©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Local and Global Structural Properties of the HIV-MN V3 Loop (*)

(Received for publication, September 21, 1994; and in revised form, November 10, 1994)

Paolo Catasti (1) (2) J. Darrell Fontenot (1) E. Morton Bradbury (2) (3) Goutam Gupta (1)(§)

From the  (1)Theoretical Biology and Biophysics Group and (2)Division LS, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 and the (3)Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The surface of human immunodeficiency virus (HIV) (^1)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 beta-turns at the N-terminal segment, (ii) a type II beta-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.




EXPERIMENTAL PROCEDURES

Materials

The linear and cyclic HIV-MN V3 loops were obtained from Peptide and Protein Research Consultants, Washington Singer Laboratories, UK, using an NIH reagent contract. The purity of the linear V3 loop (TRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAH)was 99% by high pressure liquid chromatography; a major peak at 3878.8 was obtained by fast atom bombardment mass spectroscopy which agrees with the average molecular weight (M(r)) of 3878.5 as required by the desired structure. The cyclic form (CTRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAHC) showed purity of [SIM]95%, i.e. a single major peak with a few minor impurities. A major fast atom bombardment mass spectroscopy peak at 4083.1 corresponded well with the M(r) of 4082.7 as required by the desired structure.

Structure Determination: NMR and Modeling

The methodology for structure determination consisted of three steps: steps 1 and 2 for theoretical analyses of the structure and flexibility of the V3 loop and step 3 for experimental verification of theoretical predictions by NMR spectroscopy.

Step 1: Prediction of Secondary Structures

The secondary structural elements were predicted for a V3 loop sequence by computing the probability S of a given residue i in the V3 loop to adopt a k-type of conformation (k = helix, h, beta sheet, b, coil, c, or turn, t), where:

(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.

Step 2: Generation of Energy-minimized S-S-bridged V3 Loop

This step involved obtaining an energetically stable S-S-bridged structure for a V3 loop sequence given the secondary structural states of the constituent amino acids residues as obtained after step 1 or from analyses of two-dimensional NMR data (discussed later). Appropriate ranges of (, ) values were assigned to all amino acids. For example,

(, ) 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(1) (= [vert] r(1) - r(1)^0 [vert] = 0) indicates distance constraints for an S-S bridge. Distances in the S-S-bridged V3 loop configuration are defined as r(1) = S(C1) - S(35), r(2) = C(C1) - S(C35), r(3) = C(C35) - S(C1), and r(4) = C(C1) - C^b(C35); corresponding equilibrium distances are r(1)^0 = 2.04 Å, r(2)^0 = r(3)^0 = 3.05 Å, r(4)^0 = 3.85 Å(7) . (1) 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 (CAC)-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.

Step 3: Use of Two-dimensional NMR Experiments

This step involved (i) sequential assignment of the protons belonging to constituent amino acid residues, (ii) extraction of sequential and medium-range inter-residue interactions by employing full-matrix NOESY simulations with respect to observed NOESY data(10) , and (iii) conformational sampling by Monte Carlo simulated annealing subject to the distance constraints derived from NOESY data(11) . Two-dimensional NMR experiments were conducted in 90% H(2)O, 10% D(2)O and in 100% D(2)O under the following solution conditions: peptide concentration = 1-3.5 mM, pH = 5.5 in phosphate buffer, temperature = 6-25 °C. NMR experiments were done over a wide range of peptide concentrations to examine whether there were complications due to inter-molecular associations. Analyses of the results of total correlation spectroscopy, double quantum filtered correlation spectroscopy, and NOESY (at two mixing times) experiments in 90% H(2)O, 10% D(2)O led to the sequential assignment of the spin system (HN, H, H) and also to the identification of secondary structural states of various residues in the V3 loop(12) . Additional NMR data in D(2)O further confirmed sequential assignment of all non-exchangeable protons. Prominent secondary structural elements emerged from the characteristic NOE pattern present in the two-dimensional NMR data. In addition to the characteristic structural features (i.e. the presence of beta-strand, a turn or a helix) a complete set of structural constraints were derived from two-dimensional NMR data: from Jalpha coupling and inter-residue HN-HN; H-HN, H-HN distances from two-dimensional NMR experiments in 90% H(2)O, 10% D(2)O; ^1, ^2 from J(H)alphabeta, J(H)beta, J(H)betaring coupling and intra-residue HH, HNH; and inter-residue HH and HH distances from two-dimensional NMR experiments of the V3 loop in D(2)O. All these structural constraints were used for structure determination. The structure determination consisted of two steps: (i) extraction of inter-proton distances and (ii) incorporation of these distance constraints for obtaining a cluster of structures in agreement with the NMR data. Full-Matrix NOESY simulations with respect to experimental data at two mixing times (150 and 300 msec) enabled us to include both primary and higher orders of NOEs. Thus, the complications in the distance estimate using a two-spin model often encountered at a high mixing time due to spin-diffusion (i.e. higher order NOEs) are avoided in the full-matrix NOESY simulations where all spins are considered in the relaxation(10) . Such a simulation at two mixing times improves the rigor in estimating structural constraints for pairwise inter-proton interactions, i.e. for each constraint, an upper and a lower limit of the distance. Two types of constraints are identified(11) .

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.

Solid Phase Peptide Enzyme-linked Immunosorbent Assay with Monoclonal Antibodies

Peptides (0.5 µg/ml) were bound to Dynatech Immulon IV 96 well plates (Chantilly, VA) by overnight incubation in 0.05 M Bicarbonate buffer. The remaining protein binding sites were blocked after one hour of room temperature incubation in 5% Carnation nonfat dry milk in phosphate-buffered saline at pH 7.4. The plates were then incubated with 50 µl of the appropriately diluted monoclonal antibodies for 1 h at room temperature. The plates were then washed three times with phosphate-buffered saline. This was followed by a 1-h incubation with 50 µl of the secondary antibody consisting of Sigma goat anti-mouse IgG conjugated to alkaline phosphatase and diluted 1/3000 in 5% carnation nonfat dry milk in phosphate-buffered saline at pH 7.4. The plates were then washed three times with phosphate-buffered saline. Detection was accomplished with 4 mg/ml phosphatase substrate in 0.25 M diethanolamine with 68 µM MgCl(2):6H(2)0 at pH 9.8. The reaction was terminated after 1 h by adding 50 µl of 3 N NaOH and the absorbance was read at 405 nm.


RESULTS

Molecular Modeling

Amino acid sequence analyses of V3 loops from various HIV-1 strains show that variability in amino acid sequence occurs mainly within specified regions of the V3 loop, leaving three regions that are fairly conserved. Fig. 1A shows the North American consensus V3 loop sequence and the variability in amino acid sequence observed at different sites(13) . The relatively conserved regions are: (i) the N-terminal segment which generally includes a site of glycosylation, (ii) the GPG crest, and (iii) the C-terminal segment. Amino acid sequence variability among different V3 loop sequences is confined mainly to the two regions flanking the GPG crest. Fig. 1B shows the HIV-MN V3 loop which, although lacking the glycosylation site at the N terminus, shows a close sequence resemblance with the North American consensus V3 loop. Previously, we have reported a method that defines secondary structural states of relatively conserved and highly variable regions of V3 loop sequences and predicts the energetically stable tertiary fold(s) of the S-S-bridged V3 loop(8, 9) . Using the same method we analyzed the secondary structural elements and the global structure of twenty different V3 loops. The set of V3 loop sequences included the MN isolate, V3 loops from different geographic locations, and V3 loops from isolates showing different tropisms. The analyses predicted the following secondary structural states for the three conserved regions: (i) an 8-residue long loop at the N terminus, (ii) a type II beta-turn at the GPG crest, and (iii) a C-terminal helix. two-dimensional NMR spectroscopy revealed that these conserved structural features were also present in the HIV-MN V3 loop.

NMR Experiments

NMR studies were performed on the control linear peptide only in the aqueous environment. A complete sequential assignment was achieved by combining the total correlation spectroscopy and NOESY data. NMR data showed that only the central principal neutralizing determinant sequence adopted a protruding loop with a flexible GPGR turn and disordered N- and C-terminal segments. The structural studies were performed on the cyclic HIV-MN V3 loop in aqueous and in mixed (water/TFE) solvents. Combination of total correlation spectroscopy and NOESY data in 90% H(2)O, 10% D(2)O was used to obtain the sequential assignment. Fig. 2, A and B, show the NOESY HN-H (fingerprint) and HN-HN regions for 300 ms of mixing time. Note the presence of continuous HN(i)-H(i - 1) sequential connectivity and a number of sequential HN-HN cross-peaks. However, for structure determination one requires relative strengths (not the mere presence) of these cross-peaks. The relative strengths of the sequential and medium range NOEs were obtained by performing NOESY experiments in 90% H(2)O, 10% D(2)O at two different mixing times (150 and 300 m). In addition, 27 -angle constraints were obtained from the double quantum filtered correlation spectroscopy data of the MN V3 loop in water. Various sequential and medium range NOEs of the cyclic MN V3 loop in aqueous solution are summarized in Fig. 3. The full-matrix NOESY analyses result in 200 pairwise inter-proton distances corresponding to sequential and medium range interactions. The 200 distance and 27 dihedral angle constraints indicate the following effects of the cyclization: (i) induction of an N-terminal loop containing residues 1-9, (ii) stabilization of the GPGR turn, and (iii) formation of two turns at residues 23-26 and 30-33 in the C terminus. The presence of these two turns in the C terminus reveals an incipient helix even in a polar solvent like water.


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(2) = 2K, t(1) = 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(2) = 2K, t(1) = 1K), relaxation delay = 1.5 s, number of transients = 32, temperature = 10 °C. A, the fingerprint HN-H region; B, the HN-HN region.



Solvent-induced Structural Changes

A Monte Carlo simulated annealing procedure (11) subject to the distance and the torsion angle constraints derived from the NMR data leads to a cluster of structures for the MN V3 loop in water and in a mixed water/TFE solvent. Fig. 5, A and B, show ribbon diagrams of two different folding patterns of the MN V3 loop in water and in a mixed water/TFE solvent, respectively. In each case, the average folding patterns are shown and averages computed over 70 low energy structures in agreement with the NMR data. Three conserved segments of the V3 loop are color coded: yellow for the N-terminal segment, red for the GPGR crest, and cyan for the C-terminal segment. Note that in both folding motifs the local secondary structures of the N-terminal segment and the GPGR crest remain the same; however, the induction of the C-terminal helix in the mixed solvent changes the spatial inter-relations of the three secondary structural elements in the two structures. In addition, a short but well defined beta-strand conformation present in aqueous solution disappears in the mixed water/TFE solvent. Therefore, solvent induced changes are also detected in the structure and presentation of the neutralizing epitope (comprising of the central GPGR and 3-4 flanking amino acids on either side) of the MN V3 loop.


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^14) 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.

Monoclonal Antibody Binding Data for the Linear and Cyclic V3 Loops

The induction of structure due to the S-S bridge between C1 and C35 has a strong bearing on the antibody binding properties of the HIV V3 loop. The lack of a well defined structure in the linear peptide is also evident from antibody binding studies. Binding of linear and cyclic V3-MN loops to three different monoclonal antibodies is compared in Fig. 6. Antibodies 1510, 1511, and 1289 bind to the V3 epitopes KRIHI, HIGPGR, and GPGRAF, respectively(15) . Note that the cyclic V3-MN loop is a better ligand than the linear analog in all three cases. This is consistent with the NMR evidence that the cyclic V3-MN loop is more structured than the linear analog. As expected, the most pronounced difference in binding occurs for the mAB 1510 which recognizes the sequence KRIHI on the N-terminal side of the GPG crest; this sequence also shows more ordered structure upon cyclization. For the other two antibodies, the difference in binding is smaller, because both of them include the GPGR which even in the linear analog shows a residual turn. Therefore, the NMR and antibody binding studies imply that vaccine attempts using the cyclic V3 loop would be more effective than the linear analog in inducing protective humoral immunity to the conserved structural features. The binding profile of human monoclonal antibodies 1510 and 1511 (both derived from HIV infected patients) reinforces the notion that the cyclic V3 loop presents the epitope structures similar to that found in native gp120. Interestingly, the Lys-Arg-Ile-His-Ile^14-Gly-Pro-Gly fragment which is a part of the neutralizing epitope of the cyclic MN V3 loop shows the same structure in water and in the mixed solvent as in the complex co-crystal of the neutralizing antibody and the MN V3 loop peptide antigen complex(16) .


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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant R01 AI32891-01A2. The NMR work was done at the NMR facility at the University of California, Davis, using the GE 500 MHz spectrometer (funded by National Science Foundation Grant DIR-88-04739 and United States Public Health Service Grant RR04795). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Theoretical Biology and Biophysics Group, T-10, M/S K710, Los Alamos National Laboratory, Los Alamos, NM 87547. Tel.: 505-665-2587; Fax: 505-665-3493; gxg{at}temin.lanl.gov.

(^1)
The abbreviations used are: HIV, human immunodeficiency virus; PND, principal neutralizing determinant; V3, third variable; NOE, nuclear Overhauser effect; TFE, 2,2,2-trifluoroethanol; NOESY, nuclear Overhauser and exchange spectroscopy.


ACKNOWLEDGEMENTS

We thank Dr. James Bradac of NIH Vaccine branch for providing us with the linear and cyclic MN V3 loop peptides. We also thank Dr. Angel E. Garcia for making technical comments and important suggestions. The antibodies were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: monoclonal antibodies 1510, 1511, and 1289 were all provided by Dr. Susan Zolla-Pazner, Veterans Administration Medical Center, New York, NY.


REFERENCES

  1. Geyer, H., Holschbach, C., Hunsmann, G. & Schneider, J. (1988) J. Biol. Chem. 263, 11760-11767 [Abstract/Free Full Text]
  2. Putney, S. D., Mathews, T. J., Robey, W. G., Lynn, D. L., Robert-Guroff, M., Mueller, W. T., Langlois, A. J., Ghraycb, J., Petteway, S. R., Weinhold, K. J., Fischinger, P. J., Wong-Staal, F., Gallo, R. C. & Bolognesi, D. P. (1986) Science 234, 1392-1395 [Medline] [Order article via Infotrieve]
  3. Goudsmit, J., Debouck, C., Meleon, R. H., Smit, L., Bakker, M., Asher, D. M., Wolff, A. V., Gibbs, C. J. & Gajdusck, D. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4478-4482 [Abstract]
  4. Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jellis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P., Herlihy, W. C., Putney, S. D. & Mathews, T. J. (1989), Proc. Natl. Acad. Sci. U. S. A. 86, 6768-6772 [Abstract]
  5. Deleage, G. & Roux, B. (1989) Prediction of Protein Structure and Principles of Protein Conformation , pp. 587-594, Plenum Press, New York
  6. Ramachandran, G. N. & Sasisekharan, V. (1968) Adv. Protein Chem. 23, 283:438 [Medline] [Order article via Infotrieve]
  7. Roterman, I. K., Gibson, K. D. & Scheraga, H. A. (1989), J. Biomol. Struct. & Dyn. 7, 1-25
  8. Gupta, G. & Myers, G. (1990) Cinquieme colloque des cent gardes , pp. 99-105, Pasteur, Paris
  9. Veronese, F. D., Reitz, M. S., Jr., Gupta, G., Robert-Guroff, M., Boyer-Thompson, C., Louie, A., Gallo, R. & Lusso, P. (1993) J. Biol. Chem. 268, 25894-25901 [Abstract/Free Full Text]
  10. Gupta, G., Sarma, M. H. & Sarma, R. H. (1988) Biochemistry 27, 3423-3431 [Medline] [Order article via Infotrieve]
  11. Gupta, G., Anantharamaiah, G. M., Scott, D. R., Eldridge, J. H. & Myers, G. (1993) J. Biomol. Struct. & Dyn. 11, 345-366
  12. Wuthrich, K., Billeter, M. & Braun W. (1984) J. Mol. Biol. 180, 715- 740 [Medline] [Order article via Infotrieve]
  13. LaRosa, G. J., Davide, J. P., Weinhold, K., Waterbury, J. A., Profy, A. T., Lewis, J. A., Langlois, A. J., Dreesman, G. R., Boswell, R. N., Shadduck, P., Holley, L. H., Karplus, M., Bolognesi, D. P., Mathews, T. J., Emini, E. A. & Putney, S. D. (1990) Science 249, 932-935 [Medline] [Order article via Infotrieve]
  14. Wyatt, R., Thali, M., Tilley, S., Pinter, A., Posner, M., Ho, D., Robinson, J. & Sodroski, J. (1992) J. Virol. 66, 6997-7004 [Abstract]
  15. Gorny, M. K., Xu, J., Gianakakos, V., Karwowska, S., Williams, C., Sheppard, H. Y., Hanson, C. V. & Zolla-Pazner, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3238-3242 [Abstract]
  16. Rini, J. M., Stanfield, R. L., Stura, E. A., Salinas, P. A., Profy, A. T. & Wilson, I. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6325-6329 [Abstract]
  17. Zvi, A., Hiller, R. & Anglister, J. (1992) Biochemistry 31, 6972-6279 [Medline] [Order article via Infotrieve]
  18. Chandrasekhar, K., Profy, A. T. & Dyson, H. J. (1991) Biochemistry 30, 9187-9194 [Medline] [Order article via Infotrieve]
  19. Rose, G. D., Gierasch, L. M. & Smith, J. A. (1985) Adv. Protein Chem. 37, 1-106 [Medline] [Order article via Infotrieve]
  20. Rusche, J. R., Javaherian, K., McDanal, C., Petro, J., Lynn, D. L., Grimaila, R., Langlois, A., Gallo, R. C., Arthur, L. O., Fischinger, P. J., Bolognesi, D. P., Putney, S. D. & Mathews, T. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3198-3202 [Abstract]
  21. Freed, E. O., Myers, D. J. & Risser, R. (1991) J. Virol. 65, 190-194, 333-336 [Medline] [Order article via Infotrieve]
  22. Hwang, S. S., Boyle, T. J., Lyerly, H. K. & Cullen, B. R. (1991) Science 253, 71-74 [Medline] [Order article via Infotrieve]
  23. Chesebro, B., Wehrly, K., Nishio, J. & Perryman, S. (1992) J. Virol. 66, 6547-6554 [Abstract]
  24. Sklenar, V. & Bax, A. (1987) J. Magnet. Reson. 74, 469-479
  25. Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids , Wiley, New York

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