSV G gene (Li et al., 1993). In contrast, both plots failed to detect
the 120SFNPGFPPQSCGYGTVT136 fusion peptide sequence, implying that, at
least according to the Wimley–White algorithm, this is not a suitable sequence to
drive the partitioning of the G protein into the target membrane.
In conclusion, our results in Fig. 5 suggest that the sequence proposed to be
the VSV fusion peptide is not likely to interact directly with the target membrane.
This observation does not rule out its participation in the fusion mechanism. In fact,
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Nieva and Suárez
the effect that mutations targeted to this G region display on the fusion phenotype
indicate that preservation of this sequence must be important for VSV fusion (Li
and Ghosh, 1994; Fredericksen and Whitt, 1995). Our results also suggest the existence of other G regions being more likely to partition into the target membranes of
VSV.
MEMBRANE TOPOLOGY
With the aim of comparing bilayer partitioning with bulk-phase partitioning,
Wimley and White also established a whole-residue hydrophobicity scale for partitioning into n-octanol (Wimley and White, 1996). The difference between the
interfacial and octanol scales can be used to distinguish residues that prefer to insert
deeply into the bilayer matrix from those that prefer associating with the interface
(White and Wimley, 1999). Consequently, these scales can be used as a theoretical
tool to infer the bilayer topology of viral fusion peptides interacting with membranes
(Nir and Nieva, 2000; Suárez et al., 2000b unpublished obserûation). The difference
of scales resulting in positive free energies of transfer from octanol to interface,
identifies membrane-core seeking residues (higher tendency to penetrate into the
hydrophobic core than to remain associated to the membrane interface). Such a
difference-scale plot for the fusion peptide of influenza hemagglutinin is displayed
in Fig. 6A.
Brunner (1989) demonstrated by means of hydrophobic photolabeling techniques that the fusion peptide at the N-terminus of bromelain-solubilized ectodomain of influenza hemagglutinin contacts only the outer leaflet of the liposome
bilayer. The plot in Fig. 6A further demonstrates the preferential location of the
peptide at the membrane interface with several residues showing propensity to penetrate into the hydrophobic core of the bilayer. Moreover, the plot suggests the
existence of certain periodicity in the positions of membrane-core seeking residues.
This might be related to the type of secondary structure eventually adopted by this
sequence upon partitioning into the membrane interface. Within the amino acid
sequence each core-preferring residue is followed by 2–3 residues showing tendency
to remain associated to the interface. This pattern would optimize the membrane
interaction if upon partitioning into the interface the peptide folded as an α -helix,
orienting its core-preferring residues towards the center of the bilayer (panel B).
The prediction, based on octanol-to-interface free energies of transfer, is therefore
consistent with the experimental determinations indicating that α -helix is the main
conformation adopted by this sequence in the presence of lipid vesicles (Rafalski et
al., 1991).
CONCLUDING REMARKS
As a primary conclusion, the results in this study reveal that the ectodomains
of envelope glycoproteins may contain more than one membrane-partitioning region
(Figs. 1, 3–5). Low-pH activation might result in an increased transfer to membrane
interfaces of defined protein regions that are already present as interfacial sequences
in fusion proteins activated at neutral pH (Fig. 4). In searching for possible common
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Fig. 6. (A) Standard free energies of transfer (Kcal mol −1) from interface (䊊) or octanol (●) to
water for individual amino acids of the influenza hemagglutinin fusion peptide. The continuous
line (difference between octanol and interfacial values) represents the free energy of transfer
from octanol to interface (positive values denote higher tendency to partition into octanol than
into membrane interfaces). (B) Proposed model for the interaction of the influenza hemagglutinin fusion peptide with membranes. Only the interface at the external monolayer of the target
bilayer appears indicated. Residues have been fixed as to adopt an α -helical conformation with
the chain main axis tilted 12° with respect to the bilayer plane (Hristova et al., 1999). The chain
has been immersed into the interface until the phenyl moiety of Phe3 almost transferred into
the hydrocarbon core. Displayed side chains correspond to Leu2, Phe3, Ile6, Phe9, Ile10, Trp14,
Met17, and Ile18. The sequence was modeled using the ‘‘Swiss-PDBviewer’’ program.
mechanisms shared by different groups of viral fusion proteins, it would be desirable
in the future to carry out detailed analyses on the spatial distribution of interfacial
hydrophobicity along these sequences. Identification of preferential distributions of
hydrophobic-at-interface regions, or similar segmentation patterns, might lead to a
better understanding of the molecular mechanism by which these proteins promote
membrane fusion.
In summary, the construction of Wimley–White plots might constitute in the
near future the basis for the subsequent development of strategies meant to identify
functional regions in viral envelope proteins.
ACKNOWLEDGEMENTS
The authors wish to thank Professors William R. Gallaher and Félix M. Goñi
and Dr. Ana R. Viguera for many helpful discussions. Financial support obtained
from DGCYT (grant PB96-0171), the Basque government (PI 96-46; EX-1998-28;
PI-1998-32) and the University of the Basque Country (UPV 042.310-EA085兾97;
UPV 042.310-G03兾98) is also acknowledged.
532
Nieva and Suárez
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