(Received for publication, September 21, 1994; and in revised form, November 10, 1994)
From the
Studies of the feasibility of a subunit vaccine to protect against human immunodeficiency virus (HIV) infection have principally focused on the third variable (V3) loop. The principal neutralizing determinant (PND) of HIV-1 is located inside the V3 loop of the surface envelope glycoprotein, gp120. However, progress toward a PND-based vaccine has been impeded by the amino acid sequence variability in the V3 loops of different HIV isolates. Theoretical studies revealed that the variability in sequence and structure of the V3 loop is confined to the N- and C-terminal sides of the conserved GPG crest. This leaves three regions of the V3 loop conserved both in sequence and secondary structure. We present the results of NMR studies that test the validity of our theoretical predictions. Structural studies are reported for the HIV-V3 loop (HIV-MN) in the linear and cyclic (S-S-bridged) forms. For the V3 loop sequence of the HIV-MN isolate, the three conserved secondary structural elements are as underlined below:
Finally, the conformational requirement of the PND in the V3 loop-antibody interaction is tested by monitoring the monoclonal antibody binding to the HIV-MN V3 loop in the linear and cyclic forms by enzyme-linked immunosorbent assay. The binding data reveal that the cyclic V3 loop is a better ligand for the monoclonal antibodies than the linear form although the latter has the same sequence. This means that the monoclonal antibodies recognize the PNDs as conformational epitopes.
The surface of human immunodeficiency virus (HIV) ()is studded with several copies of the glycoprotein
gp120(1) . A segment of gp120 is being considered as a
potential antigenic target for protective humoral immunity. This
segment is located inside the third variable loop, called the V3 loop (Fig. 1). Monoclonal antibodies raised against the V3 loop can
neutralize the viral infection by specifically binding to the principal
neutralizing determinant (PND) located inside the V3 loop (2, 3, 4) . However, the HIV V3 loop
undergoes sequence mutations at a rapid rate in order to escape the
immune surveillance of the host cell. But certain segments of the V3
loop remain fairly conserved among different HIV isolates; these
conserved segments of the V3 loop are probably essential in the
life-cycle of the virus. Therefore, the virus mutates the V3 loop only
enough to escape the immune pressure without risking its own life
inside the host cell. The elusive nature of the V3 loop calls attention
toward two important structural aspects in relation to V3 loop-antibody
interaction: (i) the global tertiary folding of the V3 loop and (ii)
the structure and presentation of the PND. This article describes the
results of a study aimed at exploring these structural aspects by
combining two-dimensional NMR spectroscopy, molecular modeling, and
antibody binding measurements of the V3 loop from the HIV isolate from
Minnesota (HIV-MN).
Figure 1:
Analyses of the amino
acid sequence and the conserved secondary structural elements of V3
loop sequences. A molecular modeling method (9, 10, 11) is used to define secondary
structural states of relatively conserved and highly variable regions
of V3 loop sequences, and to predict the resulting energetically stable
tertiary fold(s) of the S-S-bridged V3 loop. We found (11) that, regardless of the neighboring amino acids, the boxed
regions retained the same secondary structural elements, i.e. (i) a loop with two consecutive -turns at the N-terminal
segment, (ii) a type II
-turn at the GPG-crest, and (iii) a
C-terminal helix. Both the North American consensus V3 loop and the HIV
V3 loop isolated from a Minnesota patient (HIV-MN) showed the same
secondary structural elements for the three conserved regions of the V3
loop. A, the most common amino acid found in each site is
shown on the top row; this row corresponds to the North
American consensus V3 loop sequence. The percent frequency with which
an amino acid occurs at each site is shown directly below, and beneath each percent frequency, a column of the amino acids
that can occupy each position is listed in descending order of their
percent frequency at the given site. (This figure is adapted from
Roterman et al. (7) .) The conserved regions are
numbered 1 through 3. B, the amino acid sequence and
the conserved secondary structural elements in the HIV-MN V3
loop.
(The summation is over 1 = - to
, where
= size of the window chosen to account for the effect of the
neighboring amino acid residues:
= 5 for h;
= 3 for b; and
= 4 for c or t). P(k, i) = potential for
the k-type of conformation of individual residue i derived from the analysis of the single crystal structures of
about 65 proteins. The highest S(k, i)
determines the conformation k for the i residue (5) . Use of any existing algorithm for secondary structure
prediction is only 60% accurate. In order to improve accuracy, we
tested our predictions by requiring an S-S bridge formation that
achieves local energy minima for the cyclic V3 loop; this led to step 2
in our method.
(,
) of residues in the coil state were set free to
choose any point in the allowed space (for definitions of different
secondary structures and corresponding (
,
) values, see
Ramachandran and Sasisekharan(6) ). We simplified the sequence
by assuming Ala for residues with side chains extending beyond the
C
atom, except for the Pro and the terminal Cys. Our
rationale for doing this was that the allowed (
,
) space of
residues with a side chain longer than Ala is only a subspace of that
allowed for Ala(6) .
We obtained an S-S-bridged
structure of a V3 loop by using a linked-atom-least-square refinement
equation that minimizes function F in the space (,
):
where G (= [vert] r
- r
[vert] = 0) indicates distance constraints for
an S-S bridge. Distances in the S-S-bridged V3 loop
configuration are defined as r
= S(C1) - S(35), r
=
C
(C1) - S(C35), r
=
C
(C35) - S(C1), and r
= C
(C1) - C
(C35);
corresponding equilibrium distances are r
= 2.04 Å, r
= r
= 3.05
Å, r
= 3.85
Å(7) .
indicates Lagrangian
multipliers; d
indicates distance between
atom i (type m) and atom j (type n); and D
indicates the contact limit between atom (type m) and atom (type n)(6) . In this refinement
(
,
) of various residues were treated as elastic variables (i.e. variables with weights) such that by appropriate choice
of weights the predicted secondary structural states of residues (after
step 1) were minimally altered(8, 9) . This method
guarantees a stereochemically orthodox structure for the
S-S-bridged (CA
C)-like sequence. Finally,
appropriate side chains were attached to generate an actual V3 loop
sequence and the potential energy of the system was minimized in the
(
,
,
,
)-space using the force-field of Sippl et al. as cited in (11) . Several energy-minimized
structures of a given V3 loop sequence were obtained by choosing a
number starting structures within the specified ranges of
(
,
) values predicted in step 1. Conformational sampling of
a V3 loop sequence belonging to a given family of tertiary fold was
performed by Monte Carlo simulated annealing(9) . If the
secondary structural states of one or more residues as predicted in
step 1 were energetically unfavorable for the cyclic V3 loop, those
states were altered in step 2. Even though wrongly predicted secondary
structural states of a residue by step 1 was corrected in this step
using the energy criteria of a cyclic V3 loop, it was necessary to
examine which secondary structural states of the residues in the V3
loop were predominantly present in solution. This led to step 3 of our
methodology.
Type I is given as
Type II is given as
This type is particularly useful for an unobserved NOE where we
can set a lowest allowable distance limit for the corresponding proton
pair. The -angle constraints are also included as 1-4
distance constraints.
The energy term, EDIST, is added to the
force-field QCEP 454 due to Scheraga and co-workers(7) . The
simulated annealing is performed in the following manner. First, a
starting energy-minimized structure is chosen and Monte Carlo
simulations are performed for 50,000 steps at 1000K in the (,
,
,
)-space; the last accepted configuration is
stored to be subsequently used as a starting configuration in the next
lower temperature-cycle. Second, 50,000 Mone Carlo steps are repeated
in several cycles of gradually decreasing temperature until a
temperature of 100K is reached. Third, the lowest energy configuration
at 100K is further energy-minimized to a low energy gradient. This is
the ``temperature quenching'' step in which thermally excited
single bond rotations around the equilibrium positions are quenched.
Finally, first through third steps are repeated for 20 different
starting configurations.
All sampled low energy structures are analyzed to define the extent of conformational variability. Although 20 starting structures chosen for Monte Carlo simulation are conformationally different, they are not included in the analyses for conformational flexibility. Because these structures obtained by NMR pattern recognition followed by energy minimization do not adequately define the population density of the energy basins to which they belong. However, after simulated annealing energy barriers are crossed and different energy basins are visited sufficient number of times. Therefore, after such a sampling analyses of conformational variants become physically meaningful.
Figure 2:
NOESY
cross-sections of the cyclic MN V3 loop in water (peptide concentration
= 3.5 mM; pH 4.5). The pulse-sequence due to
Sklenar-Bax (24) was used for solvent suppression. Acquisition
parameters: data matrix (t = 2K, t
= 1K), relaxation delay = 1.5 s,
number of transients = 32, temperature = 10 °C. A, the fingerprint HN-H
region; B,
the HN-HN region. Sequence specific assignments (25) were
obtained starting from F20, which represents a unique residue
in the sequence, and moving backward and forward along the connectivity
route until completion of the assignments.
Figure 3:
Summary of the NMR data for the HIV-MN V3
loop in water and in water/TFE (7:3) mixed solvent. Nomenclature of
various sequential and medium range NOEs are taken from Wuthrich et
al.(12) . In addition to sequential H-HN,
H
-HN, and HN-HN NOEs about 10 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 NOE
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.
NMR experiments are, therefore, conducted in a less polar water/TFE
(7:3) mixed solvent to promote the formation of a C-terminal helix. Fig. 4, A and B, show the corresponding NOESY
HN-H (fingerprint) and HN-HN regions for 300 ms of
mixing. Spectra in Fig. 4, A and B, indicate
that the cross-peaks are better resolved in the mixed solvent although
the one-dimensional signals in the mixed solvent are slightly broader
than in water. NMR data of the HIV-MN V3 loop in the mixed solvent
allow identification of 220 NOESY cross-peaks including the
intra-residue HN-H
NOEs containing the
-angle
information. The induction of the C-terminal helix in the mixed
water/TFE solvent is supported by the following NMR data characteristic
of a helix(12) : (i) the chemical shift of the H
protons belonging to the residues in the 23-33 segment
shows a solvent-induced high field shift, (ii) the sequential NH-NH
cross-peaks for the residues in segment showed an appreciable
solvent-induced increase in the intensity relative to the corresponding
sequential H
-HN intensities, (iii) emergence of the
H
(i)-HN(i + 3/4) and
H
-H
(i + 3/4) NOESY
cross-peaks for the C-terminal residues. Various sequential and medium
range NOEs of the cyclic MN V3 loop in the mixed solvent are summarized
in Fig. 3.
Figure 4:
NOESY
cross-sections of the cyclic MN V3 loop in water/TFE (7:3) mixture
(peptide concentration = 2.5 mM; pH 4.5; temperature
= 10 °C). The HDO signal was pre-saturated for 1 s during
the relaxation delay. Acquisition parameters: data matrix (t = 2K, t
=
1K), relaxation delay = 1.5 s, number of transients = 32,
temperature = 10 °C. A, the fingerprint
HN-H
region; B, the HN-HN
region.
Figure 5: A and B, the ribbon diagram showing the average folding patterns of the structures the MN V3 loop in water and in mixed water/TFE solvent. In each case, the average is done over 70 sampled low energy structures. Note that, in each case, the neutralizing epitope containing the central GPGR sequence forms a protruding loop even though the local structure and presentation of the loop in two cases are noticeably different. All the sampled structures in A and B showed rms deviations of 0.25 ± 0.01 with respect to 140 specified distance constraints. The structures that satisfy the NMR constraints of the V3-MN loop in water show greater degree of flexibility than those in agreement with NMR data in the mixed water:TFE solvent; this is due to the formation of the C-terminal helix in the mixed solvent.
The flexibility of the V3 loop in
two solvents is markedly different. The nature of flexibility of the
two structures is identified by examining the standard deviations
(S.D.) of the backbone and side chain torsion angles in these two
structures around their average values. These deviations (observed by
analyzing energy minimized structures) reflect the lowest possible
values because the thermal motions (particularly for the side chains)
are filtered off by energy minimization. Table 1and Table 2show the S.D. values in the torsion angles for the V3 loop
structures in the aqueous and mixed solvents, respectively. Note that
the structure in the aqueous solvent is more flexible than the
structure in the mixed solvent. The flexibility of the residues
(Lys, Arg
, His
,
Ile
) in the aqueous V3 loop structure is greatly reduced
in the mixed solvent structure because of the induction of a C-terminal
helix involving residues 23-33. The helix for residues
23-33 of the V3 loop in the mixed solvent is a distorted one i.e. the continuous stretch of C=O(i)
(i + 4)HN H-bonds is weakened where (
,
)
values deviate from the ideal helix values (for examples, residues
Ile
and Gln
in Table 1and Table 2). Also note that the residues 20-23 and 28-34
on the C-terminal side of the V3 loop in the aqueous solvent are more
flexible than the corresponding residues of the V3 loop in the mixed
solvent.
The biological relevance of TFE-induced structural change is often questioned. However, it may be pointed out that water molecules are largely excluded from the surface of the V3 loop in its active form when it is interacting with antibodies or the host-cell receptor or with other domains of gp120(14) . Therefore, TFE-induced structural changes may shed some light on the process that accompanies the activation of the V3 loop. In addition, the physico-chemical observation that the C-terminal residues of the V3 loop adopt a helical structure in the mixed solvent is a testimony that the same residues have intrinsic helix forming propensity which is masked in water due to competing water-peptide H-bonds.
Figure 6: An enzyme-linked immunosorbent assay showing the preference of monoclonal antibodies for the cyclic over the linear form of the HIV-MN V3 loop. Human monoclonal antibodies 1510 (top) and 1511 (center), and mouse antibody 1289 (bottom) all bind to a greater extent to the cyclic V3 loop peptide. The recognized epitopes 1510 (top), 1511 (center), and 1289 (bottom) are shown on the right. In the schematic representations of the HIV-MN V3 loop shown on the right, solid circles depict hydrophobic residues, open circles charged residues, and outlined circles polar uncharged residues.
The following conclusions can be drawn based on the data of the HIV-MN V3 loop presented in this article. (i) The S-S bridge between C1 and C35 introduces structure in the V3 loop. (ii) The overall tertiary folding of the V3 loop as well as the local structure at the PND are critical in deciding the affinity of the V3 loop for antibody binding. (iii) The structure of the HIV V3 loop is intrinsically flexible and structural transitions of the loop are possible due to a subtle change in the environment (for example, the effect of TFE)(17) . In the total correlation spectroscopy and NOESY spectra of the MN V3 loop in water/TFE, we observed a broadening of the NMR line due to the mixed solvent. However, unlike the suggestion made in a previous work (18) , the broadening was not big enough to hinder complete sequential assignments and NOE determination. We assume this to be due to the fact that we used a low peptide concentration of 2.5 mM in the mixed solvent experiments which prevented the formation of aggregates. (iv) The amino acid sequence variability of the V3 loop is restricted on the two sides of the GPG crest(19) . Amino acid sequence variability in the regions flanking the conserved GPGR turn can alter the stability of the turn and/or alter (camouflage) the surface accessibility of this conserved secondary structural element. Therefore, structural studies such as ours on this type of sequence variability will be useful in the PND-based vaccine design and also in deciphering the role of the V3 loop in cell fusion (20, 21) and cell tropism (22, 23) where structure and presentation of the V3 loop might be crucially important.