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Journal of Virology, February 2000, p. 1961-1972, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Oligomeric Modeling and Electrostatic Analysis of
the gp120 Envelope Glycoprotein of Human Immunodeficiency
Virus
Peter D.
Kwong,1,*
Richard
Wyatt,2
Quentin J.
Sattentau,3,
Joseph
Sodroski,2,4 and
Wayne A.
Hendrickson1,5
Department of Biochemistry and Molecular
Biophysics1 and Howard Hughes Medical
Institute,5 Columbia University, New York, New
York 10032; Department of Cancer Immunology and AIDS,
Dana-Farber Cancer Institute, and Department of Pathology, Harvard
Medical School,2 and Department of
Immunology and Infectious Diseases, Harvard School of Public
Health,4 Boston, Massachusetts 02115; and
Centre d'Immunologie de Marseille-Luminy, 13288 Marseille
Cedex 9, France3
Received 25 June 1999/Accepted 17 November 1999
 |
ABSTRACT |
The human immunodeficiency virus envelope glycoproteins, gp120 and
gp41, function in cell entry by binding to CD4 and a chemokine receptor
on the cell surface and orchestrating the direct fusion of the viral
and target cell membranes. On the virion surface, three gp120 molecules
associate noncovalently with the ectodomain of the gp41 trimer to form
the envelope oligomer. Although an atomic-level structure of a
monomeric gp120 core has been determined, the structure of the oligomer
is unknown. Here, the orientation of gp120 in the oligomer is modeled
by using quantifiable criteria of carbohydrate exposure, occlusion of
conserved residues, and steric considerations with regard to the
binding of the neutralizing antibody 17b. Applying similar modeling
techniques to influenza virus hemagglutinin suggests a rotational
accuracy for the oriented gp120 of better than 10°. The model shows
that CD4 binds obliquely, such that multiple CD4 molecules bound to the
same oligomer have their membrane-spanning portions separated by at
least 190 Å. The chemokine receptor, in contrast, binds to a
sterically restricted surface close to the trimer axis. Electrostatic
analyses reveal a basic region which faces away from the virus, toward
the target cell membrane, and is conserved on core gp120. The
electrostatic potentials of this region are strongly influenced by the
overall charge, but not the precise structure, of the third variable
(V3) loop. This dependence on charge and not structure may make
electrostatic interactions between this basic region and the cell
difficult to target therapeutically and may also provide a means of
viral escape from immune system surveillance.
 |
INTRODUCTION |
The human immunodeficiency viruses
(types 1 [HIV-1] and 2 [HIV-2]) and related simian viruses (SIVs)
cause the depletion and functional dysregulation of CD4+
lymphocytes, resulting in the development of AIDS. The HIV envelope contains two glycoproteins: gp120, the exterior receptor-binding component, and its noncovalently interacting partner, gp41, the transmembrane envelope glycoprotein. Only half of gp41 is exposed in
the ectodomain; the other half, separated by a transmembrane region, is
thought to anchor the envelope complex to the underlying matrix. New
infections are initiated by interaction of gp120 with the N-terminal
membrane-distal domain of CD4, a glycoprotein on the surface of
specific cells of the immune system (12, 29). A second
interaction of gp120, with a member of the chemokine receptor family,
primarily CCR5 or CXCR4, is believed to trigger conformational
changes in gp41, which ultimately mediates virus-cell membrane
fusion (20, 38).
HIV receptor binding takes place in the context of an oligomeric viral
spike. Atomic-level structures have been determined for many of the
component molecules: the entire extracellular portion of CD4
(60), the complex of monomeric core gp120 (a truncated
version of gp120 with deletions of the gp41-interactive region at the N
and C termini as well as of the variable V1/V2 and V3 loops) with the
two N-terminal domains of CD4 and the antigen binding fragment (Fab) of
the neutralizing antibody 17b (32), and a final
fusion-active state of the gp41 trimer (10, 52, 56). Despite
extensive effort, the structure of the oligomeric spike has resisted
atomic-level investigation and is only known from electron microscopy
(21, 22).
Accumulating evidence suggests that the HIV viral spike is a trimer of
gp120-gp41 heterodimers. The most convincing evidence comes from the
structural resemblance of the fusion-active state of gp41 to other
fusion-active trimeric coiled-coils, including the equivalent
transmembrane envelope proteins from Moloney murine leukemia virus
(19), influenza virus (6), and Ebola virus (55). Other suggestive evidence comes from the introduction of cysteines into the coiled-coil to create disulfide-stabilized trimers (16), the trimeric nature of the underlying HIV
matrix which interacts with gp41 (25), the trimerization of
various ectodomain constructs of gp120-gp41 (X. Yang, L. Florin, M. Farzan, P. Kolchinsky, P. D. Kwong, J. Sodroski, and R. Wyatt,
submitted for publication) and the therapeutic success of a peptide
which appears to work by stabilizing an intermediate trimeric state in
the gp120-gp41 fusion process (11, 28, 58).
Although the conformation of core gp120 is known by antigenic studies
to be similar in the gp120-gp41 complex, molecular docking of gp120
onto the trimeric gp41 is not feasible because gp41 undergoes large
conformational changes (6, 10, 56). Nonetheless, by
correlating the antigenic map of gp120 with its atomic structure, a
preliminary model of oligomeric gp120 was defined (62).
Because this model was based on antibody binding, which occurs in the context of a ~600-Å2 epitope, it was of relatively low
resolution. Still it showed that the regions of the oligomer facing the
target cell after CD4 binding consisted of two components: a conserved
portion of the core and a sequence-variable excursion, the V3 loop.
These components have been shown by mutational analysis to interact with the chemokine receptor CCR5 (45).
Here we more precisely model the HIV-1 envelope glycoprotein oligomer,
using quantifiable criteria based on carbohydrate exposure, occlusion
of conserved surface residues that are solvent exposed on the gp120
protomer, and steric constraints imposed by the binding of the 17b
antibody. We have applied the same modeling techniques to influenza
virus hemagglutinin to estimate the modeling precision, since the
structures of both the monomeric "HA top" of hemagglutinin (the
gp120 core equivalent) and the HA1-HA2 heterotrimer (the gp120-gp41
complex equivalent) have been determined (4, 59). We have
also investigated the electrostatic nature of the gp120 region facing
the target cell, examining in particular the dependence of the
potential on the structure and overall charge of the V3 loop. In a
companion study (39), we tested the electrostatic predictions of our model on the binding of heparan sulfate and dextran
sulfate to different variants of gp120. Finally, we examine the
consequences of our results with regard to initial virus-cell attachment, viral mechanisms of immune evasion, and the feasibility of
anion-based therapeutic strategies.
 |
MATERIALS AND METHODS |
Structural modeling of the exterior envelope oligomer.
Superpositions, center-of-mass calculations, model rotations and
translations, radius of gyration analysis, and solvent exposure calculations were performed with the software XPLOR (5). The structure of the monomeric core gp120 complexed with the two
membrane-distal domains of CD4 (D1D2) and the neutralizing antibody 17b
(Protein Data Bank [PDB] accession code 1gc1) was superimposed onto the corresponding D1D2 domains (residues 1 to 178) of the structure of
the four-domain CD4 (the entire extracellular region) (PDB accession
code 1wio) (60). The superposition gave an RM5 deviation of
2.12 Å for all atoms in residues 1 to 178.
The coordinate system used for modeling the gp120 oligomer (Fig.
1) had the following properties: (i) the
z axis was coincident with the gp120 trimer axis; (ii) all
three rotational degrees of freedom were permitted; (iii) since the
coordinate system was threefold symmetric, only one other translation
axis, chosen here to be the x axis, was independent
(translations along the y axis were thus related by a
Z rotation and x axis translation); and (iv) the
initial position of the gp120 protomer was oriented by the
superposition described above and placed with its center of mass at
x = 35 Å and y = 0. (To distinguish
between translations and rotations, lowercase letters x,
y, and z are used to specify both the axis and
the translational position along each axis and uppercase letters
X, Y, and Z are used to specify the
rotations about each axis.)

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FIG. 1.
Coordinate system used to model the envelope oligomer
shown in the context of the various interactions of gp120 during
virus-cell attachment. CD4 (yellow) is shown reaching from the target
cell membrane (gray) to bind gp120 (120; brownish red), which is
arranged in a symmetrical fashion around the ectodomain of the trimeric
gp41 (41; blue), shown jutting out of the viral membrane (orange). The
V3 loop (green) can be seen on gp120 just above the cell membrane. The
coordinate system used for modeling has the z axis
coincident with the gp41 trimer axis, the x axis connecting
the center of mass of a gp120 protomer with the z axis, and
the y axis mutually perpendicular to the x and
z axis and sharing a common origin. Thus, X and
Y rotations (that is, about the x axis and
y axis, respectively) specify the orientation of a gp120
protomer with respect to the membranes, and rotations about the
z axis determine the orientation with respect to the
envelope oligomer.
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The gp120 protomer was rotated around its center of mass. The distance
from the
z axis of each of the various criteria chosen
for
quantification was calculated at 10° intervals. These calculations
were performed with the program GRASP, with the normal to the
z axis being determined by calculating the minimum of the
distance
matrix between the criterion being analyzed and a set of
pseudoatoms
placed at 1-Å intervals along the
z axis.
Three criteria were chosen for quantification: carbohydrate exposure,
conserved and exposed residues, and steric constraint
of the 17b Fab
fragment. For calculations of carbohydrate exposure,
the structure
of gp120 used was a model described previously (
62),
which
comprised the crystallographic core (
32), the modeled
residues 88 and 89 and the V4 loop, and the central
(
N-acetylglucosamine)
2(mannose)
3 glycan moiety of all N-linked glycosylations. Because the positions
of
the mannose residues were poorly defined in the model (
62),
the
N-acetylglucosamine residues, which are more proximal to
the
protein and appear less conformationally flexible, were used to
represent the carbohydrate. The molecular surface of the
N-acetylglucosamine
residues was constructed, and the
distance of this surface to
the
z axis was calculated as a
function of gp120
orientation.
Conserved residues were conserved across all HIV-1 isolates
(
32). The fractional solvent accessibility for individual
amino
acids of core gp120 which were extracted from the
1gc1 complex
was calculated as the ratio of the solvent-accessible surface
area for
atoms of an amino acid residue X in the protein to that
area obtained
after reducing the structure to a Gly-X-Gly tripeptide
(
47).
Residues were considered exposed if they had a solvent
accessibility of
more than 40%. The resultant set of conserved
and exposed residues on
the gp120 core (33 residues) was further
delineated by removal of those
within van der Waals radii of either
CD4 or the 17b Fab (10 residues
excluded) or those that were glycosylated
(2 residues excluded).
Finally, a clustering analysis which excluded
outliers more than 5 Å from the main cluster of conserved solvent-exposed
residues (six
residues excluded) was performed. The residues chosen
by imposing the
above criteria were 102, 103, 113, 114, 204, 208,
209, 211, 213, 214, 216, 221, 250, 439, and 491. The molecular
surface of these residues
was constructed, and the distance of
this surface to the
z
axis was calculated as a function of the
gp120
orientation.
To quantify steric constraint of the 17b Fab, its molecular surface was
calculated as oriented by the position of the gp120
protomer, and the
distance from this surface to the
z axis was
calculated.
This criterion was less strict than the others since
it was dependent
on translational positioning, for which there
was little constraint. To
account for this, an orientation was
considered sterically forbidden
only if the distance from the
17b surface to the
z axis was
less than 3 Å for both the orientation
being considered and the
previous 10° rotation, effectively adding
a 10° buffer zone to the
sterically forbidden
region.
Determination of modeling accuracy.
The accuracy of the
surface criterion optimization procedure was tested with the monomeric
HA top of influenza virus hemagglutinin complexed with Fab HC19 (PDB
accession code 2vir) (4). The "correct" oligomeric
orientation was defined by the HA1-HA2 heterotrimer (PDB accession code
5hmg) (54, 59), positioned with its trimer axis coincident
with the z axis. Superposition of the HA top onto a protomer
of the oriented oligomeric HA1-HA2 heterotrimer gave an RMS deviation
of 1.09 Å for all atoms in residues 43 to 309.
Quantification of surface criteria described above for gp120 was
performed on the HA top structure with several modifications.
For the
carbohydrate criterion, a BLAST search (
1) of the GenBank
database (release 113.0) with the HA top sequence enabled 485
HA1
sequences to be aligned. All sites of potential N-linked glycosylation
in the aligned sequences were identified. These mostly nonconserved
sites of glycosylation were at residues 45, 63, 81, 122, 126,
133, 144, 165, 246, 276, and 285. A model of the molecular surface
of the
nonbackbone portions of these residues in the HA top structure
(
2vir)
was constructed, and the distance from this surface
to the
z
axis was calculated as a function of the HA top
orientation.
For the conserved exposed residue criterion, a threshold of 99%
identity was used with the same 485 sequences. A 40% solvent
exposure
criterion was used, as calculated for the HA top (
2vir)
structure.
Because the receptor (sialic acid) is small and no
main cluster of
conserved exposed residues could be identified,
receptor distance
exclusion and outlier rejection were not used.
Conserved exposed
residues identified were 55, 57, 104, 107, 110,
129, 165, 169, 187, 208 to 210, 212, 221, 222, 225, 238, 240,
263, 269, 271, 285, 289 to 291, 293, 304, and 308. A model of
the molecular surface of the nonbackbone
portions of these residues
in the HA top structure was constructed, and
the distance of this
surface to the
z axis was calculated as
a function of the HA top
orientation.
Finally, for the steric constraint criterion, the Fab fragment of the
influenza virus-neutralizing antibody HC19 was used
in place of the
HIV-neutralizing antibody
17b.
V3 loop modeling.
In addition to the typical modeling
criteria, such as avoiding steric clashes and maximizing hydrogen
bonding, the position of the 17b antibody was used to provide
additional constraints since it is known that this antibody binds to
both native gp120 and V3 loop-truncated gp120. Three different models
of the V3 loop were constructed by using the program O (27).
Using the helix model, called alpha, the g and h helices of sperm whale myoglobin, residues 106 to 137 of 1mbo, were extracted and substituted for the GAG residues of the V3 loop in 1gc1. The myoglobin sequence was
replaced by the HXBc2 sequence, and successive rounds of stereochemical
optimization coupled to manual rebuilding were performed to remove
steric clashes. Using the beta-strand model, called beta, the f and g
strands of the immunoglobulin Bence-Jones protein, residues 80 to 107 of 1rei, were grafted onto the core gp120 in the same manner as
described for the alpha model. The nmr model was derived from nuclear
magnetic resonance (NMR) analyses of V3 loops from several different
HIV-1 isolates (7, 8) and consisted primarily of random-coil
secondary structure. Five different NMR structures were examined in
relation to the core gp120. Only two, from the mn and Haiti isolates,
both refined from H2O-trifluoroethanol (TFE) mixtures,
could be grafted onto the core without extensive clashes. Upon
completion of structure building, clashes with the carbohydrate at
position 332 of the gp120 core could not be resolved with the model
derived from the Haiti isolate, and so this isolate was not used in
further analyses. The nmr model thus derives solely from the NMR
analysis of the mn isolate in H2O-TFE (8).
Electrostatic analysis.
Electrostatic analyses were
performed analytically with the program DelPhi (41) with a
protein dielectric of 2.0, a solvent dielectric of 80, an ion exclusion
radius of 2.0 Å, a probe radius of 1.4 Å, and an ionic strength of
0.14 M. For pictorial display, the precise Delphi potentials were read
into the program GRASP (42), with the local potentials
displayed at the solvent-accessible surface.
Homology modeling.
Homology modeling was carried out with
the program PrISM (64), with sequence alignments and
homologous models constructed based on the 1gc1 gp120 structure.
 |
RESULTS |
Modeling.
A three-dimensional model of the gp120 portion of
the trimeric HIV envelope glycoprotein complex was developed subject to the conditions that the surface of gp120 that is occluded in the oligomer interface should (i) maximize carbohydrate exclusion, (ii)
minimize conserved residue exposure, and (iii) be sterically compatible
with binding of the 17b antibody. Since gp120 interacts with gp41,
which is a symmetric trimer in its isolated form and remains trimeric
when in the complex, the model was constrained to be threefold
symmetric. The occluded interface in the oligomer is expected to
comprise gp120-gp41 contacts, gp120-gp120 contacts, and occluded
surfaces that large ligands such as antibodies cannot access. The
constraining conditions for the model were chosen because, a priori,
they represent reasonable expectations about the oligomer interface and
because they could be reduced to quantifiable criteria.
Glycosylation sites occur almost exclusively on the exposed surfaces of
protein molecules, and they occur near protein interfaces
only at the
periphery. Carbohydrate residues in N-linked glycans
tend to be both
flexible and highly hydrated; although they can
be secured by protein
contacts, the resultant entropic loss is
large, making such
interactions generally unlikely. As a consequence,
we could expect the
oligomeric interface to be free of
glycosylation.
We would also expect exposed surface residues on gp120 to be variable,
a consequence of immune pressure. Possible factors
of conservation are
limited primarily to occlusion at the oligomer
interface, involvement
with receptor binding, or constraints of
folding topology. Conservation
due to the last two criteria could
be eliminated by examining residues
for proximity to either the
CD4 or the 17b Fab binding site (the 17b
site here served as a
surrogate for the chemokine receptor binding site
[
61]) and
by performing a clustering analysis to
remove statistical outliers
(constraints on exposed residues for
structural purposes should
be
rare).
Several ligands are known to bind to gp120 in the context of the
oligomeric complex as well as to isolated gp120. The sites
for such
ligands must be oriented on the oligomer interface and
also
appropriately oriented for biological interactions. The 17b
antibody is
known to bind to oligomeric gp120 (
53) and does
not cause
appreciable dissociation of the gp120 from the oligomer
(
44). Therefore, any valid oligomer model must be sterically
compatible with 17b binding. Similarly, productive binding of
HIV to
CD4-positive cells is known to involve intact glycoprotein
oligomers.
Therefore, one expects the CD4 binding site to be both
free on the
oligomer surface and appropriately oriented for attachment
to CD4 on
the cell
surface.
The crystal structure of core gp120 was first elaborated with a modeled
completion of two N-terminal residues (one of which
is glycosylated),
of the V4 loop, and of the two
N-acetylglucosamine
residues
at each site of N-linked glycosylation. This elaborated
core was then
oriented as a rigid body relative to a coordinate
system established
with a threefold-symmetry axis perpendicular
to the viral surface (Fig.
1). The initial orientation of gp120
was set such that the D1D2 portion
of CD4 in the core gp120-CD4
complex would be superimposed onto a
promoter of D1D2 in the structure
of dimeric soluble CD4, oriented with
its diad axis perpendicular
to the hypothetical cell surface. (The
dimeric soluble CD4, which
consists of the entire extracellular portion
of CD4, crystallizes
as a dimer in three different space groups
[
60]; its orientation
is physiologically relevant and
thus serves to position the hypothetical
cell surface.) The gp120
protomer was then reoriented about its
center of mass, displaced from
the triad axis sufficiently to
avoid collisions with other protomers.
First, all rotational orientations
about the
z axis were
tested with respect to the quantifiable
criteria, and the optimal value
was found to be at 30° (Fig.
2a,
left
panel). Then, with the
Z orientation at the optimum,
rotations
were made successively about the
X and
Y rotational axes (Fig.
2a, middle and right panels). A
protomer in the optimized model
can be obtained from the
1gc1 PDB
coordinates by the Euler
rotation (
1 = 6.90,
2 = 112.34,
3 = 22.60) followed
by the
translation (
tx =

25.91,
ty =

71.24,
tz = 30.90). This
procedure
of successive rotations, which was used for computational
economy,
does not sample all of the rotational space; nevertheless, we
expect the result to be close to the optimum for the three conditions
since it preserves the initial orientation relative to CD4 on
the cell
surface. In addition, visual inspection of the final
orientation
confirms that it is close to, if not at, the global
optimum.


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FIG. 2.
Quantitative modeling of the gp120 oligomer.
Quantitative modeling employed three criteria: carbohydrate exposure
(open circles), occlusion of conserved residues which are solvent
exposed on a gp120 protomer (filled symbols), and steric considerations
of 17b binding (forbidden regions [shaded]). These criteria have been
graphed with respect to their distances from the trimer axis (depicted
here on the vertical axis) as the rotational parameter (horizontal
axis) was varied. The coordinate system was as defined in the legend to
Fig. 1. (a) Rotational orientation of the HIV gp120 core. (Left panel)
The orientations of X and Y in this panel were
determined by the superposition of CD4 in the gp120-CD4 complex
(32) with its orientation in the four-domain CD4 structure
(60). The maximum (filled squares) and minimum (filled
triangles) distances of the conserved, solvent-exposed residues from
the trimer axis were determined at 10° intervals in Z;
minimum values correspond to regions of greater oligomer occlusion. The
distance of the carbohydrate (open circles) from the trimer axis is
shown at 10° intervals; peaks correspond to carbohydrate-free regions
at which the overall exposure of carbohydrate is greatest. The highest
peak in this panel corresponds to a large carbohydrate-free region
centered about the first helix in the inner domain of core gp120
(32). The second-highest peak corresponds to the CD4 binding
site, which is also free of carbohydrate. (Middle panel). Given the
optimal Z orientation, rotations were made around the
x axis. Here the minimum and maximum distances for the
conserved, solvent-exposed residues have been averaged (filled
triangles). The 0 of the X rotation corresponds to the
orientation of X as determined by the initial superposition
with CD4. (Right panel) Given the optimal orientation for X
and Z as determined in the previous two panels, the
remaining rotational axis was varied. Again, the minimum and maximum
distances for the conserved, solvent-exposed residues have been
averaged (filled triangles). The extended carbohydrate-free region
corresponds to the overlap between the carbohydrate-free chemokine
receptor region and the presumed oligomer interface. Steric constraints
from binding of the 17b antibody (forbidden regions [shaded]) helped
to distinguish these regions. As with the X rotation, the 0 of the Y rotation corresponds to the orientation of
Y as determined by the initial superposition with CD4,
independent of the modeling criteria used here. (b) Surface criterion
optimization of the influenza virus hemagglutinin HA top. The origin of
each panel corresponds to the orientation of the HA top superimposed on
the HA1-HA2 heterotrimer (59). The symbols used for the
criteria are the same as described for panel a. (c) Surface criteria
depicted from the perspective of the viral membrane at the (0° 0°
30°) orientation for the gp120 core and the (0° 0° 0°)
orientation for the HA top. (Left image) the gp120 core is depicted as
a copper-brown C backbone worm. The molecular surfaces of all of the
N-acetylglucosamine residues used in the modeling are
colored cyan. The molecular surfaces of the side chains of the
conserved exposed residues on the monomeric gp120 core are colored
magenta. The gp120 protomer has been positioned such that the distance
of its center of mass from the trimer axis is proportional to that
observed in hemagglutinin. (Right image) The HA top is depicted as a
black C backbone worm. The molecular surfaces of side chains that
are sites of glycosylation in at least 1% of the 485 aligned
hemagglutinin sequences are shown in blue. The molecular surfaces of
side chains which are conserved and exposed on the HA top monomer are
colored magenta. The orientation shown here corresponds to the
superposition of the HA top onto the HA1-HA2 heterotrimer.
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The carbohydrate criterion and the exposed and conserved surface
residue criterion were completely independent. Nonetheless,
quantification of these two criteria led to maxima and minima
within
20° of each other for all independent axial rotations (with
the 17b
steric criterion used bifunctionally to eliminate incompatible
orientations) (Fig.
2). Taken together, the three criteria produced
well-defined peaks with all three independent rotational parameters,
thereby allowing a "best" orientation to be derived at an
X rotation
of 0°, a
Y rotation of 0°, and a
Z rotation of 30° (0° 0° 30°).
This best orientation
actually corresponds to two possible alignments
with respect to the
viral membrane, "up" and "down." One of these
alignments could
be eliminated due to CD4 (or 17b) steric constraints;
the binding of
CD4 (or 17b) in this orientation would bury it
in the viral membrane
(Fig.
3), thus permitting a single unique
best orientation to be derived.

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FIG. 3.
Trimeric model of gp120. Three orientations of the model
are shown. The images at the top depict the view from the orientation
of the viral membrane. The middle images depict the view from the side,
in between the viral and target cell membranes. The images at the
bottom depict the view from the target cell membrane. The left-most
three images are C worm representations of core gp120 (copper brown)
and the two membrane-distal domains of CD4 (yellow). Also shown are the
gp120 carbohydrate cores (blue), the
(N-acetylglucosamine)2-(mannose)3
cores shared by both high-mannose and complex N-linked glycan moieties.
The carbohydrate shown here represents approximately half the
carbohydrate on gp120, with the rest extending further from the gp120
surface. The middle images show the electrostatic surface of gp120 for
the core. The electrostatic potential is depicted at the
solvent-accessible surface, which is colored according to the local
electrostatic potential, ranging from dark blue (most positive) to red
(negative). The right-most images show the gp120 core with
carbohydrate, with the solvent-accessible surface colored cyan for
carbohydrate, yellow for the surface of gp120 less than 3 Å from CD4,
green for the surface of gp120 less than 3 Å from the 17b antibody,
and copper brown for the remaining surface of core gp120. The degree to
which carbohydrate covers all of the solvent-accessible trimer surface
is remarkable. Other than a small region at the viral proximal portion
of the oligomer (where the missing N and C termini most likely reside),
the only carbohydrate-free surfaces large enough to serve as an
antibody epitope correspond to regions of receptor binding.
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The resultant model of the core oligomer (Fig.
3) displayed several
relevant features that were not included in the modeling
criteria. The
observed and deduced binding sites for CD4 and neutralizing
antibodies
were all on exposed surfaces. The variable loops (V1
to V5) were all
well exposed on the oligomer. The N and C termini
were clustered at the
end pointing toward the viral membrane (with
the C terminus proximal to
the oligomer axis poised to interact
with the trimeric gp41).
Additionally, the region identified by
substitutional mutagenesis as
interacting with chemokine receptors
(
45) was free of
carbohydrate and pointed directly at the target
membrane. These
features are consistent with the biology of the
gp120 trimer and with
the qualitative characteristics of the rough
model obtained previously
(
62).
Modeling accuracy.
To estimate the precision of the surface
criterion quantification procedure, we turned to the influenza virus
hemagglutinin system. Atomic-level structures are known for both the
HA1-HA2 heterotrimer (59) (equivalent to the gp120-gp41
oligomeric complex) and a monomeric proteolytic fragment, the HA top,
complexed with the Fab of a neutralizing antibody (4)
(equivalent to the gp120-17b complex). In addition, the
fusion-activated forms of gp41 and HA2 are structurally similar, and
although core gp120 and the HA top show no sequence similarity, they
have almost identical radii of gyration (20.6 and 20.8 Å,
respectively), making rotations about a position displaced the same
distance from the trimer axis reasonable.
The HA top contains much less glycosylation than core gp120. Only
three sites are present, although no carbohydrate is modeled
in the
2vir coordinates (X31 isolate). In contrast, 18 sites
are present on
the gp120 core model (HXBc2 isolate). To increase
the accuracy of this
criterion for the HA top, all nonconserved
sites of glycosylation were
identified and then used to enhance
overall surface coverage,
increasing the residues used in the
carbohydrate criterion to 11. Even
so, this was less than two-thirds
of that used for gp120 (compare the
structures in Fig.
2c). In
addition, because side chains, instead of
glycan moieties, were
used to mark the positions of the carbohydrates,
criterion uncertainty
increased. Nevertheless, the residues identified
tended to be
on the outside of the trimer, and for rotations in
Y and
Z this
criterion produced well-defined
maxima close to the known trimer
orientation (Fig.
2b and
c).
The hemagglutinin sequence is much less divergent than the gp120
sequence. This makes it difficult to find a reasonable set
of conserved
exposed residues. (If there were no divergence, for
example, this
criterion would be meaningless.) Using 485 aligned
HA1 sequences and a
minimum solvent exposure of 40%, 39 residues
showed 98% identity, 28 showed 99% identity, and 12 showed 100%
identity. The 100% criterion
was judged too strict because sequencing
errors might account for
some divergence, and so a 99% criterion
was used. This stringent
99% criterion selected approximately
the same number of surface
residues as the less-restrictive gp120
criterion (for which all
single-atom substitutions [e.g., Gln
to Glu] were included, as well
as larger substitutions as long
as they did not change the character of
the amino acid [e.g.,
Lys to Arg or Tyr to Trp]). While these
residues tended to cluster
at the known oligomer interface, the minima
produced by this criterion
were not well-defined (Fig.
2b and c). For
example, the minimum
Z rotation of the furthest conserved
residues appears close to
the maximum for the nearest (Fig.
2b, left
panel), and the
X rotation
showed very little change in
parameter distance (Fig.
2b, middle
panel). This lack of definition may
be related to the packing
of the hemagglutinin trimer, with conserved
residues clustering
at two discrete interfaces, as opposed to gp120,
for which one
central cluster was observed (Fig.
2c). Such clustering
would
account, for example, for the local maximum at the origin of the
Z rotation for the minimum conserved residue
distance.
Finally, because the HC19 Fab binds further from the trimer axis than
17b, it does not produce as much of a steric clash.
Indeed, for
rotations in
X, no angles are
restricted.
Judging from the poor shape of the quantification curves, the
surface criteria optimization did not work as well with the
HA top as
with the gp120 core. Overall results of the optimization
procedures are
shown in Table
1. The internal agreement
of the
criterion optimizations, as judged by the peak orientational
difference
between the carbohydrate and conserved-residue extrema, was
much
better for gp120. Despite the poor agreement, the mean of the
extrema for the HA top was within 20° of the correct orientation,
suggesting that averaging these independent criteria reduced the
overall error. In the case of gp120, the 17b steric criterion
reduced
the mean deviation even further, to one-fourth that observed
for the HA
top. This suggested that the error associated with
the rotational
parameters of the resultant gp120 oligomeric model
was only 5°.
While it is conceivable that the deletion of the gp120 N and C termini
(57 and 19 amino acids, respectively) could alter the
modeling results,
we feel that this is unlikely. With respect
to the carbohydrate and 17b
steric criteria, the missing termini
are carbohydrate free and 17b
binds to core gp120. Thus, both
criteria should be unaffected. With
respect to the conserved and
exposed amino acid criterion, similar
extrema were observed for
both the furthest and closest residues (Fig.
2a, left panel).
If some of the 15 exposed and conserved amino acids
are covered
by the termini, a subset should give similar results. In
the event
that all or nearly all 15 are covered, this would localize
much
of the missing termini in the same region as the previous exposed
amino acids; since the missing termini correspond to the epitopes
of
virtually all of the nonneutralizing antibodies, this would
again place
the expected oligomer interface in a similar region.
Finally, the
agreement between the carbohydrate and exposed-residue
criteria was so
good that even if the exposed-residue criterion
were omitted, the
extrema would change by only 5° on average.
Thus, we feel that the
presence of the missing termini is unlikely
to substantially alter the
results obtained
here.
Although the modeling precisely defined the rotational parameters of
the trimer, the translational parameter was only partially
determined.
Steric constraints defined a minimum approach, and
distance constraints
between the C terminus of gp120 and the N
terminus of gp41 defined a
maximum, but these two criteria did
not discriminate sharply. With the
center of rotation for a protomer
placed at 35 Å from the trimer axis,
which was close to the sterically
constrained minimum approach, the
C

-C

diameter of the
model was ~110 Å;
if the (mannose)
3 carbohydrate extensions were
included,
the diameter increased to ~150 Å. These dimensions agreed
with
electron microscopic observations of the viral spike, although
these
observations vary widely, from 100 Å (by negative staining
of gp120
from SIV [
22]) to 150 Å (by ultrathin section tannic
acid cytochemistry and surface replica electron microscopy of
HIV-1
[
21]). If the distance from the trimer axis is
proportional
to the dimensions of the protomer in the
x and
y directions, using
the relative proportions of
hemagglutinin as a guide would place
the gp120 core 30.5 Å from the
trimer axis (Fig.
2c). A protomer
of this hemagglutinin proportionate
model can be obtained from
the
1gc1 coordinates with the Euler rotation
specified previously
followed by the translation
(
tx =

30.43,
ty =

71.24,
tz = 30.90).
At this distance, steric clashes
occur between neighboring gp120
protomers with the carbohydrate at
position 197, although these
are easily resolved by minor movements of
this flexible portion
of the model. (This proportionate model is shown
in Fig.
2c and
7; the rest of the figures depict the gp120 protomer
with a center
of mass at
x = 35 Å.)
Electrostatic analysis of the oligomeric core gp120.
Electrostatic analysis of our model of the oligomeric core gp120
defined an electropositive surface which would face the target cell
membrane. We tested the robustness of this basic region to changes in
the modeling parameters. As can be seen in Fig.
4, rotations of ±30° and translations
of ±5 Å maintained the electropositivity of the region.

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FIG. 4.
Robustness of the basic cell-facing surface of core
gp120 to variations in modeling parameters. The oligomer modeling was
dependent on four independent parameters, one translational (trans) and
three rotational (rot). The effect of varying these parameters on the
basicity of the cell-facing surface of the proposed envelope oligomer
is shown here. All of the images in this figure are depicted from the
view of the target cell membrane. A C worm diagram of core gp120
(copper brown) with carbohydrate (blue) is shown to aid in orienting
each variation in modeling parameter. The surface diagrams depict the
local electrostatic potential. A dark blue (basic) surface can be seen
on core gp120 (HIV-1, HXBc2 isolate) in all modeling parameter
variations.
|
|
We used sequence analysis combined with homology modeling to test the
conservation of this basic region throughout the primate
immunodeficiency viruses. Although the charge of the region differed
among viruses, its basic nature was conserved in different clades
as
well as with HIV-2 and SIV (Fig.
5).

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FIG. 5.
Conservation of basic cell-facing surface on core gp120.
Homology modeling was used to construct the corresponding structures of
core gp120 for HIV-1 clades C and O as well as HIV-2 and SIV, starting
with the crystal structure of HIV-1 clade B core gp120 (32)
modeled as the envelope oligomer. The electrostatic potentials are
depicted at the solvent-accessible surface, from the perspective of the
target cell membrane. A basic (dark blue) surface can be seen in all
isolates, although the precise charge distribution and the degree of
overall basicity show variation.
|
|
Analysis of the V3 loop.
The V3 loop comprises roughly 30 amino acids. It is too large to be modeled correctly without
experimental constraints. We used the steric constraints inherent in
connecting the V3 loop to core gp120 as well as those consistent with
17b Fab binding. Nevertheless, very different V3 loop structures could
be successfully built (Fig. 6a).


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FIG. 6.
Modeling and electrostatic contribution of the V3 loop.
(a) Different V3 loop models were constructed: alpha (green), beta
(dark green; toward the back left of gp120 in this orientation), Haiti
(forest green; depicted as the front right model in this orientation),
and nmr (light green) (see text for details). These are displayed as
C worm diagrams in the context of core gp120 (copper brown) and the
17b Fab fragment (purple violet). As can be seen, steric constraints,
primarily from 17b, separate the V1/V2 stem from the V3 loop on a gp120
protomer, although quite diverse V3 loop structures can be successfully
grafted onto the gp120 core. The position of the trimer axis (as
determined by quantitative modeling) and the N and C termini of core
gp120 are labeled for reference. (b) C worm diagrams for the
different V3 loops (green) are depicted by themselves (top) and with
core gp120 (copper brown) (bottom) for the HXBc2 strain of HIV-1 gp120
as arranged in the envelope oligomer. The orientation of each image is
shown from the perspective of the target cell membrane. (c) Analysis of
the electrostatic potential of the cell-facing region. Models of the
trimeric gp120 core have been constructed with different V3 loops
(described above for panels a and b), and the charge on the V3 loop for each of these structure has been varied between 0 and 9. For each of these 15 different models, the electrostatic
potential has been calculated at various positions, either along the
trimer axis x = 0 or below the center of mass of a
protomer x = 35. Increasing numbers in z
correspond to increasing distances away from gp120 (moving away from
the virus). The position in z varies from z = 0, which corresponds to 0.5 Å below the gp120 core, to
z = 40, which is 40.5 Å below the core but only
approximately 25 Å below the V3 loop. The difference between core and
V3 loop distances occurs because the V3 loop models extend roughly 15 Å below the core (as can be seen in panel a). (Left panels) The
electrostatic potential from each model, that is, the three different
V3 loop models shown in panel b, is graphed as a function of structure.
No simple correspondence is seen. (Center and right panels) The
electrostatic potential from each model is graphed as a function of
charge. A rough linear correspondence is seen. This has been
least-squares fitted (solid line), and the r2
fit, which shows how well the data correlate to the line, is depicted
(an r2 of 1.0 corresponds to a perfect fit). The
correlation is poor close to the gp120 core but increases as the
distance from the virus increases. The dotted line corresponds to the
least-squares fit if the potential is calculated for only the V3 loop.
For the top three panels on the right, in order to use a reasonable
scale, two outlier points, for charges of 6 and 9, are not depicted,
although they have been used in the least-squares fitting.
|
|
The overall charge of the V3 loop ranges from +2 to +10, with that of a
CCR5-using isolate generally in the range of +3 to
+5 and that of a
CXCR4-using isolate (often a T-cell-line-adapted
strain) being from +7
to +10. With the HXBc2 sequence (+9 charge
on the V3 loop), we analyzed
the electrostatics of three very
different V3 loop structures, as well
as the effect of varying
the overall net V3 loop charge (Fig.
6c).
Analysis of the potential near the cell-facing region of the oligomer
showed a correlation between the potential and the overall
V3 loop
charge, especially at distances further than 20 Å from
the gp120 core
(Fig.
6c, middle and right panels). In contrast,
the potential did not
seem to correlate with the precise structure
of the V3 loop (Fig.
6c,
left
panels).
The relatively low electrostatic potential for a zero V3 loop net
charge suggested that the contribution of the core to the
overall
potential was small. We tested this explicitly by calculating
the
electrostatic potential as a function of only the V3 loop
(Fig.
6c). At
long distances and with a high charge (for example,
CXCR4-using
isolates), the V3 loop potential dominated, approximating
closely the
potential for the entire core with V3 loop. At short
distances and with
a low V3 loop charge, the core contributed
significantly to the
electrostatic potential. This was especially
true at
x = 35,
y = 0, directly below the protomer, close to the
above-described basic conserved region on the
core.
 |
DISCUSSION |
From the earliest days of structural biology, for example, with
myoglobin and hemoglobin, biologically important results have been
extracted from low-resolution oligomeric models of more highly resolved
protomers. The construction of such models often requires little
additional information; if the protomer conformation does not change,
only three angles and one distance are needed to specify a symmetric
oligomer. Here, optimization of quantifiable surface criteria was used
to precisely determine the orientation of gp120 in the oligomeric viral
spike. The orientational precision of better than 10° corresponded to
an average positional error of less than 3.5 Å for a molecule with a
20-Å radius of gyration. Because we did not have precise experimental
limits on the translation component, however, our analysis of the model
was limited to properties that depend primarily on rotational
parameters. Still, the resultant model defined several interesting
features, and here we explore their functional, immunological, and
therapeutic implications.
Receptor binding.
Studies of the effect of soluble CD4 on
virus entry suggest that more than one CD4 molecule must bind the
envelope glycoprotein oligomer to initiate virus-cell fusion (33,
37, 50). The model derived here demonstrates that three CD4
molecules can bind without CD4:CD4 steric interference (in the
hemagglutinin proportionate model, the closest CD4-to-CD4 contact is
over 35 Å). CD4 binds obliquely to the sides of the oligomer, with the
third and fourth domains of CD4 being almost parallel to the cell
membrane. Because CD4 is an extended molecule and it binds gp120 at the
N-terminal membrane-distal domain, the membrane-spanning portion of CD4
is positioned far from the oligomer axis. With the hemagglutinin proportionate model, the end of the second CD4 domain (residue 178)
would be 127 Å from the same position on an adjacent CD4 and the last
ordered extracellular residue (363) would be 198 Å away (Fig.
7).
Even accounting for a tighter
protomer packing or for the known segmental flexibility between domains
2 and 3 of CD4, membrane-spanning portions of adjacent CD4 molecules
would be separated by at least 190 Å.

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FIG. 7.
Selected features of the oligomeric gp120 model. (a) The
entire extracellular portion of CD4 (yellow C worm) is shown binding
to the oriented gp120 oligomer (copper-brown C worm). Carbohydrate
on the gp120 is colored cyan. Distances between adjacent CD4 molecules
are shown for the C of residue 178 at the end of the second domain
and the C of residue 363, the last ordered residues prior to the
transmembrane-spanning region. The contact region highlighted in panel
b has been circled. The hemagglutinin proportionate model is used here,
and thus the gp120 is close to the minimum protomer packing distance.
(b) An enlargement of the contact site between gp120 core protomers in
the oligomer. The coloring for one protomer is the same as in panel a;
the other protomers are colored black. In addition, the molecular
surfaces of side chains of conserved exposed residues in this
contacting region (residues 195 to 210) are colored magenta. Starting
from the top of the figure, these include residues 196, 197, 198, 201, 204, 208, and 209. Residue 197 is glycosylated, and its carbohydrate
has been depicted in magenta. This region is sensitive to the binding
of CD4, which is seen to the right of the figure in close proximity.
The orientation of both images is shown from the perspective of the
target cell membrane.
|
|
The chemokine receptor, in contrast, binds close to the trimer axis, at
a gp120 surface which is almost 20 Å closer to the
target cell
membrane. This arrangement is consistent with simultaneous
CD4 and
chemokine receptor binding to a protomer. In terms of
multiple
chemokine receptors binding to the oligomer, our data
show that the 17b
Fab fragment, whose epitope overlaps the chemokine
receptor binding
site (
45), lies at the edge of the sterically
restricted
"forbidden zone" in all three rotational parameters
(Fig.
2). This
suggests that multiple chemokine receptors bound
to the same oligomer
may be sterically strained. Since the chemokine
receptor is an integral
membrane protein and
z translations are
constrained, such
strain could only be relieved by movement of
the gp120 protomer away
from the trimer axis. Such movement may
serve to signal the binding
state of gp120 at the target cell
membrane to the gp41-interactive
regions, roughly 50 Å distal,
triggering gp41 fusion-related
conformational changes at the appropriate
moment. In this regard, it
may be relevant that some of the substitutions
that effect chemokine
receptor binding map closer to the trimer
axis than the 17b epitope,
and many of these axis-proximal substitutions
induce dissociation of
gp120 from the oligomer (
45).
Multivalent CD4 molecules have been constructed with the intent of
binding multiple gp120 molecules to enhance avidity. Our
results show
that this is only possible with an extremely long
and flexible linker,
thus ruling out enhanced affinity with, for
example, the two N-terminal
domains of CD4 in an antibody format
(
9). Indeed, the
dimensions are such that although the position
of viral spikes is fixed
by the underlying matrix, binding to
a neighboring spike (which is
roughly 380 Å away, assuming 72
viral spikes per virion and a virion
diameter of 1,000 Å) would
be almost equally feasible. (Such
orientational steric restrictions
should also apply to antibodies that
bind to the CD4 binding site.)
Finally, with regard to the known segmental flexibility of CD4, the
optimal orientations of the
x and
y axes of gp120
in the
quantitative modeling were found to coincide with the dimer
orientation
of CD4. Given that the orientations were based on
completely different
criteria, this coincidence suggests that the
orientation of CD4
on the cell surface may be more rotationally
constrained with
respect to the plane of the membrane than previously
thought (
60).
Oligomeric contacts.
A central mechanistic question is how CD4
activates gp120 on the virion, transforming the stable envelope into a
reactive fusogen. CD4 is known to cause conformational changes in
gp120, including most prominently the formation of the bridging sheet (32, 53). Interestingly, the edge of this conserved sheet can be seen as a pivotal site of contact in the oligomer; although the
specific details of the contact are dependent on the ill-defined translation component, it is the well-defined rotation parameters which
position the bridging sheet in its prominent location. In the
hemagglutinin proportionate model, steric clashes are found with
residue 197 in this region. Moreover, deletion of the outer two strands
of the bridging sheet generates an oligomer to which CD4 binds without
inducing shedding (63), and mutations in this region,
especially of the glycosylation site at residue 197, correlate with a
CD4-independent infection phenotype (30). All of these lines
of evidence are consistent with the notion that activation of the
trimer may involve close contact of the bridging sheet with the
neighboring protomer (Fig. 7).
Another region of potential protomer:protomer contact involves the
sequence-variable V1/V2 and V3 loops, which were truncated
in the core
gp120 structure. In the V3 loop modeling it was clear
that 17b acts as
a barrier between the V1/V2 stem and the V3 loop
(Fig.
6a). Unless 17b
binding substantially alters the positioning
of the V1/V2 and/or V3
loop, this suggests that these loops probably
do not contact each other
within a protomer. Data from experiments
in which revertants were
obtained subsequent to mutation of the
V3 loop, however, show that
changes in the V1/V2 loop can rescue
changes in the V3 loop
(
46). Moreover, some neutralizing antibodies
in simian-human
immunodeficiency virus-infected monkeys apparently
recognize a
discontinuous V2-V3 determinant (
15), and a functional
interaction of the V2 and V3 loops is able to determine coreceptor
choice and neutralization resistance (
50). These data
suggest
that, in the context of the oligomer, the V1/V2 loop and the V3
loop are near one another. In this regard, it may be relevant
that the
model derived here for the trimer displayed the base
of the V3 loop
juxtaposed to the V1/V2 stem from the neighboring
protomer.
Biological implications of the basicity of the cell-facing surface
of the gp120 oligomer.
Nonspecific electrostatic interactions of
the basic cell-facing surface may affect entry of virus into a cell in
several ways. With respect to initial attachment, these interactions
may play a role in binding of virus to polyanions such as heparan
sulfate. This conjecture agrees with biochemical results which show
that heparan sulfate influences the binding of HIV virions to some cells (36). In addition, the model derived here allows us to analyze the electrostatic properties of a wide variety of HIV strains.
In a companion study (39), we found that the observed binding of polyanions to different isolates of gp120 as well as to
mutants of gp120 with and without the V3 loop correlates extremely well
with model-based electrostatic predictions.
Other acidic surfaces may interact with the basic cell-facing region
described here. For example, negatively charged lipid
head groups may
create an acidic zone in the target membrane itself.
The degree of
membrane acidity is dependent on the composition
of the lipid head
groups, which may differ in different cells
as well as locally, from
patch to patch, on the surface of an
individual cell. The target
membrane compositional variation may
explain the ability of heparinase
to affect initial binding in
some cell types and not in others
(
36,
43).
Numerous studies have documented the binding of membrane-associated
proteins through both a hydrophobic interaction and an
electrostatic
attraction to membrane head groups (
34,
40).
A theoretical
and experimental analysis of the binding of the
polylysine peptides
(Lys)
3, (Lys)
5, and (Lys)
7 to a
33% acidic
membrane (a 2:1 mixture of phosphotidylcholine and
phosphotidylglycerol)
showed that each charge enhances the binding of
the polymer to
the membrane by roughly 1 order of magnitude
(
3). While it
is difficult to correlate the polylysine
results with the V3 loop/core
basicity determined here, that analysis
does suggest that if the
cell-facing surface of oligomeric gp120 is
highly basic, direct
interactions with the membrane will be enhanced.
Moreover, our
results suggest that the wide variation in overall charge
on the
V3 loop will be a primary determinant of the magnitude of this
electrostatic interaction. (We note, however, that the relatively
poor
correlations between potential and charge at shorter distances
suggest
that the precise structure of the V3 loop will influence
short-range
interactions.)
After gp120 is bound to CD4, nonspecific electrostatics may play a role
in virus entry and chemokine receptor binding. CD4
itself is quite
basic, with a net overall charge for the first
two domains of +5, which
should serve to increase the overall
basicity of the CD4-gp120 complex.
In addition to the membrane
head group interactions detailed above,
nonspecific electrostatics
may effect binding to the sulfonated, acidic
N terminus of the
chemokine receptor (particularly CXCR4) (
14,
17,
18). Relevant
to this, mutagenesis of the chemokine receptor
binding site on
gp120 showed that substitutions of negatively charged
amino acids
for positively charged ones often have substantial effects
on
binding (
45).
Implications of electrostatics for therapeutic development and
immune surveillance.
Our results suggest that the biological
effects of polyanions are primarily the consequence of nonspecific
electrostatic interactions. If so, this may explain the dramatic
differences in in vitro and in vivo effectiveness of such polyanions as
dextran sulfate. Since the primary interaction appears to be dependent
on charge density rather than a particular structural motif
(57), it seems unlikely that the specificity essential for
effective therapeutic treatment is present. This does not bode well for
drug development based on polyanions such as sulfated polysaccharides
(2, 24, 35), carboxylated albumins (26, 51),
porphyrins (13, 49), or DNA or RNA derivatives
(31). Nevertheless, the unusual basicity observed for the
CXCR4-using viruses may, in this particular case, permit anion-based intervention.
Because generation of broadly neutralizing antibodies depends on
detection of conserved sequences and nonspecific electrostatic
interactions can be achieved through variable regions like the
V3 loop,
the nonspecific binding predicted here, which does not
appear to be
sensitive to the precise structure of the V3 loop,
may serve as a
mechanism of gp120 immune system evasion. Additionally,
the structures
of several neutralizing antibodies complexed with
their V3 loop
epitopes revealed that the V3 loop exists in at
least two quite
different conformation (
48). This suggests that
the V3 loop
may adopt different structures, adding yet another
layer of difficulty
to its recognition by the immune
system.
Initial nonspecific electrostatic attachment may help to conceal the
conserved chemokine receptor site, which is on the cell-facing
surface.
Studies have shown that polyanions compete effectively
with antibodies
for binding to the V3 loop (
23,
39). The chemokine
receptor
binding site, which in any case may only be exposed after
a
conformational change is induced by CD4 (
32,
53), may thus
be multiply shielded from immune system
surveillance.
Last, we note that although electrostatic potentials are calculated
from the exact positions of the atoms in the atomic structure,
the
effect of electrostatics on biological processes such as initial
viral
attachment and virus-cell membrane fusion depends on
much-lower-resolution
parameters. Since the modeling criteria used here
are not well
constrained with respect to protomer translations, the
relative
insensitivity of the electrostatics to this parameter bodes
well
for the analysis. Indeed, the basic surface defined here appears
to be robust to relatively large changes in modeling parameters,
both
for the core (Fig.
4) and for the V3 loop (Fig.
6). This
robustness,
coupled with the low resolution of the effects, increases
our
confidence in the biological significance of our
conclusions.
 |
ACKNOWLEDGMENTS |
We thank An-Suei Yang for assistance with the program PrISM and
homology modeling, Barry Honig and Ben Hitz for help with the programs
DelPhi and GRASP, Lawrence Shapiro for a thorough reading of the
manuscript, and a reviewer for suggesting the use of influenza virus
hemagglutinin to test the modeling procedure.
This work was supported by grants from the National Institutes of
Health (AI 31783 and AI 39420); by the ANRS, INSERM, and CNRS of
France; and by a Center for AIDS Research grant to the Dana-Farber
Cancer Institute (AI 28691). The Dana-Farber Cancer Institute is also
the recipient of a Cancer Center Grant from the National Institutes of
Health (CA 06516). Columbia University is a participant in the Center
for AIDS Research (AI 42848). This work was made possible by gifts from
the late William McCarty-Cooper, from the G. Harold and Leila Y. Mathers Charitable Foundation, and from the Aaron Diamond Foundation as
well as Douglas and Judi Krupp. R.W. was a fellow of the American
Foundation for AIDS Research, and P.D.K. is a recipient of a Burroughs
Wellcome Career Development award.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biophysics, Black Building, Room 204, Columbia University, 650 W. 168th St., New York, NY 10032. Phone: (212) 305-1846. Fax: (212) 305-7379. E-mail:
kwong{at}convex.hhmi.columbia.edu.
Present address: The Sir William Dunn School of Pathology,
University of Oxford, Oxford OX1 3RE, England.
 |
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Journal of Virology, February 2000, p. 1961-1972, Vol. 74, No. 4
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