Structure and Polymorphism of HIV-1 Third Variable Loops
1996; Elsevier BV; Volume: 271; Issue: 14 Linguagem: Inglês
10.1074/jbc.271.14.8236
ISSN1083-351X
AutoresPaolo Catasti, E. Morton Bradbury, Goutam Gupta,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe 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. 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. INTRODUCTIONA neutralizing determinant(1.Gyer H. Holschbach C. Hunsmann G. Scheider J. J. Biol. Chem. 1988; 263: 11760-11767Google Scholar, 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. Science. 1986; 234: 1392-1395Google Scholar, 3.Goudsmit J. Debouck C. Meleon R.H. Smit L. Bakker M. Asher D.M. Wolff A.V. Gibbs C.J. Gajdusck D.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4478-4482Google Scholar, 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. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6768-6772Google Scholar, 5.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. Science. 1990; 249: 932-935Google Scholar, 6.Mathews T.J. Langlois A.J. Robey W.G. Chang N.T. Gallo R.C. Fischinger P.J. Bolognesi D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9709-9713Google Scholar, 7.Gorny M.K. Xu J. Gianakakos V. Karwowska S. Williams C. Sheppard H.Y. Hanson C.V. Zolla-Pazner S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3238-3242Google Scholar, 8.Steimer K.S. Scandella C.J. Skiles P.V. Haigwood N.L. Science. 1991; 254: 105-108Google Scholar) located inside the V3 ( 1The abbreviations used are: V3third variableHIV-1human immunodeficiency virus, type 1DQF-COSYtwo-dimensional double-quantum filtered correlated spectroscopyrmsroot mean squareNOEnuclear Overhauser effectNOESYNOE spectroscopyTFEtrifluoroethanol.) 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.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. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6768-6772Google Scholar, 5.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. Science. 1990; 249: 932-935Google Scholar). Antibodies against the V3 loop generally exhibit type-specific neutralization profiles(6.Mathews T.J. Langlois A.J. Robey W.G. Chang N.T. Gallo R.C. Fischinger P.J. Bolognesi D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9709-9713Google Scholar, 7.Gorny M.K. Xu J. Gianakakos V. Karwowska S. Williams C. Sheppard H.Y. Hanson C.V. Zolla-Pazner S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3238-3242Google Scholar), 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.Gorny M.K. Xu J. Gianakakos V. Karwowska S. Williams C. Sheppard H.Y. Hanson C.V. Zolla-Pazner S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3238-3242Google Scholar, 8.Steimer K.S. Scandella C.J. Skiles P.V. Haigwood N.L. Science. 1991; 254: 105-108Google Scholar). 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.Gupta G. Myers G. Cinquieme Colloque des Cent Gardes. Pasteur, Paris1990: 99-105Google Scholar, 28.Veronese F.D. Reitz M.S. Gupta G. Robert-Guroff M. Boyer-Thompson C. Louie A. Gallo R. Lusso P. J. Biol. Chem. 1993; 268: 25894-25901Google Scholar) and two-dimensional NMR(10.Gupta G. Anantharamaiah G.M. Scott D.R. Eldridge J.H. Myers G. J. Biomol. Struct. & Dyn. 1993; 11: 345-366Google Scholar, 11.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar) 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.Fontenot J.D. Gatewood J.M. Mariappan S.V.S. Pau C.-P. Parekh B.S. George J.R. Gupta G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 315-319Google Scholar) 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.Gupta G. Anantharamaiah G.M. Scott D.R. Eldridge J.H. Myers G. J. Biomol. Struct. & Dyn. 1993; 11: 345-366Google Scholar, 11.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar, 12.Fontenot J.D. Gatewood J.M. Mariappan S.V.S. Pau C.-P. Parekh B.S. George J.R. Gupta G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 315-319Google Scholar). 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.Milich L. Margolin B. Swanstrom R. J. Virol. 1993; 67: 5623-5634Google Scholar):(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).MATERIALS AND METHODSSynthesis and PurificationThe cyclic Haitian V3 loop was synthesized and purified by Dr. Anita Hong (Anaspec, CA). The cyclic RF V3 loop was synthesized and purified by Dr. G. M. Anantharamaiah, University of Alabama at Birmingham. Both Drs. Hong and Anantharamaiah provided their services under a contract with the AIDS Division of the National Institutes of Health.NMR SpectroscopyAll NMR experiments on the RF V3 loop were carried out on the 600 MHz Bruker spectrometer at the University of Alabama at Birmingham, whereas the data on the Haitian V3 loop were collected on a 500 MHz Bruker AMX spectrometer at Chemistry, Science, and Technology-4, Los Alamos National Laboratory. NMR spectra were collected at 10°C with 3 mM peptide concentration (pH 5.5) for the Haitian V3 loop and at 5 mM peptide concentration (pH 5.5) for the RF V3 loop. All two-dimensional data were acquired in the phase-sensitive mode with the presaturation of the HDO signal during the relaxation delay. DQF-COSY data (14.Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Google Scholar) were collected with the following acquisition parameters: t2 = 4096, t1 = 1024, relaxation delay = 1.5 s, number of transients = 32 for the Haitian V3 loop and number of transients = 48 for the RF V3 loop. Total correlated spectroscopy data (14.Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Google Scholar) were collected with the following parameters: t2 = 2048, t1 = 1024, relaxation delay = 1.5 s, number of transients = 32/48, isotropic mixing = 70 ms. NOESY (14.Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Google Scholar) data were collected with similar acquisition parameters and with 75 and 250 ms of mixing time (tm) for the Haitian V3 loop and 200 and 400 ms for the RF V3 loop. The sequential assignments ( 2Authors on request will provide additional data containing proton NMR assignments, Jα coupling constants, NOE volumes, NOE-derived distances, cartesian coordinates, torsional angle data of the the three structures: V3 Haiti in water and in the mixed water/TFE solvent and V3 RF in water. The surface accessibility data of the three structures in Figure 6:, Figure 7:, Figure 8: are also available from the authors on request.) were performed by combining the total correlated spectroscopy, DQF-COSY, and NOESY data, processed on a SGI Indigo instrument using the Felix software (BioSym Inc.).Structure DerivationTwo-dimensional NMR data of the Haitian V3 loop in water and in TFE/water mixture and for the RF V3 loop in water were analyzed with the aid of full-matrix NOESY simulations, associated R-factor test, and energy calculations(10.Gupta G. Anantharamaiah G.M. Scott D.R. Eldridge J.H. Myers G. J. Biomol. Struct. & Dyn. 1993; 11: 345-366Google Scholar, 11.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar). This produced a set of distance constraints(9.Gupta G. Myers G. Cinquieme Colloque des Cent Gardes. Pasteur, Paris1990: 99-105Google Scholar, 10.Gupta G. Anantharamaiah G.M. Scott D.R. Eldridge J.H. Myers G. J. Biomol. Struct. & Dyn. 1993; 11: 345-366Google Scholar, 11.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar, 12.Fontenot J.D. Gatewood J.M. Mariappan S.V.S. Pau C.-P. Parekh B.S. George J.R. Gupta G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 315-319Google Scholar, 28.Veronese F.D. Reitz M.S. Gupta G. Robert-Guroff M. Boyer-Thompson C. Louie A. Gallo R. Lusso P. J. Biol. Chem. 1993; 268: 25894-25901Google Scholar). The energy term (EDIST defining the distance constraints) is added to the force field as in the work of Scheraga and co-workers(15.Roterman I.K. Gibson K.D. Scheraga H.A. J. Biomol. Struct. & Dyn. 1989; 7: 1-25Google Scholar). Monte Carlo simulated annealing (10.Gupta G. Anantharamaiah G.M. Scott D.R. Eldridge J.H. Myers G. J. Biomol. Struct. & Dyn. 1993; 11: 345-366Google Scholar, 11.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar, 16.Kirkpatrick S. Gelatt Jr., C.D. Vecchi M.P. Science. 1983; 220: 671-680Google Scholar) is performed to obtain a set of structures in agreement with NMR data. The maximum step size of the torsion angles was set at 15°, which produced an acceptance ratio of 0.20-0.30 for the 50,000 step MC cycle at each temperature. Full-matrix NOESY calculations were repeated for the final 50 low energy structures of the V3 loop. We define distances from the NOE data in terms of the upper and lower limits (range is above 0.5 Å). However, in our sampled structures the violations of these distances are ~0.22 Å. This implies that the uncertainty in distance estimate is always above 0.7 Å.RESULTSNMR Experiment on the Haitian V3 LoopFig. 1A shows the NOESY fingerprint HN-Hα region of the Haitian V3 loop in water at 10°C for 250 ms of mixing. Fig. 1B shows the DQF-COSY fingerprint HN-Hα region of the Haitian V3 loop in water at 10°C; the ranges of φ values are estimated from JHN-Hα coupling data. Note the presence of intra-residue HN-Hα and inter-residue HN(i+1)-Hα(i) cross-peaks. Proline residues (Pro5, Pro14, and Pro17) show Hδ(Pi+1)-Hα(i) cross-peaks. Note that residues Cys1, Cys2, Thr3, and Arg4 do not show any cross-peaks. Fig. 1C shows the NOESY HN-HN region of the Haitian V3 loop in water at 10°C for 300 ms of mixing. Although a set of sequential HN-HN cross-peaks are observed in the C-terminal stretch (residues 28-33) of the V3 loop, no medium range NOEs like Hα(i)-Hβ(i+3) or HN(i)-Hβ(i+3) cross-peaks are seen as a corroboratory evidence of a helical stretch. Fig. 3 summarizes the NOE data of the Haitian V3 loop in aqueous environment from the analyses of the data at 100 and 300 ms of mixing. Note that only a few Hβ(i)-HN(i+1) cross-peaks are observed for the Haitian V3 loop in the aqueous environment. Seven nonsequential NOESY cross-peaks are observed (see panel of Fig. 3B).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 Hα-HN, Hβ-HN, and HN-HN NOEs, six sequential Hβ-HN NOEs were obtained in both the solvents. The sequential Hβ-HN connectivity for Pro is missing, but the sequential Hα-Hδ 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 Hα-HN NOEs, indicative of an induction of a helix.2 In a previous work(24.Chandrasekhar K. Profy A.T. Dyson H.J. Biochemistry. 1991; 30: 9187-9194Google Scholar, 25.Zvi A. Hiller R. Anglister J. Biochemistry. 1992; 31: 6972-6979Google Scholar), 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.de Lorimier R. Moody M.A. Haynes B.F. Spicer L.D. Biochemistry. 1994; 33: 2055-2062Google Scholar), the authors studied two 24 linear peptides containing the neutralizing determinant of the IIIB isolate of HIV-1. They reported (26.de Lorimier R. Moody M.A. Haynes B.F. Spicer L.D. Biochemistry. 1994; 33: 2055-2062Google Scholar) 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 #x0201C;nascent" helix formed by a stretch if interconnected turns was observed.View Large Image Figure ViewerDownload (PPT)Fig. 2A shows the NOESY fingerprint HN-Hα 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. 3 summarizes 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 Hα(i)-Hβ(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-Hαand HN-HN cross-peaks, there is an enhancement in the relative HN-HN/HN-Hα cross-peak intensities for residues 26-34, which is again indicative of a helical structure in this segment. (iii) A few sequential Hβ-HN cross-peaks are observed in this stretch that are either weak or absent in the aqueous solvent. (iv) Finally, the Hα 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.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar).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-Ha region. B, the HN-HN region. In the fingerprint region note the double population of the residue Gly18. One of the populations has an additional medium range NOE between Hα-G18 and HN-F21, indicating a conformational equilibrium between two forms, one extended and one bent at the fragment Gly18-Lys19-Ala20-Phe21. Interestingly such a double population for Gly18 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 Hα resonances in the C-terminal region of the peptide that is a strong indicator of the helical conformation.View Large Image Figure ViewerDownload (PPT)The residues in the neutralizing epitope, Ile13-Pro14-Met15-Gly16-Pro17-Gly18-Lys19-Ala20-Phe21-Tyr22, are unequivocally assigned in water and in the solvent. Two conformational states of Gly18 (i.e. Gly18 and Gly18*) are clearly evident. Gly18 not only shows two populations in terms of the chemical shift values of (Hα,HN) but also in terms of the NOESY connectivities. As shown in Fig. 2A, a second population of Gly18* is observed in mixed populations; Gly18 shows a Hα(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 Hα(G18)-HN(F21) cross-peak suggests that such an interaction perhaps is not stabilized in a polar environment.NMR Experiment on the RF V3 LoopWe have chosen the RF V3 loop to examine the effect of sequence variation on the overall folding of the V3 loop and the local structure at the GPG crest. The RF V3 loop is different from the Haitian V3 loop at eight positions and is more positively charged. Fig. 4A shows the NOESY fingerprint HN-Hα region of the RF V3 loop in water at 10°C for 400 ms of mixing. Note the presence of intra-residue HN-Hα and inter-residue HN(i+1)-Hα(i) cross-peaks except for the residues Cys1, Cys2, Thr3, Arg4, Pro5, and Asn6, possibly due to inherent flexibility in the N-terminal region. Fig. 4B shows the NOESY HN-HN region of the RF V3 loop in water at 10°C for 300 ms of mixing. Although a set of sequential HN-HN cross-peaks are observed in the C-terminal stretch (residues 29-34) of the V3 loop, no medium range NOEs indicative of helical fragments are seen. Fig. 5 summarizes the NOE data of the RF V3 loop in the aqueous environment from the analyses of the data at 200 and 400 ms of mixing. Only intra-residue and sequential NOEs are observed. The JHN-Hα coupling constants from the DQF-COSY spectrum (Fig. 4C)) provide the φ values, which are converted into HN-Hα intra-residue distances.2 The lack of solubility of the highly polar RF V3 loop prevented us from carrying out NMR experiments in the mixed water/TFE (7:3) solvent.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-Ha 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 Val20 (only Val in the sequence) and moving backward and forward along the connectivity route until completion of the assignments. Assignments in the fragment Cys1-Asn7 were not possible, presumably due to the intrinsic flexibility in the region Asn6-Asn7-Asn8.View Large Image Figure ViewerDownload (PPT)Figure 5:Summary of the NMR data for the HIV RF V3 loop in water. Sequential Hα-HN, Hβ-HN, and HN-HN NOEs. The sequential Ha-HN connectivity for Pro is missing, but the sequential Hα-Hδ NOEs provide the NOE link.2 Lorimier et al.(26.de Lorimier R. Moody M.A. Haynes B.F. Spicer L.D. Biochemistry. 1994; 33: 2055-2062Google Scholar) 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.Chandrasekhar K. Profy A.T. Dyson H.J. Biochemistry. 1991; 30: 9187-9194Google Scholar, 25.Zvi A. Hiller R. Anglister J. Biochemistry. 1992; 31: 6972-6979Google Scholar), 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.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar).View Large Image Figure ViewerDownload (PPT)Structures of the Haitian V3 LoopWe have previously reported structural studies on the Thailand and MN V3 loops(10.Gupta G. Anantharamaiah G.M. Scott D.R. Eldridge J.H. Myers G. J. Biomol. Struct. & Dyn. 1993; 11: 345-366Google Scholar, 11.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar). The NMR data for those two sequences revealed that the structure of the V3 loop contained a few reasonably well defined secondary structural elements, i.e. a GPGR(Q) turn and a nascent C-terminal helix. However, the V3 loops are considerably flexible within the constraints of these secondary structural elements and the Cys2-Cys2 disulfide bridge. Therefore, we developed and applied a method to explore the extent of conformational flexibility of the V3 loop that is consistent with the NMR data(10.Gupta G. Anantharamaiah G.M. Scott D.R. Eldridge J.H. Myers G. J. Biomol. Struct. & Dyn. 1993; 11: 345-366Google Scholar, 11.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar). Briefly, we employ the following steps. (i) We analyze the NMR data to assign secondary structural states, i.e. ranges of φ and ψ values, to the residues of the V3 loop. The NMR data for the aqueous form include the NOEs in Fig. 3 and the DQF-COSY data (Fig. 1C); the JHN-Hα coupling constants from the DQF-COSY spectrum provide the φ values, which are converted into HN-Hα intra-residue distances.2 Line broadening in the mixed solvent prevented us from obtaining a high quality DQF-COSY spectrum in the water/TFE (7:3) mixture. (ii) We obtain a set of starting (energy minimized) structures of the V3 loop subject to the φ and ψ values and disulfide bridge constraints. (iii) We then use these starting structures for Monte Carlo simulated annealing and energy minimizations for sampling the conformational space. (iv) We finally select a set of low energy structures (50 for the Haitian V3 loop) and analyze the conformational parameters to examine the nature of the flexibility.Fig. 6 shows 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 β-strand flanking the GPG crest, magenta for the central β-turn at position G16-P17-G18-K19, yellow for the C-terminal extended β-strand flanking the GPG crest, and blue for the C-terminal segment D26-H35, which can form an α-helix. In water the C-terminal segment consists of consecutive β-turns centered around Ile28-Gly29 and Ile31-Arg32, whereas in the water/TFE (7:3) mixture it further folds into a well defined α-helix as evidenced by the presence of sequential HN-HN NOEs (Fig. 2B) and medium range Hβ(i+3)-Hα(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 α-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 dαβ(i,i+3) and dαN 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. 6 is 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. 6 is 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 β-strand flanking the GPG crest, magenta for the central β-turn at the GPG crest, yellow for the C-terminal extended β-strand flanking the GPG crest, and blue for the C-terminal segment, which can form an α-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.View Large Image Figure ViewerDownload (PPT)Fig. 7 shows 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 Ile13-Pro14-Met15-Gly16-Pro17-Gly18-Lys19-Ala20-Phe21-Tyr22 is shown. The following color coding was used in these skeleton models: red for the central Gly16-Pro17-Gly18-Lys19 crest, green for the N-terminal Ile13-Pro14-Met15 residues in extended conformation, and yellow for the C-terminal Ala20-Phe21-Tyr22 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.Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Google Scholar), whereas in the other, the GPG crest forms the typical type II β-turn followed by a type III β-turn involving residues Gly18-Arg18-Ala20-Phe21, as evidenced by the presence of a medium range NOE between α-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.Ghiara J.B. Stura E.A. Stanfield R.L. Profy A.T. Wilson I.A. Science. 1993; 264: 82-85Google Scholar), and it will be referred as #x0201C;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 so
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