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PROTEINS: Structure, Function, and Genetics 34:197–205 (1999)
Molecular Dynamics Study of the Proposed ␤-Hairpin Form
of the Switch Domain From HIV1 gp120 Alone and
Complexed With an Inhibitor of CD4 Binding
Andreas Graf von Stosch,1 C.W. von der Lieth,2 and Jennifer Reed1*
1Department of Pathochemistry, German Cancer Research Center, Heidelberg, Germany
2Department of Spectroscopy, German Cancer Research Center, Heidelberg, Germany
The strong tendency of ␤-hairpin
peptides to aggregate can prevent their structural
resolution. The polar form of the switch peptide
(LAV 15mer) at the CD4-binding domain of HIV1
gp120 is such a peptide, and NMR investigations of
its interaction with a class of CD4-binding inhibitors developed in this laboratory have been hindered. Detailed knowledge of the interaction is
required for the development of more potent switch
inhibitors, that act by disrupting the cooperative
folding transition necessary for binding to the CD4
receptor. In carrying out molecular dynamics simulation of the free peptide under polar conditions, we
found that the properties of the resulting structure
agree closely with those observed by circular dichroism. The same conditions, used to model the peptide/
inhibitor complex, produced a stable bimolecular
structure with specific interactions between the
inhibitor and side chains on the peptide, (e.g., Trp12
and the LPCR tetrad), known to control the folding
transition. These help explain existing data on the
relative potency of inhibitor derivatives and provide a basis for improved inhibitor design. Proteins
1999;34:197–205. r 1999 Wiley-Liss, Inc.
Key words: gp120; switch domain; molecular dynamics; peptide
The process involved as the human immunodeficiency
virus 1 (HIV1) env glycoprotein gp120 binds to the CD4
receptor on host cells presents an interesting problem in
protein structure/function from both the theoretical and
the practical point of view. Previous work in this laboratory has established that a 15-residue sequence from the
principal CD4-binding domain of gp120 conserves the
ability to refold abruptly and cooperatively from ␤-sheet to
helix, triggered by changes in medium polarity across a
very narrow range.1 This phenomenon was observed in the
corresponding sequence from all strains tested despite
50% variability and appears to be closely involved in the
ability of gp120 to bind to the CD4 receptor, i.e., destroying
the cooperativity of the refolding process—the conformational switch—also stops binding to CD4-expressing cells.1,2
Although certain aspects of the mechanism behind this
process have been reported,3,4 and a nuclear magnetic
resonance (NMR) structure of the helical form could be
obtained,5 all attempts to date at determining the fine
structure of the ␤-sheet form observed by circular dichroism have been frustrated by the extreme tendency of the
peptide to aggregate at the concentrations necessary for
NMR. A more accurate idea of this form than can be
obtained by CD spectroscopy is desirable, since the exogenous switch inhibitors that have been developed as
possible therapeutic agents against HIV12,6 appear to
interact with the ␤-conformation.
Oligopeptides that have a preference for adopting a
␤-hairpin conformation in solution present considerable
difficulties for researchers interested in obtaining an accurate three-dimensional structure. The two most advanced
techniques, NMR and X-ray crystallography, can both be
frustrated by the tendency of ␤-hairpin peptides to aggregate.5,7–18 Indeed, the few cases in which NMR structure
determination of ␤-hairpins has been reported concern
artificial peptides purposely designed to avoid aggregation.19–21 On the other hand, the precise structure of
natural ␤-hairpins can be important, as several are implicated in pathogenic processes: the ␤-amyloid and the prion
protein PrPsc, for example. The polar or unbound form of
the switch domain from the env glycoprotein gp120 from
HIV1 is a case in point, one where molecular modeling
offers the only possibility at present of obtaining a better
idea of the structure of this 15-residue sequence and its
potential interaction with switch inhibitors. Molecular
dynamics simulation of the 15-residue switch domain
results in a stable structure that conforms to the conformational parameters established by physical measurements,
e.g., circular dichroism. Parallel runs in the presence of a
functional inhibitor molecule display a spontaneous and
specific association between the two that should prove
useful in improving inhibitor design.
All calculations were carried out on a Silicon Graphics
Indigo II (Extreme) workstation (R4400) coupled with an
SP2-IBM parallel processing array. Molecules (the LAV
Grant sponsor: Deutsche Forschungsanstalt für Luft- und Raumfahrt; Projektträger Gesundheitsforschung des BMBF.
*Correspondence to: Dr. Jennifer Reed, Department of Pathochemistry, German Cancer Research Center, Im Neuenheimer Feld 280,
D-69120 Heidelberg, Germany. E-mail:
Received 7 April 1998; Accepted 5 October 1998
peptide and the inhibitor BM 50.0311) were constructed
with the Insight II molecular modeling program package
(Builder and Biopolymer modules22). The LAV peptide
(LPCRIKQFINMWQEV) was constructed with unblocked
N- and C-termini to correspond with the peptide used for
physical measurements. (The structure of BM 50.0311 and
the nomenclature used here are given in the Insert to
Fig. 4a.)
Energy minimization and calculations of dynamic trajectories were carried out with the Discover program (versions 2.9.5 and 94.0) as part of the Insight II program
package. Structures were initially minimized using the
conjugate gradient algorithm with the gradient convergence criterium set to 0.0001 kcal/mol Å.
The consistent valence force field (CVFF) without any
cross-terms was used22 for the peptide as well as for the
peptide/inhibitor complex. The potential type and the
partial charges were assigned using the automatic assignment procedure of Insight II. The total charge of peptide as
well as the peptide/inhibitor complex was zero. No cutoff
distance for the nonbonded interaction terms (van der
Waals and Coulomb) was introduced for the initial simulation of the LAV peptide structure in the absence of
inhibitor (simulation I) or with inhibitor (simulation II).
No explicit solvent was included for the initial simulations. An effective dielectric constant of 4.0 was chosen as
the closest approximation to the conditions obtaining
during the physical measurements, as discussed in van
Gunsteren and Berendsen.23 Time steps of 1 fs were chosen
for both calculations. The temperatures were set to 400K
(I) and 300K (II), respectively. Both simulations were run
for a cumulative time of 1.02 ns with images recorded
every 250 fs. All simulations were carried out under
constant temperature conditions. The temperature and
the total energy was monitored to check that the molecular
system was equilibrated during the production of the
simulation. (This was true for all simulations.)
An extended conformation of the LAV peptide was
chosed as starting structure in both simulation runs in
vacuo. For simulation II, the inhibitor was arbitrarily
placed 20–25 Å away from the closest peptide backbone
atoms before the molecular dynamics run was initialized.
Being aware that the chosen conformation might be
dependent on the force field selected and the dielectric
constant used, we repeated simulation I applying two
different force fields (AMBER and CFF91), three additional dielectric constants (e⫽1,8,80), and reduced the
electrostatic contributions scaling them by a factor of 0.5.
Visual inspection of series of conformations from the end of
the simulations demonstrated—in spite of the inherent
flexibility of a peptide of this size—quite similar overall
shapes under all tested simulation conditions: a double
␤-hairpin showing the first turn starting after Arg4 and a
second one at Ile9 and Asn10. The second turn is more
pronounced than the first one. The results confirm that the
selected conformation is representative and not artificial
due to the use of specific force field or other simulation
Ideally all simulations should be performed using explicit solvent molecules. This would require roughly a 60 Å
box with about 25,000 atoms in the primary cell. To enable
proper folding of the peptide, the production time should
probably cover several nanoseconds (ns). Such a simulation is not feasible even with large supercomputers within
a reasonable time. Therefore, simulations III and IV were
run with explicit solvent (H2O), using as starting structure
a representative frame after stabilization of the in vacuo
MD run with LAV peptide and the inhibitor/peptide
complex, respectively. The molecules were placed in the
middle of a 34 ⫻ 34 ⫻ 34 Å large box, which was filled with
1263 water molecules using the SOAK option of the
Biopolymer option in Insight II. The same partial charges
as for the simulation without explicit water molecules
were applied. A simple point charge model is used for the
solvent, where additionally all vibrations of the water
molecule are allowed. No SHAKE option was used. Periodic boundary conditions were applied using a double
cutoff of 12/13 Å for the evaluation of the nonbonded
interactions. After a minimization of 1,000 steps using the
conjugate gradient algorithm, an equilibration period of
50,000 steps was followed by a production time of 1,000,000
steps. The integration step was 1 fs, the simulation
temperature 300K. Every 500 steps the coordinates were
Our aim in performing a molecular dynamics simulation
of the LAV peptide/inhibitor complex was twofold; we
wished to see whether the two molecules would associate
spontaneously (and specifically), and we wished to find out
which inhibitor/peptide interactions were important for
the association. Ideally, we wanted the starting configuration to consist of the peptide in extended form and the
inhibitor placed at a sufficient distance from it to ensure
that no interaction existed at the beginning of the run
(⬃25 Å). As a calculation of this ensemble with explicit
water molecules would require prohibitive computation
time, we adopted the compromise of carrying out the
simulation first in vacuo and then checking the stability of
the complex formed—if any—by subjecting it to a further
MD simulation with explicit solvent. To test whether this
compromise was capable of producing biologically relevant
results, we planned to use the technique first on the LAV
peptide alone. Simulation conditions that produced a
conformation in the isolated peptide that corresponded to
the secondary structure characteristics measured by circular dichroism spectroscopy might reasonably be expected
to produce a realistic approximation of the behavior of the
peptide/inhibitor ensemble.
For simulation I, of the LAV peptide in vacuo, stabilization of all main-chain ␾ and ␺ angles was reached after 350
ps, although most were already established by 100 ps.
Visual inspection of a representative frame showed the
peptide adopted the form of a double ␤-hairpin with a turn
connecting the LPCR tetrad at the N-terminus to the main
structure and a second turn at Ile9-Asn10 reversing chain
direction. Estimates of the secondary structure content of
Fig. 1. Ramachandran plot of the ␾ and ␺ angles of LAV 15mer residues during the MD simulation in vacuo. Values were taken from 106
frames at 20 frame intervals after stabilization was reached. Shown are
the average ␾ and ␺ angle for each internal residue surrounded by an
ellipse whose major and minor axes are the standard deviations for these
values. Pro2 ⫽ 䊊; Cys3 ⫽ 䊐; Arg4 ⫽ 䉭; Ile5 ⫽ 䉮; Lys6 ⫽ L; Gln7 ⫽ °;
Phe8 ⫽ 䊉; Ile9 ⫽ 䊏; Asn10 ⫽ 䉱; Met11 ⫽ 䉲; Trp12 ⫽ ♦; Gln13 ⫽ ¢; Glu14 ⫽ 䉺.
the polar form of the LAV 15mer derived from its far UV
circular dichroism spectrum yield values of 50–60% ␤-sheet
and roughly two reverse turns.1 A plot of the ␾ and ␺ angles
adopted by each residue during the MD simulation (after
full stabilization has been reached) places nine residues in
the ␤-region of the Ramachandran map and four in the
region typical for 310 helix and reverse turn, in full
agreement with the physical measurements (Fig. 1). Once
attained, the conformation is quite firm, as can be seen
from the relatively small deviations of ␾ and ␺ angles in
Figure 1.
A frame from the MD simulation in vacuo after a stable
structure had formed was used as the starting configuration for further simulation of the LAV 15mer peptide with
solvent (H20). No significant difference was seen in the
Ramachandran space on addition of solvent molecules.
Figure 2 shows an overlay of the backbone trace at 20
frame intervals during the ca. 1.0-ns run. The simulation
continues to depict an orthogonal double ␤-hairpin with a
high degree of conservation in the main chain secondary
structure. This stability, together with the close correspondence between the properties of the simulated structure
and the secondary structure content estimated from the
far-ultraviolet (UV) CD spectra, suggest that the MD
simulation represents a reasonably realistic model for the
conformation of LAV 15mer under polar conditions. The
position of the two reverse turns, located at residues 5 and
Fig. 2. Stereo pair of backbone traces (106, taken at 20 frame
intervals) of the LAV15 peptide simulation in the presence of simulated
solvent. Alignment was to the Gln7 amide nitrogen, C␣-carbon and
carbonyl carbon. Note the double orthagonal ␤-hairpin form is stable
throughout the run.
Fig. 3. A single frame from the MD simulation of the LAV 15mer peptide in the presence of
solvent showing the typical 90° ring to ring interaction between Pro7, Phe8 and Trp12.
9, is in agreement with the location of these turns in an
NMR-derived structure of the peptide in methanol, where
partial ␤-structure persists (unpublished results, A. Lindemann, J. Reed). It is also in agreement with the recently
published crystal structure of a truncated deglycosylated
form of gp120 complexed with a CD4 fragment and an
anti-CD4 Fab,24 where the LAV 15mer corresponds to the
C-terminal portion of ␤-sheet 19, all of ␤-sheet 20, and the
N-terminal portion of ␤-sheet 21.* The modeled structure
places the conserved Trp12 side chain in a central position
(Fig. 3), a location well suited for participation in the
multiple interactions with neighboring side chains that
charged-to-alanine experiments indicated are necessary
for stabilization of the ␤-type form.3
There are some unexpected aspects of the MD structure
that may be relevant to the mechanism of cooperative
refolding in this peptide. The LPCR tetrad at the Nterminus of the peptide has been proposed to function as a
nucleation site for the polarity-triggered refolding of the
switch domain into a 310 helix; it has theoretical potential
*The region around Trp427 (Trp12 in the LAV 15mer) has been
predicted to be helical in the CD4-bound form of gp120.1,24 In the
crystallized complex, however, this region interacts with an antiparallel ␤-sheet array of of the Fab fragment, which may serve to
maintain the ␤-structure in solution.
for forming a reverse turn, it adopts a well-defined turn
structure in isolation that is compatible with the ␾ angles
of a 310 helix25 and, in its absence, no helix is formed.3 All
this would lead one to expect that the model would exhibit
a turn centering on these residues. Instead, their ␾ and ␺
angles map to the extended region of Ramachandran
space, and the reverse turn occurs immediately C-terminal
to them. A possible explanation may be that the modeled
Pro2 residue tends to exist in alignment with the interacting ring systems of Trp12 and Phe8 in the interior of the
molecule (Fig. 3). Similar favorable interactions between
prolines and aromatic residues have been reported to form
the basis of the SH3 domain polyproline binding site.26
Whether this is representative of the situation in vivo
must await NMR resolution of the peptide structure, but it
is interesting that the helical nucleation site structure
adopted spontaneously by the TLPCRI fragment in solution24 is not the dominant form in the modeled ␤-type
An MD simulation repeated using the same in vacuo
conditions as before, but with the peptide in the presence
of the known switch inhibitor BM50.0311 (Fig. 4), resulted
rapidly in an extremely stable and specific association
between the two molecules. The inhibitor is enclosed in a
loop of the peptide, and structural variation in the latter is
Fig. 4. Starting configuration of the LAV 15mer peptide and the switch inhibitor molecule for the
MD simulation (without explicit solvent) of their interaction. Inset: the inhibitor substance BM
50.0311 with nomenclature used in the text.
reduced from that in the free state. Again, a frame from
this MD run after stabilization was used as the starting
configuration for an MD simulation with solvent. The
inhibitor remains in position, inserted in the center of a
single ␤-hairpin (Fig. 5a–c). A cluster of favorable interactions that persist, once established, throughout the run
appear to be responsible for this (Fig. 6). The principal
interactions are as follows:
1. Aromatic/aromatic interactions between the indole ring
of Trp12 and the heterocyclic ring of the inhibitor. This
association is meta-stable, i.e., the orientation of the
two rings varies throughout the run but the distance
between their centers is held at 3.5–6.0 Å. Interestingly,
there is no tendency after the initial collapse of the
peptide for aromatic/aromatic interactions between the
inhibitor ring system and the Phe8 residue. As a Trp at
position 12 is strictly conserved among HIV 1 strains
while the Phe is not, this is encouraging.
2. Charge interactions between the inhibitor heterocyclic
C-8 carbonyl and the backbone amides of Gln13 (2.49 ⫾
0.52 Å) and Arg4 (3.62 ⫾ 0.89 Å).
3. H-bonding between the inhibitor C-2 carbonyl and the
indole N of the Trp imidazole (4.02 ⫾ 0.97 Å). This is one
of the later associations to form but is not broken once
4. H-bonding between the inhibitor side chain amide (N-a)
and the peptide backbone carbonyls of Lys6 (2.80 ⫾ 0.71
Å) and Ile9 (2.84 ⫾ 0.54 Å).
5. H-bonding between the inhibitor side chain amide (N-c)
and the peptide backbone carbonyls of Ile9 (3.60 ⫾ 0.84
Å) and Lys6 (3.01 ⫾ 0.79 Å).
6. H-bonding between the inhibitor side chain carbonyl (b)
and the backbone amides of Lys6 (4.14 ⫾ 0.94 Å) and
Met11 (4.39 ⫾ 0.72 Å).
It is not surprising that in any MD simulation without
explicit water molecules favorable intermolecular H-bond
and ionic interactions between the inhibitor and the
peptide should occur. The close association between inhibitor and peptide persists, however, when the simulation is
carried out with water ‘‘soaking.’’ Moreover, there are
several aspects of the modeled complex that suggest it may
be representative of the actual relation between the two
molecules. The first is the remarkable rapidity with which
the peptide and its ligand associate and the stability of
that association when formed. More relevant is the correspondence between the favorable interactions listed above
Fig. 5. Overlaid backbone traces (106, taken at 20 frame intervals)
from the MD simulation in the presence of simulated solvent of the LAV
15mer/inhbitor interaction. Alignment was to the C4-C10-C3 atoms of the
heterocyclic ring in the inhibitor, not to atoms within the peptide. a: View
from above the plane of the single ␤-hairpin, showing central placement of
the inhibitor molecule. b: Side view, showing perpendicular orientation of
the inhibitor. c: View from the open end of the hairpin. The N-terminal
LPCR reverse turn can be seen in this orientation.
Fig. 6. Stabilizing interactions between inhibitor and peptide. Frame
is taken from the simulation of the inhibitor/peptide complex with solvent.
Note the displacement of the Trp12 residue from the central position
occupied in the absence of inhibitor, the association of the heterocyclic
ring system of the inhibitor with the tryptophan imidazole, and the
hydrogen bond network connecting the pseudopeptide tail of the inhibitor
to the two antiparallel strands of the LAV 15mer backbone.
and chemical groups found to be necessary to the activity
of switch inhibitors in general. For example, all successful
switch inhibitors tested so far have had a minimum of
three carbonyl groups. One of these interacts in the MD
simulation with a residue of the switch sequence that is
totally (Trp12) conserved in HIV1 strains, while others are
involved in H-bonding with the peptide backbone. Substitution of the inhibitor side chain carbonyl with a sulfur
atom abolishes activity, and this carbonyl is one of the first
groups to interact with the peptide. Finally, all successful
inhibitors have a double ring system, often aromatic, that
has been postulated to interact with the critical Trp
residue. The association between the Trp indole and the
inhibitor heterocyclic ring is a prominent feature of the
modeled complex.
The MD simulation has helped explain some aspects of
the mechanism by which the switch inhibitor disrupts
cooperative refolding of the LAV 15mer. A major effect is
that the bulk of the inhibitor heterocyclic ring occupies the
central space in what is now a single ␤-hairpin, displacing
the Trp indole ring that occupies this area in the simulated
free peptide. At the same time, the Trp residue is no longer
in a position to maintain the multiple interactions with
side chain groups that experimental data have implicated
in stabilizing the ␤-form until a critical polarity is reached.
This could explain why the response of the peptide to
decreasing polarity in the presence of the inhibitor is a
linearly proportional loss of ␤-sheet and gain of helix.
It should be noted that in the inhibitor-complexed model
the reverse turn at the N-terminus is centered on the
LPCR tetrad, as expected from the 1H-NMR data,5,25
rather than on Ile5 and Lys6 as in the MD model of the free
peptide. The ␾ and ␺ angles of these last now map to the
extended region of the Ramachandran plot (Fig. 7). If the
tendency to adapt an extended conformation for LPCR in
the model of the free peptide is correct, an additional effect
of the inhibitor may be to permit formation of the nucleation site even in the presence of the Phe and Trp side
inhibitor activity. The molecular dynamics simulation has
given us some idea as to why these groups are necessary.
Additionally, it has provided us with an hypothesis as to
the mechanism by which the switch inhibitors disrupt the
cooperativity of the ␤-sheet = helix transition; this in turn
can be used to optimize inhibitor design. The most recently
synthesized substances show an improvement in ID50
(cell-binding test2) of greater than two orders of magnitude. While rational drug design should ideally proceed
from actual physical data, here molecular dynamics simulation has been able to function as a practical tool in a case
where these could not be obtained.
We thank Dr. Rüdiger Pipkorn for preparation of the
LAV 15mer peptide and Angelika Lampe-Gegenheimer for
her expert assistance with the manuscript.
Fig. 7. Ramachandran plot of the ␾ and ␺ angles of the LAV 15mer
peptides in the presence of inhibitor during the MD simulation in vacuo.
Values were calculated as in Fig. 1; key as in Fig. 1.
chains under high polarity conditions, thus lowering the
threshold for formation of helix at the same time as it
destabilizes the ␤-sheet.
In two chemically unrelated families of switch inhibitor,
the modified dipeptide CPF and the compound used here
with its pseudo-peptide side chain, the presence of any
aliphatic group larger than a methyl at a position corresponding to the N-terminus abolished activity.2 It was
proposed that this might be due to the ability of methyl
(but not larger) groups to establish nonbonded resonance
structures, i.e., that a degree of rigidity in this area
allowed inhibitor activity. Certainly the heterocyclic ring
devoid of side chain has no significant activity as a switch
inhibitor (unpublished results). The simulation shows that
the extended chain of the inhibitor rotates as a rigid unit
around the Na-C6 linkage to the ring system, adopting two
well-defined rotational positions (Fig. 5b,c). In each of
these two positions it can serve as an ersatz peptide
backbone, forming hydrogen bond linkages to the main
chain carbonyl of Ile9 and Met11 amide on one side of the
loop and the Lys6 carbonyl and amide on the other. These
are established early in the run and serve to fix the
heterocyclic ring in a central position, where its displacement of and interaction with the Trp residue is favored.
We wished to know more about the interaction between
the LAV 15mer and switch inhibitors in order to gain
specific information on possible improvements in inhibitor
design. It had already been discovered empirically that the
presence of certain functional groups was necessary for
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