 |
INTRODUCTION |
Fusion between enveloped viruses and host cells is an essential
step for viral infection (1). The fusion domain within the
transmembrane (TM)1 proteins
has been shown to be responsible for virus-cell fusion (2). For a
majority of viruses, the fusion peptide, which penetrates into the
target membrane, has been found at the NH2 terminus of the
TM subunit (3). A conformational change in the fusion domain of the
virus, triggered by low pH (4) or binding to the receptors (5) leading
to exposure of the fusion peptide, has been proposed to be an important
intermediate step for virus-mediated fusions (for review, see Ref. 6).
As suggested by many experiments, the fusion proteins oligomerize in
the fusogenic state (7), and the fusion pore is believed to be composed
of several oligomeric units of fusion proteins (8-10). Thus,
accumulation of the virus glycoproteins has also been found at the
cell-cell contact region (11). Recently an early fusion active
conformation of HIV-1 TM gp41 has been detected using an inhibitory
synthetic peptide (12).
Recent structural studies by x-ray crystallography on the hemagglutinin
of influenza virus (7) and the TM glycoprotein gp41 of human
immunodeficiency virus revealed that the core of the fusion domain
consists of an inner triple-stranded coiled-coil buttressed by three
helices formed by the COOH-terminal region of the ectodomain of the
envelope glycoproteins (13-15). An anti-parallel core complex of the
fusion domain of fusion-mediating virus has also been deduced from a
recent solution NMR study on a truncated protein derived from the
ectodomain of simian immunodeficiency virus (16). The common features
of these helix bundles are that the central coiled-coil trimer is
formed by the leucine zipper-like heptad repeat sequence and that the
height of the cylinder of the complexes is at least 60 Å. Thus, it is
of interest to examine how the fusion complex containing several long
rigid cylinders initiates approach of the merging membranes.
The function of the NH2-terminal fusion peptide of gp41 has
been characterized, and the ensuing more polar region has been shown to
associate with the surface subunit, gp120, of the external domain of
HIV-1 env glycoprotein. These two regions of gp41 have also
been implicated in the viral fusion by mutational studies (17). Despite
elucidation of the core of the viral fusion domain complex by the x-ray
diffraction and NMR studies, the structural role in the fusion event of
the fusion peptide and the intervening sequence between it and the
zipper-like region has not been addressed.
To better define the role of a given domain in the functions of a
protein, a peptide corresponding to that segment can be studied
(18-20). In particular, the NH2-terminal hydrophobic
peptides have been found to cause cell lysis, to induce electric
current, and to promote lipid mixing in liposomes (21, 22). A peptide corresponding to amino acids 5 through 16 of gp41 has been found to
induce current under external applied voltage (21). The interaction with SDS micelle, on the atomic scale, of the 23-amino acid peptide corresponding to the NH2-terminal region of gp41 has been
studied recently (23). It was found that Ala-Gly dipeptide, located 15 and 16 residues from the NH2 terminus of gp41, is at the
micellar-aqueous boundary and the region COOH-terminal to it is flexible.
In view of the highly conserved nature of the NH2-terminal
region of gp41 implying its important structural role in the fusion (24), we report here the structure and conformations of a peptide encompassing amino acids 5 through 55 of gp41, under varying solution conditions. Using NMR, circular dichroism spectroscopies, light scattering, and gel filtration chromatography we attempt to further unravel the virus-mediated cell fusion in light of the reported oligomeric core structure of gp41 (13, 14, 16). The NMR methods are
particularly suited for our purposes because they afford atomic
resolution, the observation of conformational change in aqueous and
membrane-mimic environments, and information regarding the flexibility
of the specific regions in the peptides or proteins under study. In
combination with the crystalline structural data on the core complex of
gp41 external domain, the results show the physical properties for the
NH2-terminal region of the TM glycoprotein gp41 and suggest
the conformational transition during the fusion event and the
hemifusion intermediate. Similar changes in conformation and assembly
state may also occur in the fusion process of other enveloped viruses
such as influenza virus.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The peptide
(formyl-GALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLL-OH,
gp41-N51) corresponding to amino acids 5-55 of gp41 and the
peptide (NH2-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-OH, gp41-C36) corresponding to amino acids 127-162 from strain
LAV1a (LAV, lymph adenopathy-associated virus) were
synthesized in an automated mode by a solid phase synthesizer from
Applied Biosystems (Foster City, CA) model 431A using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry. The peptides
were cleaved from the resins by trifluoroacetic acids and purified by
reverse phase high-performance liquid chromatography using a C8
column. The primary sequence of the peptide was ascertained by electrospray mass spectrometry as well as amino acid analysis.
SDS (sodium dodecyl sulfate) was acquired from Boehringer Mannheim
(Mannheim, Germany) and d25-SDS from Cambridge Isotope
(Andover, MA).
1,2-Ditetradecanoyl-sn-glycero-3-phosphocholine (PC) and
1,2-dihexadecanoyl-sn-glycero-3-phospho-L-serine
(PS) were acquired from Calbiochem (San Diego, CA). Lysozyme (molecular mass: 14.4 kDa) was purchased from Sigma. All reagents were used in the
experiments without further purification. Solutions containing vesicles
are prepared by solubilizing the lipids in chloroform:methanol (4:1,
v/v) mixture and drying the sample under nitrogen stream before
dissolving in buffer solution. Mixtures of peptides and SDS or
phospholipids were sonicated for at least 0.5 h before measurements.
NMR Experiments--
Micellar solutions containing 1 mM gp41-N51 and 60-100 mM d25-SDS
were used for NMR measurements. One-dimensional and two-dimensional nuclear Overhauser effect spectroscopy and total correlation
spectroscopy NMR experiments were performed in a Bruker AMX500
spectrometer as described previously (23). The NOE data
were converted into interproton distance using 2H/3H cross-peak of the
aromatic ring of phenylalanine as reference. A range of 0.6-1.0 Å was
allowed to vary in the distance constraints in the structural computation.
1 mM each of lysozyme and gp41-N51 in D2O
solutions was used in pulsed field gradient experiments for the
diffusion coefficient (Ds) determination.
Temperature was kept at 298 K. The signal attenuation at time 2
relative to that at time 0 is given by the equation,
|
(Eq. 1)
|
where
is the 1H gyromagnetic ratio,
is pulse
field gradient duration, G is the gradient strength in gauss
cm
1,
is the diffusion delay time. The diffusion
coefficient of methanol was used to calibrate the gradient strength. In
the experiments,
and
were kept at 11 and 14 ms, respectively,
while G was varied systematically. The maximum G
was set at 45 gauss cm
1.
According to Stokes-Einstein equation, Ds is
inversely proportional to the hydrodynamic radius of a spherical
particle, hence it is inversely proportional to the molecular mass to
the one-third power.
Circular Dichroism Experiments--
For CD measurements, cells
with path lengths of 0.5-1.0 mm were employed in final 10-50
µM peptide concentrations. Vesicular samples were
adjusted to pH 7.0 by phosphate buffer. All the CD experiments were
carried on a Jasco 720 spectropolarimeter at ambient temperature. The
spectra were recorded from 190 to 260 nm at scanning rate of 10-50
nm·min
1 with a time constant of 2 or 4 s. Each of
the CD data was obtained from an average of three to four scans with
step resolution of 0.05 nm and bandwidth of 1 nm.
CD data are represented by the mean residue ellipticity [
]
(degree·cm2·mol
1), obtained from the
observed ellipticity (
) according to the following equation [
] =
·l
1·c
1·N
1
where l is the cell length in mm, c is the molar
concentration, and N is the number of amino acid residues in
the peptide. Quantitative prediction of the secondary structure was
accomplished by fitting CD data with Hennessey-Johnson algorithm (25),
using 33 proteins of known secondary structure as the basis set.
Light Scattering Experiments--
Measurements were performed on
a DLS-7000 light scattering photometer (Photal Otsuka Electronics,
Osaka, Japan) with control unit LS-71 using He-Ne laser light source of
10 milliwatts at 632.8 nm. The instrument was operated with a software
controlled stepping motor driven goniometer. The measurements were made
at 10° interval in the scattering angle over the range of
40°-140° at room temperature. Samples were dissolved in water and
filtered twice before measurements. The concentration used in the study was 1.4 mg·ml
1. Lysozyme was used as the molecular mass
reference. The molecular weight of samples measured at low
concentration can be obtained from the following equation,
|
(Eq. 2)
|
where K is the optics
constant,2 c is
the concentration, Mw is the average molecular weight,
R
is the Rayleigh ratio, RG is the radius of gyration of the molecule,
is scattering angle, and
is the wavelength of incident light
(for review, see Ref. 26).
Gel Filtration Chromatography--
The experiments were
performed in a Hitachi L-6000 liquid chromatography using a
Superdex-75 HR 10/30 column (Pharmacia). The peptide and protein
samples of 0.4-0.8 mg·ml
1 concentrations were eluted
with solution containing 22% acetonitrile and 0.1% trifluoroacetic
acid with a flow rate of 0.5 ml·min
1. Data were
recorded at a wavelength of 214 nm. Transferrin, ovalbumin, ribonuclease A, and aprotinin were used as molecular mass markers (molecular mass: 81, 43, 13.7, and 6.5 kDa, respectively).
Structure Calculations--
A total of 480, including 232 nonsequential, NOE interactions and 50 intraresidue dihedral angles,
were utilized in the structural computations using DG/simulated
annealing protocols of Biosym programs InsightII, Discover, and
NMRchitect (version 2.3) from Biosym Technologies, Inc. (San Diego,
CA). In simulated annealing protocol, the temperature was raised to
1000 K and a molecular dynamics run was carried out for 8 ps to allow
more conformational space to be explored. The system was subsequently
annealed to 300 K in 10 steps for a total 35 ps and minimized by the
steepest-descent and conjugated gradients methods before final refined
structures were obtained.
 |
RESULTS |
NMR Data Indicate Enhanced Helical Structure for gp41-N51 in the
Presence of SDS Micelles--
Sequential assignment of 1H
NMR chemical shift of gp41-N51 in the SDS micellar dispersion was
accomplished from the fingerprint (Fig.
1A) and NH/NH regions (Fig.
1B) to resolve overlapping cross-peaks. The 1H
chemical shift assignment of the peptide in aqueous solution devoid of
the detergent dispersion was made with the same strategy. The NOE
interactions in these two solutions are summarized in Fig. 1,
C and D (chemical shifts of 1H
resonances for the peptide in SDS micellar solution and in water are
tabulated in Table I). Resonance peaks of
the
H of Gly1, Gly6, Gly12, and
Gly16 in SDS micellar solution are split, suggesting a
certain degree of structural rigidity in the NH2-terminal
region of the peptide. In contrast, in water
H of glycine residues,
with the exception of Gly6, give rise to degenerate
chemical shifts, implying more flexible conformation for the peptide in
aqueous solution. This is likely due to the fact that Gly6
is in the FLG motif shown to form a type I
-turn in the fusion peptide of gp41 in aqueous solution (27).

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Fig. 1.
The fingerprint region
(A) and NH/NH region (B) of NOE
spectrum of gp41-N51 in SDS micellar solution and summary of
the NOE interactions for the peptide in SDS micellar solution
(C) and in water (D). The
leucine zipper region starts with Ile44. Potential NOE
peaks due to spectral overlap are designated by the dotted
line. The NOE peaks are more sparse in the region
Ala12-Leu22.
dNN(i,j) denotes the NOE cross-peak
arising from NH of residue i and NH of residue j;
dSX(i,j) denotes the NOE cross-peaks
of the side chain protons of residues i with any protons
from residue j.
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|
To identify the secondary structure, the chemical shifts are compared
with the random coil values as compiled by Wishart and Sykes (28), and
the result is presented in Fig. 2. In
water (Fig. 2B), the helical structure can be detected in
the Gln25-Leu30 stretch and is more prominent
in the region COOH-terminal to Gln35. This result is in
agreement with the NOE results shown in Fig. 1D where
d(i,i + 3) interactions, characteristic of
helical form, are observed in these regions. In the presence of SDS
micelles (Fig. 2A), on the other hand, the regions adopting
helical structure encompass Leu2-Gly12,
Val24-Val34, and
Asn38-Leu50. The sequence following
Ile44 has the zipper 4-3 repeat pattern and thus is
expected to have high propensity of forming helical structure. The
NH2-terminal fusion peptide of gp41 has been shown to
insert into membranous structure primarily as a helix (23). In addition
to the helix formation of the NH2-terminal fusion peptide
in SDS solution, enhancement of the helical form in the micellar medium
is also observed for the region Gln25-Ser31
and the region following Gln35. This is evident from the
larger chemical shift deviation of
H and
H from the random values
in SDS solution than those obtained in water as shown in Fig. 2. The
present data for gp41-N51 thus corroborate with the idea that helix is
induced in the micellar environment.

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Fig. 2.
Chemical shift deviation of
(solid circles) and
(empty circles) protons for each
of the residues of gp41-N51 in SDS solution (A) and in
water (B). Negative values for H and positive
values for H represent the helical structure, while the reverse
indicates a -strand structure.
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|
CD Data Also Reveal Enhancement in
-Helix for gp41-N51 in
Vesicular and Micellar Solutions--
The CD results displayed in Fig.
3 are consistent with the NMR data in
that the helical content for the peptide is significantly increased
upon association with SDS micelle. The helical structure is also
enhanced in both PS and PC vesicular solutions. From the CD data, the
secondary structural element analyzed by the algorithm of Hennessey and
Johnson (25) for gp41-N51 is summarized in Table
II. The result clearly indicates an
increase in the helix population in the membrane-mimic environment,
especially in dispersions that carry negatively charged headgroups.

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Fig. 3.
Far-UV-CD spectrum of gp41-N51 in water
( ) and in SDS micellar (- - - -), PS-PC
vesicular (molar ratio of peptide:PS:PC = 1:75:25,· · · · ; = 1:25:75, · · ·) solutions.
It appears that the helicity of the peptide is higher in the
presence of negatively charged micelle or vesicle.
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Table II
Secondary structure analysis from CD data of gp41-N51 in aqueous, SDS,
and lipidic solutions (in %)
Data were obtained from CD spectra shown in Fig. 3.
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|
Oligomerization of gp41-N51 in Water Is Indicated by Light
Scattering Experiments--
The results of light scattering
measurements on gp41-N51 and lysozyme are presented in Fig.
4. The intercept for gp41-N51 is
5.71 × 10
5, which yields a particle mass of 17.5 kDa. The value is approximately three times the monomeric mass of 5.4 kDa for gp41-N51. The molecular weight determined by light scattering
experiments is a weight-averaged value, hence the data indicate that
the peptide molecules are predominantly trimeric or they are in an
equilibrium between dimers and tetramers. The error estimated from the
difference between the experimental and reported values of lysozyme is
9%.

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Fig. 4.
Light scattering data on gp41-N51 (×) and
lysozyme ( , molecular mass: 14.4 kDa) in water. The
intercepts of Kc/R
versus sin2( /2), representing the reciprocal
of the average molecular mass, are 5.71 × 10 5 and
6.37 × 10 5, respectively, for gp41-N51 and lysozyme
at room temperature. The data thus yield molecular masses of 17.5 ± 1.4 and 15.7 ± 1.0 kDa, respectively, for gp41-N51 and
lysozyme, indicating that the NH2-terminal portion of gp41
exists in aqueous solution approximately as trimers.
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|
Gel Filtration Experiment and Diffusion Coefficient Measurement
Support Oligomerization of gp41-N51 Molecules in Aqueous
Solution--
The gel filtration data on gp41-N51 displayed in Fig.
5A may arise from dimerization
of the peptide. Oligomerization of the NH2-terminal peptide
of gp41 is also demonstrated by diffusion coefficient measurements from
NMR pulsed field gradient experiments shown in Fig. 5B. The
Ds of the peptide is slightly larger than that of
lysozyme with a molecular mass of 14.4 kDa. Thus, as in the light
scattering experiments, diffusion coefficient measurements indicate
that the peptide molecules exist in the aqueous solution as trimers, or
there is equilibrium between dimers and tetramers for the peptide. It
is of interest to observe that a particle mass of about 15 kDa is
obtained for the peptide in the concentration on the order of 1 mg·ml
1 for light scattering and NMR experiments, while
smaller size is detected at lower concentration employed in gel
filtration experiment. The oligomerization of the
NH2-terminal peptide of shorter length encompassing 33 amino acids has also been reported by Kliger et al.
(29).

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Fig. 5.
A, gel filtration trace of gp41-N51 in
aqueous solution. The positions of molecular mass standards are marked.
B, diffusion coefficient measurements of gp41-N51 (×) and
lysozyme ( , molecular mass: 14.4 kDa) in aqueous solution at 298 K. Ds values as determined from the slope for gp41-N51
and lysozyme are 1.09 × 10 6 and 1.05 × 10 6 cm2·s 1, respectively.
Self-assembly for the peptide is clearly indicated by the light
scattering result demonstrated in Fig. 4 and these data.
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Three-dimensional Structures of gp41-N51 Derived from NOE Data
Suggest a Flexible Segment Adjacent to the Hydrophobic
NH2-terminal Region--
The three-dimensional structure
of gp41-N51 in the presence of SDS micelles was calculated by employing
the constraints derived from NOE data and hydrogen bonds suggested by
the deuteron-hydrogen exchange experiments. Fig.
6A displays a typical computed
structure and the superposition of 10 structures excluding residues
between Ala10 and Val24. As expected, the
helical form can be observed in the NH2-terminal region up
to Ala11, and the segments
Leu22-Val34 and
Asn38-Leu50. The sequence between
Ala11 and Val24 exhibits substantial
flexibility as indicated by Fig. 6B, which shows larger root
mean square deviation values of backbone atoms for these residues than
residues in the remainder of the peptide as analyzed from the 10 conformers used in Fig. 6A. This flexible region overlaps
with the gp120-associating site implicated in the mutagenesis studies
(1).

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Fig. 6.
A, superposition of 10 structures of
gp41-N51 calculated based on NOE constraints derived from NMR data of
the peptide in SDS solution. The sequence between Ala10 and
Val24, represented by two ribbons, is disordered. A
310 helix can be discerned for
Gln35-Asn38 segment. B, root mean
square deviation of backbone atoms for residues of gp41-N51 analyzed
from the 10 structures displayed in A. Considering the
central Leu5-Gln47 portion free of fraying-end
effect, Ala10-Thr23 section exhibits large
fluctuation, indicating a flexible region. The large root mean square
deviation value for Asn38 suggests that the
Gln35-Asn38 310 helix is readily
transformed into an -helix.
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|
The root mean square deviation values of atomic fluctuation for
Asn38 is substantially larger than those of the adjacent
residues. In addition, a 310 helix is found for the
Gln35-Gln37 stretch (
(35) =
71 ± 9°,
(35) =
30 ± 7°,
(36) =
82 ± 11°,
(36) =
37 ± 14°, and
(37) =
79 ± 22°,
(37) =
35 ± 13°), which is readily transformed into
-helix. The
structures are thus convergent except for the segment spanning
Ala11-Leu22. Aside from both ends of gp41-N51
that are subject to the fraying effect, the larger fluctuation is seen
for the Ala11-Leu22 region. The result concurs
with the idea that the Ala11-Gly12 dipeptide
is localized at the micellar-aqueous boundary, as noted previously
(23), and determines the depth of penetration for the fusion peptide
into the micelle. As will be discussed in the next section, the
flexibility of this segment may have implication in the fusogenic
activity of the NH2-terminal portion of gp41.
 |
DISCUSSION |
Conformational Change of the Hydrophobic Fusion Peptide in
Membrane-mimic Environment and the Flexibility of the Adjacent Polar
Region of gp41 Suggest an Essential Role for These Two Regions in the
Virus-mediated Fusion Process--
Our CD data in water, the micellar
and vesicular solutions for both the 23-amino acid fusion peptide
sequence of gp41 in a previous report (23), and a longer gp41-N51
peptide indicate that, in the membranous environment, the helix form is
induced at the expense of
-form or random coil for the
NH2-terminal hydrophobic stretch of gp41. As shown
previously (23), these data suggest that the fusion peptide penetrates
into the membrane primarily as a helix. In the present study,
conformational adaptability of gp41-N51 on the amino acid level
exhibited in the presence and absence of membrane-mimic environment is
provided by NMR data. However, conformation of the more polar sequence
following the fusion peptide, Ala12-Leu22, was
found more flexible from structural analysis of NMR data (Fig. 6). This
conclusion is also consistent with scarcity of NOE cross-peaks
involving protons in the residues of
Ala12-Leu22 segment, likely due to the smaller
motional correlation times and/or more disordered structure, both of
which are compatible with the idea of a flexible region. Because the
segment was found to be essential for association with gp120 (2, 30),
the flexibility of this region in the membranous medium suggests that
it plays a role in the conformational change during the
native-to-fusogenic transformation of gp41, which is likely triggered
by the dissociation of gp120 from gp41 upon binding to CD4 and the
secondary receptor (for example, CCR5 or CXCR4).
As indicated in Figs. 2, A and B, the FLG motif
(Phe4-Gly6) undergoes a
to
transition
as the peptide interacts with the SDS micelle. Moreover, the motif has
been shown to serve as an initiation site for helix formation (27).
Together with insertion into the micelle of the
NH2-terminal Gly1-Ala10 region, as
suggested by the spin label attenuation data (23), these results
suggest that the FLG motif may make initial contact with, and penetrate
into, the membrane as part of the conformational change of gp41
following binding of the HIV envelope glycoprotein to its receptors in
the fusion event (31, 32).
The Ala11-Gly12 region where the structures
begin to diverge as shown in Fig. 6B coincides with the site
found at the micellar-aqueous interface from spin label attenuation
experiment for the fusion peptide in SDS solution. These results are
reasonable in that the segment of the peptide in the micellar interior
is more restricted than the region lying on the external surface of the
micelle. Since the hydrocarbon chains of the cellular membrane are
generally longer than that of SDS micelle, our data indicate that the
inner leaflet of the target membrane bilayer is not likely to be
reached by the viral fusion peptide during fusion. Therefore, it is
probably that the direct effect of viral fusion peptide is to cause the hemifusion, as first suggested by White and co-workers on the fusion
mediated by hemagglutinin of the influenza virus (33).
The increase in helicity for the segment
Gln25-Ser31 and the stretch
Ser19-Val24 (Fig. 2) in the presence of SDS
micelles strongly suggests that these changes are part of the
structural transition in gp41 as it transforms from fusion-inactive to
fusion-active state. As already mentioned, the
Gln35-Asn38 stretch is likely to be helical in
the fusogenic state. The enhancement of helical form for gp41-N51 in
the membranous medium suggests that helix is the dominant form during
membrane fusion.
Oligomerization of gp41-N51 in Water Detected by Light Scattering,
Gel Filtration, and Diffusion Coefficient Measurements Indicates the
Propensity of Self-assembly of the Peptide and Suggest a Functional
Role of Merging Viral and Target Cell Membranes for the
NH2-terminal Portion of gp41--
The oligomerization
state of the envelope glycoprotein of HIV-1 has been examined by
various techniques, which yielded differing results of dimeric,
trimeric, and tetrameric structures (13, 14, 34). It is, however,
agreed that the membrane-anchoring subunit gp41 is primarily
responsible for the oligomerization. In the present work, the peptide
corresponding to the NH2-terminal region of gp41 was found
to be oligomeric in water. Although the mass values deduced from gel
filtration and diffusion coefficient data depend somewhat on the
molecular shape, the experiments point to a non-monomeric state for
gp41-N51. However, more definitive result on the oligomerization is
provided by light scattering data which give the absolute molecular
mass from the intercept of a typical Zimm plot (26, 35). Dimers are
apparently present in the more dilute solution for the gel filtration
experiment, whereas trimers or mixtures of dimers and tetramers can
account for data obtained in the more concentrated solution for NMR
diffusion coefficient measurements. The latter conclusion is in
agreement with the result obtained from the light scattering experiment in the same concentration range. Hence it is probable that different association states found in various studies arise from different conditions under which experiments are performed. Different
oligomerization and assembly states might also be present at various
stages of fusion event. Since gp41-N51 peptide molecules oligomerize in aqueous environment, it is likely that the NH2-terminal
portion of gp41 penetrates into the target membrane in oligomeric form during some stage of fusion process.
Analogous examples for the zipper-like domain to induce oligomerization
have been reported for the protein A/gp41(538-593) chimeric protein
(36) and for the maltose-binding protein/gp41(558-595) chimeric
protein (34). The zipper-like domain encompassing amino acids 555-583
of gp41 is responsible for oligomerization of the chimeric proteins,
since both protein A and maltose-binding protein are monomeric (34,
36). The stability of oligomerization is apparently critical in merging
the phospholipids of the two apposing membranes. This is supported by
the observation that a mutation in gp41 fusion peptide dominantly
interfered with fusion and infectivity (37), which can be rationalized
by participation of the defective mutant protein in the oligomer,
rendering the fusion complex ineffective.
It has been shown by a number of investigators that the peptide
corresponding to the hydrophobic NH2-terminal fusion
peptide sequence is sufficient to cause leakage and phospholipid mixing of the vesicular bilayers (22, 38). The vesicle and micelle are more
prone to fuse than planar cellular membrane because of their smaller
radius of curvature (39). Thus, to effect the fusion of planar bilayers
of the viral and cellular membranes, it is possible that the core
oligomeric complex formed by the zipper-like coiled-coil and
COOH-terminal helices of gp41 ectodomain acts as the joint of a
molecular nipper to hold the oligomeric structure, while the fusion
peptide segments that penetrate into the membranes and the flexible
polar border sequences would bring the two opposing membranes into
contact by acting as the arms of a nipper. More explicitly, since the
extended rod-like cylinder is at least 60 Å in length, the helical
length would be extended to more than 90 Å if the intervening sequence
between zipper-like domain and the hydrophobic fusion peptide also
assumes rigid rod conformation, making approach of the two apposing
membranes impossible. The flexibility of the
Ala11-Leu22 stretch, illustrated in Fig. 6,
A and B, and the propensity of oligomerization of
the NH2-portion of gp41 render possible direct contact of
phospholipids of the opposing membranes (Fig.
7). This is done by bending the
Ala11-Leu22 segment and the base of the gp41
ectodomain to make the core oligomeric complex parallel to the membrane
surfaces. Flexibility of the sequence near the base of gp41 ectodomain
was suggested by the crystallographic study of the NH2- and
COOH-terminal helices of gp41.

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Fig. 7.
Proposed model for the interaction of gp41
with target and viral membranes during fusion process. Top
panel, the core oligomeric coiled-coils associating with the
COOH-terminal helices of gp41 ectodomain. The NH2-terminal
fusion peptide is primarily in - and random forms and the sequence
between the fusion peptide and zipper-like domain, which may have a
loose structure, is not completely dissociated from gp120 and/or the
co-receptor on the target membrane. This is due to the highly
hydrophobic nature of the fusion peptide, which does not favor its
complete exposure to the polar solvent. Initial contact of gp41 with
the target membrane is probably through the FLG motif.
Middle, complete dissociation of gp41 from other components
of fusion complex exposes the fusion peptide, which subsequently
inserts into, and destabilizes, both the target and viral membranes
primarily as a helix. Helical structure is also enhanced for the
sequence NH2-terminal to the zipper-like region. According
to the determined depth of penetration of fusion peptide, only the
outer leaflet of the membrane bilayer of target cell has direct contact
with the viral fusion peptide. Driven probably by the oligomeric
coiled-coil zipper-like domain, the NH2-terminal region of
the gp41 molecules self-associate. Propensity of the
NH2-terminal region of gp41 to oligomerize, in turn,
induces bulged surfaces on the two approximate membranes when the
fusion peptides insert into these two membranes, thus facilitating the
membrane merger. Alternatively, the interaction of the flexible region
of gp41-N51 with the base region of the gp41 ectodomain can promote
membrane fusion. Note that some hydrophobic NH2-terminal
sequences may insert into the viral membrane. The straddling of the
NH2 termini from a core complex of gp41 over the target and
viral membranes and the tendency to oligomerize by the
NH2-terminal regions of gp41 provide the energy required to
dehydrate the membranes to be merged. As a consequence, the two
opposing membranes are brought into contact and dehydration occurs as
the two membranes begin to attach. From the figure it can be visualized
that the importance of the pliability of the segment
Ala11-Leu22 to allow the core coiled-coil
rod-like structure with length greater than 60 Å to perform its function in bringing the two
membranes into contact. Bottom, the two membranes merge, and
several units of fusion core oligomer of gp41 assemble to form the
fusion pore to allow the flow of nucleocapsid of the virus. Initial
lipid mixing probably involves only the outer leaflets of the two
membranes, i.e. stalk formation, as suggested by the finding
that the fusion peptides of gp41 do not reach the inner leaflet of
either the target or viral membrane bilayer.
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Energetically, a driving force to merge the two approaching membranes
is provided by the tendency for the NH2-terminal portion of
gp41, represented by gp41-N51, to self-assemble and/or by the interaction between the NH2-terminal region and the
COOH-terminal region of the gp41 external domain. The latter hypothesis
is based on the fluorescence data on gp41-C36 incubated with gp41-N51
(see Fig. 8). The model illustrated in
Fig. 7 emphasizes the essential role in bringing the two fusing
membranes into approximation played by the segment
NH2-terminal to the zipper-like domain of gp41. Because the
core antiparallel helix bundle of gp41 functions as the joint or hinge
of a nipper, it cannot exert force on the target or viral membranes.
The latter function is then furnished by the arm of the nipper, the
polar intervening sequence (Ala11-Leu22) of
gp41, and/or by its interacting with the segment near the base of gp41
ectodomain. It must be noted that the rigid structure such as the core
hetero-helix bundle cannot directly exert force on the membranes.
Rather, closing of flexible arms (Ala11-Leu22
of gp41-N51), due to the tendency to oligomerize for the
NH2-terminal portion of gp41 extending from the core
structure and/or the base region of the gp41 ectodomain can promote
membrane coalescence, as illustrated in Fig. 7. It is stressed that the
fusion peptide, because of its hydrophobicity, is not likely to be
completely exposed to solvent during the entire fusion process. The
fusion peptide may be ushered by other component(s) of the fusion
reaction, such as gp120 or the receptor or the co-receptor, to the
target membrane.

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Fig. 8.
Fluorescence spectra of gp41-C36 and its
complex with gp41-N51 in aqueous solution. Molar ratio of the two
peptides is indicated for the complexes. The right panel
shows the second order derivative of the traces of the left
panel. A second component with maximum at 329 nm is more evident
for higher ratio of gp41-N51, suggesting an interaction of the
COOH-terminal region of gp41-C36 with gp41-N51. Method: the
fluorescence experiments on gp41-C36 were performed on a Jasco FP-777
spectrofluorometer at concentrations of 5-15 µM using a
cell of 1 cm in length at ambient temperature. An excitation wavelength
of 280 nm was used to record spectra from 300 to 450 nm with a scan
rate of 100 nm·min 1, response time of 1 s, and
data interval of 0.1 or 0.2 nm. The bandwidths for excitation and
emission were 5 and 1.5 nm, respectively.
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Membrane fusion is an energy costing event, since it involves
destabilization of the bilayers and dehydration of the interface regions of merging membranes (40). This notion is also consistent with
the low efficiency of productive entry by the virus into the target
cell (41). The insertion of the hydrophobic fusion peptide makes
initial perturbation of the target membrane. Propensity of the region
encompassing gp41-N51 for oligomerization and the interaction between
the flexible, more polar NH2-terminal region outside the
membranes and the segment preceding the membrane-anchoring sequence of
gp41 probably helps to overcome the hurdle of membrane dehydration
during the membrane merging.
In summary, study of the NH2-terminal region of gp41
complements the structural investigation of core complex formed by the oligomers of the zipper-like domain and the COOH-terminal helix. The
findings of flexibility of the Ala11-Val24
segment and the tendency of forming oligomers by gp41-N51 and its
interaction with the region near the base of the ectodomain of gp41
along with increased helicity in the membranous medium for the peptide
suggests an structural role of the fusogenic activity of the
NH2-terminal portion of gp41. Our data also imply a stalk and a hemifusion intermediate for the fusion reaction mediated by HIV.
Similar mechanism may be used by other enveloped viruses in mediating
cell fusion.