(Received for publication, May 8, 1995; and in revised form, December 27, 1995)
From the
The third variable (V3) loop of HIV-1 surface glycoprotein, gp120, has been the target of neutralizing antibodies. However, sequence variation inside the V3 loop diminishes its effectiveness as a potential vaccine against HIV-1. The elusive nature of the V3 loop structure prompted us to carry out a systematic study on different isolates in an attempt to identify a common structural motif in the V3 loop regardless of the amino acid sequence variability. We have previously determined the structural features of two V3 loops: V3 Thailand and V3 MN. In this paper, we present the structure of two other variants: V3 Haiti and V3 RF. Our results show that similar secondary structures are observed in all the four V3 loops: a GPG(R/K/Q) crest in the center of the neutralizing domain, two extended regions flanking the central crest, and a helical region in the C-terminal domain. For the Haitian V3 loop, we also show how the conserved structural features are masked through a conformational switch encoded in the amino acid sequences on the C-terminal side of the GPGK crest.
A neutralizing determinant(1, 2, 3, 4, 5, 6, 7, 8) located
inside the V3 ()loop of the envelope glycoprotein, gp120,
has been the target for protective immunity against the human
immunodeficiency virus, type 1 (HIV-1). However, the amino acid
sequence variation within the V3 loop has eluded the effectiveness of
V3-based vaccine design(4, 5) . Antibodies against the
V3 loop generally exhibit type-specific neutralization
profiles(6, 7) , although a subset of anti-V3
antibodies specific for the less variable elements of the V3 loop show
a broader range of neutralizing activity (7, 8) . To
better understand the effect of sequence variation on the structure and
antigenicity of the HIV-V3 loop, we developed a method combining
molecular modeling (9, 28) and two-dimensional
NMR(10, 11) to analyze the global structure of the
entire cyclic V3 loop and the local structure at the neutralizing
determinant. We attempted to answer two specific questions: (i) Are
there conserved secondary structural elements inside the V3 loop in
spite of sequence variation? and (ii) Can the sequence variation inside
the V3 loop mask this conserved secondary structure? Recently, we have
shown (12) that in spite of the observed sequence variation, a
conserved secondary structure is located inside the V3 loop. This
structure consists of a solvent-accessible protruding motif (or a knob)
spanning 8-10 residues with a central GPG(Q/R/K) type II turn at
the crest of the knob(10, 11, 12) . In this
article, we demonstrate how amino acid sequence variation flanking the
GPG crest can camouflage an otherwise conformationally pure epitope.
For this purpose, we performed two-dimensional NMR and molecular
modeling studies on two cyclic V3 loops from the Haitian and RF
isolates(13) :
(Cysteines at position 2 and 36 are S-S bridged; the first cysteine that is underlined in the sequence has a protective group on S; site-specific differences in sequence are marked in bold).
Figure 1:
NOESY (mixing time = 250 ms) and
DQF-COSY cross-sections of the cyclic Haitian V3 loop in water (peptide
concentration = 3.0 mM, pH 5.5, temperature = 10
°C). A, the fingerprint HN-H region. B, DQF-COSY HN-H
cross-section. C,
the HN-HN region. For NOESY experiments, the acquisition parameters
were as follows: t2 = 2048 data points, t1 = 1024 data
points, relaxation delay = 1.5 s, number of transients =
32. The same acquisition parameters were used for the DQF-COSY
experiment except for t2, which was increased to 4096 data points.
Sequence specific assignments (14) were obtained starting from
Phe
(only Phe in the sequence) and moving backward and
forward along the connectivity route until completion of the
assignments. Note the resonance doubling of the residue
Gly
, indicating a conformational equilibrium between the
two forms. However, no additional NOEs to discriminate between the two
conformations were observed.
Figure 3:
Summary of the NMR data for the HIV
Haitian V3 loop in water (under panel 1) and in water/TFE
(7:3) mixed solvent (under panel 2). In addition to sequential
H-HN, H
-HN, and HN-HN NOEs, six
sequential H
-HN NOEs were obtained in both the
solvents. The sequential H
-HN connectivity for Pro is
missing, but the sequential H
-H
NOEs
provide the link. Note the solvent-induced change in the sequential NOE
pattern in the C-terminal segment; in the mixed solvent, there is an
enhancement of the sequential HN-HN NOEs relative to the corresponding
H
-HN NOEs, indicative of an induction of a helix.
In a previous work(24, 25) , the NMR assignments
of the V3 loops of MN and IIIB isolates in aqueous solution were
obtained. The authors also performed CD studies on TFE mixed solutions
but did not succeed in assigning the NMR spectra in such mixed
solvents. In another NMR work(26) , the authors studied two 24
linear peptides containing the neutralizing determinant of the IIIB
isolate of HIV-1. They reported (26) the presence of a
transient turn at the GPGR crest, and the ability of mixed TFE
solutions to induce helical conformation in the C-terminal domain of
the peptides, whereas in water only a ``nascent'' helix
formed by a stretch if interconnected turns was
observed.
Fig. 2A shows the NOESY fingerprint HN-H region of the Haitian V3 loop in a water/TFE (7:3) mixture at 10
°C for 250 ms of mixing. Although the cross-peaks of the Haitian V3
loop are broader in the mixed solvent than in the aqueous environment,
we are able to assign residues 5-36. Fig. 2B shows the NOESY HN-HN region of the Haitian V3 loop in the mixed
solvent at 10 °C for 250 ms of mixing. Due to the broadness of the
peaks it is not possible to decipher the interaction of two HN protons
that are close to the diagonal. However, quite a number of distinct
HN-HN cross-peaks are observed in this cross-section. Fig. 3summarizes the NOE data of the Haitian V3 loop in the
mixed solvent from the analyses of the data at 75 and 250 ms of mixing.
The NOE data of the Haitian V3 loop in the mixed solvent is distinct
from that in water in the following respects. (i) Medium range
H
(i)-H
(i+3) cross-peaks are
observed for the residue pairs (27, 30), (31, 28), (32, 29), and (33,
30) which are indicative of a helical core spanning residues
27-33. (ii) Although there is a decrease in the absolute
intensities of sequential HN-H
and HN-HN cross-peaks,
there is an enhancement in the relative HN-HN/HN-H
cross-peak intensities for residues 26-34, which is again
indicative of a helical structure in this segment. (iii) A few
sequential H
-HN cross-peaks are observed in this
stretch that are either weak or absent in the aqueous solvent. (iv)
Finally, the H
protons of the residues 27-34 in
the C terminus of the Haitian V3 loop are high field shifted in the
mixed solvent as also observed in the case of the MN V3
loop(11) .
Figure 2:
NOESY cross-sections of the cyclic Haitian
V3 loop in water/TFE (7:3) mixture (peptide concentration = 3.0
mM, pH 5.5, temperature = 10 °C). The HDO signal
was presaturated for 1.2 s during the relaxation delay. Acquisition
parameters: data matrix (t2 = 2048 data points, t1 = 1024
data points), relaxation delay = 1.2 s, number of transients
= 32, temperature = 10 °C. A, the
fingerprint HN-H region. B, the HN-HN region. In
the fingerprint region note the double population of the residue
Gly
. One of the populations has an additional medium range
NOE between H
-G18 and HN-F21, indicating a
conformational equilibrium between two forms, one extended and one bent
at the fragment
Gly
-Lys
-Ala
-Phe
.
Interestingly such a double population for Gly
is also
observed in water, although the medium range NOE with F21 absent in the
aqueous environment (see Fig. 1). Note that uniform upfield
shift of the H
resonances in the C-terminal region of
the peptide that is a strong indicator of the helical
conformation.
The residues in the neutralizing epitope,
Ile-Pro
-Met
-Gly
-Pro
-Gly
-Lys
-Ala
-Phe
-Tyr
,
are unequivocally assigned in water and in the solvent. Two
conformational states of Gly
(i.e. Gly
and Gly
*) are clearly evident. Gly
not
only shows two populations in terms of the chemical shift values of
(H
,HN) but also in terms of the NOESY connectivities.
As shown in Fig. 2A, a second population of
Gly
* is observed in mixed populations; Gly
shows a H
(G18)-HN(F21) cross-peak (Fig. 3). The chemical shift of other residues in the second
population are indistinguishable from the first. Although this
conformational variant is also present in the aqueous solvent, the
absence of the H
(G18)-HN(F21) cross-peak suggests that
such an interaction perhaps is not stabilized in a polar environment.
Figure 4:
NOESY (mixing time = 400 ms) and
DQF-COSY cross-sections of the cyclic RF V3 loop in water (peptide
concentration = 5.0 mM, pH 5.5). A, the
fingerprint HN-H region. B, the HN-HN region. C, DQF-COSY cross-sections of the cyclic RF V3 loop. For NOESY
experiments, the acquisition parameters were as follows: t2 =
2048 data points, t1 = 1024 data points, relaxation delay
= 1.5 s, number of transients = 32. Same acquisition
parameters were used for the DQF-COSY experiment except for t2, which
was increased to 4 K. Sequence-specific assignments were obtained
starting from Val
(only Val in the sequence) and moving
backward and forward along the connectivity route until completion of
the assignments. Assignments in the fragment Cys
-Asn
were not possible, presumably due to the intrinsic flexibility in
the region
Asn
-Asn
-Asn
.
Figure 5:
Summary of the NMR data for the HIV RF
V3 loop in water. Sequential H-HN,
H
-HN, and HN-HN NOEs. The sequential H
-HN
connectivity for Pro is missing, but the sequential
H
-H
NOEs provide the NOE link.
Lorimier et al.(26) studied a 40-residue-long
peptide containing a T-helper epitope 16 residue long in the N-terminal
region and a 24-residue-long segment derived from the V3 loop of the
HIV-RF isolate studied in this work. In the V3 loop region the authors
observed the GPGR turn, whether the rest of the C-terminal fragment was
mostly disordered. However, we would like to stress that in the study
of the linear peptides(24, 25) , the authors did not
consider the importance of the disulfide bridge locking the V3 fragment
in a closed loop. We have previously shown that the cyclic V3 loops are
better ligands for V3-specific
antibodies(11) .
Fig. 6shows the ribbon models of the Haitian V3 loop in water (left) and water/TFE (7:3) mixture (right). The
following color coding was used in these ribbon diagrams: gray for the N-terminal protruding loop at position T3-R10, green for the N-terminal extended -strand flanking the GPG crest, magenta for the central
-turn at position
G16-P17-G18-K19, yellow for the C-terminal extended
-strand flanking the GPG crest, and blue for the
C-terminal segment D26-H35, which can form an
-helix. In water the
C-terminal segment consists of consecutive
-turns centered around
Ile
-Gly
and
Ile
-Arg
, whereas in the water/TFE (7:3)
mixture it further folds into a well defined
-helix as evidenced
by the presence of sequential HN-HN NOEs (Fig. 2B) and
medium range H
(i+3)-H
(i) NOEs (Fig. 3B, panel 2). Due to the intrinsic
conformational flexibility of the V3 loop, side chains are quite
mobile, and they do not sample a single rotamer conformation. In these
representations only average positions for the side chains are shown.
However, in the
-helical region for the mixed solvent structure,
the side chains are organized in a cylindrical array as experimentally
observed by the presence of a network of
d
(i,i+3) and d
sequential
connectivities (Fig. 3B). Nonetheless, in both the
structures (Fig. 6) the neutralizing epitope containing the
central GPGK sequence forms a protruding loop even though the local
structure and presentation of the loop in the two cases are noticeably
different. The aqueous structure of the Haitian V3 loop in Fig. 6is the average of 50 sampled configurations that exhibit
rms deviations below 1.5 Å with respect to the backbone atoms.
Out of 50 sampled structures of the Haitian V3 loop in the TFE/water
mixture, a small subset of six structures shows a large
(>2.6Å) rms deviation of the backbone atoms from the rest of
the structures. The remaining 44 structures are within 1.6 Å rms
deviations of the backbone atoms. The average structure in Fig. 6is taken over these 44 structures.
Figure 6:
The ribbon diagrams describe
representative folding patterns for the structures of the Haitian V3
loop in water (left) and in mixed water/TFE solvent (right). The following color coding was used in these ribbon
diagrams: gray for the N-terminal protruding loop, green for the N-terminal extended -strand flanking the GPG crest, magenta for the central
-turn at the GPG crest, yellow for the C-terminal extended
-strand flanking the
GPG crest, and blue for the C-terminal segment, which can form
an
-helix. In each case, the average is done over 50 sampled low
energy structures. Ribbon models in the two cases correspond to the
average structure. All the sampled structures of the Haitian V3 loop in
water showed rms deviations of 0.24 ± 0.02 Å with respect
to 95 independent distance constraints. All the sampled structures of
the Haitian V3 loop in mixed water/TFE solvent showed rms deviations of
0.27 ± 0.02 Å with respect to 123 independent distance
constraints. The structures of the Haitian V3 loop in water show a
greater degree of flexibility than those in the mixed water/TFE
solvent; this is due to the formation of the C-terminal helix in the
mixed solvent.
Fig. 7shows
two conformations representing the aqueous environment (left)
and the mixed solvent forms (right). The central region of the
Haitian V3 loop containing the neutralizing determinant residues
Ile-Pro
-Met
-Gly
-Pro
-Gly
-Lys
-Ala
-Phe
-Tyr
is shown. The following color coding was used in these skeleton
models: red for the central
Gly
-Pro
-Gly
-Lys
crest, green for the N-terminal
Ile
-Pro
-Met
residues in extended
conformation, and yellow for the C-terminal
Ala
-Phe
-Tyr
residues, which show
a solvent-induced effect. In the water structure the C-terminal
fragment is in an extended conformation (open state). In the mixed
solvent, two types of chain folding are observed: one folded form is
similar to that of the MN-V3 loop(11) , whereas in the other,
the GPG crest forms the typical type II
-turn followed by a type
III
-turn involving residues
Gly
-Arg
-Ala
-Phe
, as
evidenced by the presence of a medium range NOE between
H
-G18 and HN-F21 (closed state). Such an S-shaped
conformation has been previously reported for a peptide containing the
V3 neutralizing determinant complexed to an antibody(18) , and
it will be referred as ``arched'' conformation for the rest
of the paper. Our NMR data (Fig. 1A and Fig. 3)
clearly indicated that these two states are simultaneously present in
mixed solvent, whereas only the open state exists in aqueous solutions.
We have not shown the open state structure of the Haitian V3 loop in
the mixed solvent because it closely resembles the already published
structure of the MN-V3 loop (11) .
Figure 7:
Two conformations representing the aqueous
environment (left) and mixed solvent forms (right).
The central region of the Haitian V3 loop containing the neutralizing
determinant residues
Ile-Pro
-Met
-Gly
-Pro
-Gly
-Lys
-Ala
-Phe
-Tyr
are shown. The following color coding was used in these skeleton
models: green for the N-terminal
Ile
-Pro
-Met
residues in extended
conformation, red for the central
Gly
-Pro
-Gly
-Lys
crest, and yellow for the C-terminal
Ala
-Phe
-Tyr
residues, which show
solvent induced arching effect. Solvent-accessible areas were
calculated using the Molecular Surface Package due to Connolly (27) with a probe radius of 1.5 Å. In the fragment
Lys
-Phe
, the aqueous structure has a lower
surface accessibility than the structure in the mixed TFE/water
solvent.
Figure 8: The ribbon diagrams describe representative folding pattern of the RF V3 loop in water. The same color coding described in the legend to Fig. 6was used here. The structural features of the V3-RF in water resemble those observed for the Haitian and the MN V3 loops in water(11) .
Previous NMR studies on the Thailand and MN V3 loops (10, 11) and the current work on the Haiti and RF V3
loops (Fig. 6Fig. 7Fig. 8) can be summarized as
follows: (i) A GPG-turn at the crest of the V3 loop is present in all
the four sequences. (ii) Stretches of -strand adjacent to the
GPG-turn on the N- and C-terminal sides are common to all the four
sequences. (iii) The residues in the C-terminal segment form a few
turns in water and a helix in the less polar mixed solvent. (iv) In
spite of the constraints of secondary structures ((i)-(iii)) and
the disulfide bridge, the V3 loop exhibits conformational flexibility
as evidenced by the absence of long range NOESY interactions commonly
observed in well folded globular proteins(14) .
However, a
``protruding knob'' formed by the central GPG turn and the
-strands on either side emerges as the secondary structural
feature conserved among diverse V3 loop sequences. The single crystal
structure of the HIV-1 neutralizing antibody (monoclonal antibody 50.1)
complexed to 16-residue-long linear MN-V3 fragment shows the hint of
such a protruding knob, although the segment on the C-terminal side of
the GPGR type II turn remains disordered(18) . The
crystallographic observation suggests that the protruding knob of the
V3 loop that includes the neutralizing epitope might well be
specifically recognized by the antibody. However, the conserved
protruding knob of the V3 loop need not always be presented in its
conformationally pure form because HIV may find a way to mask this
conserved secondary structural element. In this work we report one such
mechanism of masking as revealed by the closed state in Fig. 7.
In this form of the Haitian V3 loop, the NMR data indicate an arching
of the residues on the C-terminal side of the GPGK turn. This is a
departure from the protruding knob motif that contains the central GPG
turn and two
-strands on either side. Such an arched conformation
of the neutralizing epitope has also been observed in an antibody
(monoclonal antibody 59.1) complexed with a linear V3
fragment(18) .
When combined with the single crystal
data(17, 18) , our NMR data ( (10) and (11) and this work) indicate that the closed or arched
conformation of the neutralizing epitope of the V3 loop is possible and
can be recognized by the antibody. In addition, our data also indicate
that an equilibrium between the closed and open state (Fig. 7)
is possible for the same V3 loop sequence. The arching around
Ala-Phe
tends to mask Lys
and
Ala
(Fig. 7). The closed form of the V3 loop may
camouflage some essential elements of the neutralizing epitope from the
immune system. For instance, this masking will interfere with the
binding of antibodies (8, 19) that recognize the PGRAF
epitope.
Most importantly such a local masking of Ala and Phe
should affect the proteolysis of the
(Arg/Gln/Lys)
-Ala
peptide bond by thrombin
and tryptase(20, 21) ; the second enzyme lies on the
T-cell surface. When gp120 is used as a substrate these two enzymes
show exceptional specificity for cleavage of the
(Arg/Gln/Lys)
-Ala
peptide bond inside the V3
loop. Most striking is the observation that the V3 loops of T-cell
tropic virus strains are 1,000 times more susceptible to cleavage by
these two enzymes than the V3 loops of macrophage tropic
strains(21) . The T-cell tropic V3 loops are more positively
charged than the macrophage tropic V3
loops(21, 22, 23) . Our studies reveal that
the open state of the neutralizing epitope of the V3 loop is
exclusively preferred for MN and RF V3 loops with net charges of more
than +5, whereas the closed state of the neutralizing epitope
begins to appear for the Haitian V3 loop with a net charge of +3.
Therefore, the proteolysis data (21) are consistent with our
structural conclusions.