The LLSGIV stretch of the N-terminal region of HIV-1 gp41 is critical for binding to a model peptide, T20

Vishwa Deo Trivedi1, Shu-Fang Cheng, Cheng-Wei Wu, Radhakrishnan Karthikeyan, Chen-Jui Chen and Ding-Kwo Chang2

Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan 1 Present address: Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX 77030-1501, USA

2 To whom correspondence should be addressed.E-mail: dkc{at}chem.sinica.edu.tw


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A number of peptides and peptide analogs derived from the membrane proximal region of gp41 ectodomain are found to be effective inhibitors of human immunodeficiency virus type 1 (HIV-1)-mediated fusion events. One of them, T20 (aa 638–673), was found disordered and sparingly soluble in water, but became soluble upon mixing with selected, structured peptides from the amino terminal heptad repeat (HR1) region of gp41 using a simple and sensitive method of reduction in the scattering of T20 suspension. From the results on mapping the locus of interaction with T20 by employing partially overlapping peptides derived from HR1, it was concluded that the LLSGIV segment was a critical docking site for the C-terminal peptide of gp41 in its putative inhibitory action consistent with a previous fluorescence study. It was also found that peptides capable of solubilizing T20 dispersion have a high content of helix, as well as ß-strand, conformation in aqueous solution. Specificity of T20/HR1-derived peptide binding was ascertained by using a scrambled sequence of a T20-active peptide and a plateau in scattering reduction of T20 suspension with variation in the concentration of a T20-active HR1 peptide. Implications on the mechanism of T20 inhibition and the sequence of folding of the gp41 core structure are discussed.

Keywords: binding site/coiled coil/conformational transition/inhibitory peptide/inner and outer helix regions


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Events of membrane attachment and fusion are crucial in the cellular infection process of all enveloped animal viruses, including human pathogens such as influenza virus and HIV-1 (White et al., 1983Go; Wiley and Skehel, 1987Go; White, 1992Go; Hernandez et al., 1996Go). HIV-1 transmembrane fusion protein gp41, non-covalently associated with the surface subunit gp120, is generated by the post-translational cleavage of the precursor, gp160 (Hunter and Swanstrom, 1990Go; Moore et al., 1993Go; Luciw, 1996Go). The gp120 subunit recognizes the target cell by binding to the CD4 glycoprotein and chemokine receptors (Dimitrov, 1997Go; Kwong et al., 1998Go; Rizzuto et al., 1998Go) followed by its dissociation from gp41 which then undergoes structural rearrangement into the fusogenic form [reviewed by Chan and Kim (Chan and Kim, 1998Go)]. A schematic representation of the primary structure of HIV-1 gp41 ectodomain is illustrated in Figure 1Go. The amino terminus of gp41 contains a hydrophobic sequence referred to as the fusion peptide for its critical role in the fusion activity (Gallaher et al., 1989Go; Freed et al., 1990Go). Another feature of gp41 is the presence of two 4–3 hydrophobic heptad repeat (HR) sequences. They are predicted to form coiled coil and are also present in various viral fusion proteins (Gallaher et al., 1989Go; Delwart et al., 1990Go). The N-terminal heptad repeat, HR1, is located near the fusion peptide, whereas the C-terminal heptad repeat, HR2, precedes the transmembrane region and is separated by a disulfide loop from the HR1 region. The protein dissection experiments have shown a soluble and thermally stable complex composed of the HR1 and HR2 peptides, N-51 and C-43 (Blacklow et al., 1995Go; Lu et al., 1995Go; Chan et al., 1997Go). The N-51 and C-43 peptides and their smaller fragments have been shown to form an {alpha}-helical trimeric complex of heterodimers known as the ‘core structure’ of the gp41 ectodomain (Blacklow et al., 1995Go; Lu et al., 1995Go; Chan et al., 1997Go; Lu et al., 1999Go). In the core structure, three N-terminal helices form a parallel, coiled coil trimer (inner core), while the other three C-terminal helices fold in the reverse direction and pack against the N-terminal trimer on the external face, as deduced from the solution and crystal structures of HIV and SIV gp41 ectodomain (Chan et al., 1997Go; Tan et al., 1997Go; Weissenhorn et al., 1997Go; Caffrey et al., 1998Go; Chan and Kim, 1998Go; Lu et al., 1999Go; Yang et al., 1999Go). Similar structure has been reported in many other viral proteins (Fass et al., 1996Go; Weissenhorn et al., 1999Go; Yang et al., 1999Go). Elucidation of the core structure, however, does not provide clues to the sequence of folding events leading to the observed conformation, such as the initial association site between HR1 and HR2 regions.



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Fig. 1. Schematic representation of HIV-1LAI gp41 ectodomain. The residues are numbered according to the positions on gp160. Fusion peptide (FP) represents the amino terminal sequence while transmembrane region (TM) is located before the cytoplasmic domain (not shown) in the gp41 ectodomain. Residues included in T20 and other peptides from the fusion peptide and HR1 domains are indicated. The scrambled sequence of gp41(541–555), gp41(541–555)sc, used for demonstrating the specific interaction of T20 with HR1-derived peptides, is indicated at the bottom row.

 
Recent studies shed some light on the molecular events underlying the receptor-triggered conformational change of the HIV-1 envelope glycoprotein (Dimitrov, 1997Go; Chan and Kim, 1998Go; Furuta et al., 1998Go; Kliger et al., 2001Go). Using T20 from the HR2 region, it has been inferred that the conformational alteration occurring in the T20 binding domain of gp41 near the fusion peptide is among the major changes within the HIV-1 envelope protein during the viral entry (Furuta et al., 1998Go). Nonetheless, a detailed molecular description of the process was not offered.

It has been speculated for some time that the potent antifusion activity of T20 may be mediated by its binding to the HR1 region of gp41 (Lu et al., 1995Go; Lawless et al., 1996Go; Chan et al., 1997Go; Chan and Kim, 1998Go; Rimsky et al., 1998Go), thereby impairing the folding of N- and C-terminal helices and hence the core structure (Weissenhorn et al., 1997Go, 1999Go; Chan and Kim, 1998Go). This view was augmented by a report of Kliger and Shai, who showed that T20 cannot perturb the six-helix bundle of gp41 core once it is formed (Kliger and Shai, 2000Go). Interaction of T20 with lipid bilayer was found to be highly cooperative with a Gibbs free energy value of -8.7 kcal/mol, whereas the peptide C34 (aa 628–661) had a 10-fold weaker affinity towards the membrane (Kliger et al., 2000Go). These data suggested the involvement of a domain outside the leucine zipper-like motif of the HR1 region for the inhibitory activity of T20.

A recent mutational study on the N-terminal heptad repeat region of gp41 (Weng et al., 2000Go) revealed that the residues near L556 at the N-terminal portion of the coiled coil domain are more critical for the viral infectivity than those forming a deep cavity in the trimeric inner helix core (near Q577). Other studies also pointed to the interaction of the HR2 region with the region near the fusion domain (Jiang et al., 1993aGo,bGo; Neurath et al., 1995Go). Several biophysical and genetic investigations showed that the region proximal to the leucine zipper-like motif (coiled coil inner core) is critical in blocking the fusion process (Wild et al., 1994Go, 1995Go; Lawless et al., 1996Go; Rimsky et al., 1998Go). Using T20 as a model peptide and assuming that the peptide fragments can reflect the interaction, particularly at its early stage, between HR1 and HR2 of gp41, we attempted to screen various peptides spanning the HR1 region to locate the strong binding site of this peptide. The poor solubility of T20 and its property of staying in suspension at low pH for a sufficiently long period allowed us to explore a simple and sensitive method to evaluate the selected peptides for their affinity and binding kinetics with the model inhibitor peptide. The use of small peptides in identifying the interaction site of T20 leads to the idea that the locus is critical to the stability of gp41 six-helix bundle and is likely to be an initiation point of association between the two HR domains. By scrambling the sequence of one of the HR1-derived peptides and varying the ratio of the interacting peptides, we showed that the association giving rise to the turbidity reduction is specific. On the basis of our findings, a model is presented for the folding of gp41 helix hairpin core and the mechanism of T20 inhibition of the gp41 core-mediated membrane fusion. Our result may aid the design of antiviral drugs and vaccine since the site may constitute a highly conserved target in HIV and SIV envelope glycoproteins.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Various peptides from the ectodomain of HIV-1 gp41, as outlined in Figure 1Go, were prepared with the procedures described previously (Chang et al., 1999Go). The peptides used in this study (Figure 1Go) span several regions, namely, the fusion peptide for gp41(512–534); the fusion peptide and an overlapping region with HR1 for gp41(516–566); the HR1 region and the abutting segment to its N-terminal side for gp41(526–540), gp41(529–543), gp41(532–546), gp41(535–549), gp41(538–552), gp41(541–555), gp41(541–555)sc, gp41(544–558), gp41(547–561), gp41(550–564), gp41(553–567), gp41(555–569) and gp41(545–587); as well as a model peptide T20, namely gp41(638–673), from the HR2 region. All the peptides were synthesized in an automated mode by means of Fmoc chemistry with a solid-phase synthesizer (Model 431A, Applied Biosystems). The synthesized peptides are N-capped by the acetyl group and C-capped with the amino group. They were cleaved from the resin and purified by HPLC on a reversed-phase C4 or C18 column. The primary sequence of peptides was ascertained by electrospray or MALDI mass spectrometry. All reagents used were of analytical grade.

Turbidity clearance (TC) assay

All turbidity measurements were made on a Hitachi double-beam spectrophotometer (U-2001) at ambient temperature at pH 4.0. The turbidity was measured at 400 nm, while the wavelength scan measurements were conducted in the range 400–240 nm. All measurements were made by using matched cells of 1 cm pathlength. A stock 46.7 µM T20 suspension was prepared by adding T20 peptide to 10% (v/v) DMSO in water with vigorous vortex mixing. A 450 ml aliquot of the suspension was mixed with 50 ml of the stock HR1-derived peptide dissolved in 10% (v/v) DMSO in water to give a final concentration of 42 µM for each of the components in the mixture. In a separate set of experiments for Figure 2Go, the fusion peptide (FP)- and HR1-derived peptides were dissolved in DMSO and mixed with T20 suspension in 10% (v/v) DMSO aqueous solution. A time scan of 3000 s was performed on the latter T20 suspension to ensure a stable absorbance reading prior to mixing experiments. The final concentrations of FP or HR1-derived peptide and T20 were 84 and 42 µM, respectively. The solutions were also used for the wavelength scan. The DMSO control experiment was performed at a final 10% concentration to ensure that there is no interference of DMSO with the results.



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Fig. 2. (A) Time scans of T20 suspension in the absence (curve a) and presence (curve b) of gp41(545–587). The T20 suspension displays high stability over the measurement time. The decrease in scattering upon addition of peptide gp41(545–587) is suggestive of the formation of an ordered state of these two peptides in solution. The concentration of T20 was 42 µM and the molar ratio of T20:gp41(545–587) was 1:2. The suspension was kept at pH 5.0 with sodium phosphate buffer at room temperature. Curve c demonstrates that gp41(541–555) stays clear in 10% DMSO aqueous medium at pH 4.0 and 42 µM concentration over the time span of the TC measurement. (B) Wavelength scans of the T20 suspension, in the absence (curve a) and the presence (curve b) of gp41(545–587). Curve c displays the scan with gp41(545–587) alone which, in contrast to curve a, does not exhibit characteristic of scattering from large particles except an absorbance due to tryptophan (residue 571, Figure 1Go). The wavelength profile d indicates that gp41(541–555) exhibits no scattering under the conditions for curve c in (A).

 
To investigate the specificity of interaction between T20 and the HR1-derived peptides, increasing amounts of gp41(535–549) were added to the T20 suspension to give final 15-mer/T20 molar ratios of 0.6, 1.1 and 1.4 at pH 4.0. In Figure 4B AGo,0 Af denotes the decrease in turbidity of the T20 dispersion upon introduction of the indicated quantity of the 15-mer peptide at the end of 3000 s time scan experiment. The leveling off of this value with gp41(535–549)/T20 around 1.0 suggests a specific association between the two peptides and their stoichiometery is 1:1.



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Fig. 4. (A) Kinetic profiles of turbidity clearance at room temperature in the presence of various peptides, as indicated. The uncertainty in the reading is 0.04 absorbance unit for the curves. The concentration of T20 was 42 µM and the molar ratio of T20 to other peptides was 1:1. For other details see Materials and methods. Curves for gp41(532–546), gp41(535–549), gp41(538–552), gp41(541–555) and gp41(544–558) indicate that the ordered structure is formed on the order of s for these peptide complexes. The profile for the gp41(541–555) sequence-scrambled peptide is marked with an asterisk. (B) Turbidity decrease, A0Af, of T20 (42 µM) suspension upon mixing with various amounts of gp41(535–549). The absorbance values were taken after 3000 s of mixing the two peptides in 10% DMSO aqueous solution, at which time the readings were steady. The saturated A0Af value is attained at the molar ratio of ~1:1 for the two peptides, supporting the idea of a specific interaction between the two peptides with 1:1 stoichiometry.

 
The turbidity data were analyzed by subtracting the data points collected at either 30 or 60 s intervals with the values obtained from the control experiment. The kinetic profile was obtained by using the corrected absorbance values in the form of A0 At against time for these scans, where A0 and At represent the initial absorbance value (at zero time) and the absorbance at any given time point (t), respectively.

Circular dichroism (CD) experiments

All CD measurements were carried out on a Jasco 720 spectropolarimeter. Spectra were recorded from 184 to 260 nm at a scanning rate of 20 nm/min with a time constant of 4 s, step resolution of 0.1 nm and bandwidth of 1 nm. Cells with a pathlength of 1.0 mm were employed for a peptide concentration of 40 µM. Phosphate buffer solution was used to adjust the solution to neutral pH. The CD trace for each of the peptides was obtained by averaging five scans. The program Varselec was used to analyze the CD spectra for secondary structure. The analysis is based on singular value decomposition and variable selection and the basis set of the analysis is the CD of 33 proteins for which the secondary structures are determined from X-ray crystallography (Manavalan and Johnson, 1987Go).

The mean residue ellipticity, [{theta}], in degree cm2 dmol-1, was calculated using the following relationship (Woody, 1995Go):


where N is the number of amino acid residues in the peptide tested, c is the molar concentration and l is the pathlength in cm.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In aqueous solution at low pH, T20 has poor solubility, forms aggregate and fails to exhibit a stable secondary structure under physiological conditions (Wild et al., 1994Go; Judice et al., 1997Go). It contains three tryptophan residues located near the carboxyl tail. The UV spectrum of the inhibitor peptide is shown in Figure 2BGo. A higher scattering (turbidity) value at 400 nm and the lack of a distinct maximum at 280 nm (curve a) indicate that the peptide exists as a suspension (Leach and Scheraga, 1960Go). Interestingly, we observed that the turbid suspension stays at least 2 h without forming larger aggregates (Figure 2AGo, curve a). Use of 100 mM phosphate buffer or introduction of salt resulted in rapid aggregation. Hence more dilute buffer (10 mM) was used to maintain the pH of the medium. Interestingly, addition of gp41(545–587) to T20 suspension results in a substantial decrease in the turbidity, as demonstrated in Figure 2AGo (curve b). At the end of time scan the complex of T20 and gp41(545–587) was subjected to a wavelength scan, the results of which are depicted in Figure 2BGo (curve b). The contribution of gp41(545–587) to total absorbance is shown as curve c, indicating a homogeneous solution for the 43-mer peptide. In contrast, the mixture represented by curve b in Figure 2BGo was characterized by a lower scattering intensity, a distinct peak at 280 nm and a minimum at 255 nm. This is in line with the notion that addition of this long, sparingly soluble peptide derived from HR1 region solubilizes the T20 suspension, possibly forming a more ordered structure.

The peptide gp41(516–566) containing the fusion peptide and part of the leucine zipper-like motif of HR1 domain was used to test the validity of this TC methodology. Our static light scattering and fluorescence data have shown the oligomeric state of this peptide in water and its ability to bind to the region overlapping the T20 sequence (Chang et al., 1999Go). The kinetic profiles are presented in Figure 3Go. It appears that the initial rate of turbidity clearance is rapid followed by a slower phase. Also shown in Figure 3Go is another peptide, gp41(545–587), which covers the HR1 domain and interacts with T20. In contrast, the putative fusion peptide gp41(512–534) failed to exhibit significant TC property, suggesting its inability to associate specifically with T20 to form an ordered structure. This prompted us to examine the role of the sequence between residues 526 and 566. To pinpoint the association site on HR1 for the T20 peptide, we used the dissection approach with an array of partially overlapping sequences. Eleven peptide sequences of 15 amino acid residues each, gp41(526–540) through gp41(555–569) as indicated in Figure 1Go, were designed in such a way that each peptide is separated from its neighboring one by three or two residues. Unlike the above-mentioned, larger peptides, these 15-mer peptides were found to be poorly soluble in aqueous medium, with the exception of gp41(553–567), which is sparingly soluble under the experimental conditions. To overcome the poor solubility, a minimal amount of DMSO was used to dissolve the peptides in the turbidity measurements. The presence of DMSO does not significantly affect the nature of the T20 suspension. Figure 4AGo shows the time profiles of turbidity clearance in the presence of these 15-mer peptides. Note from these curves that the initial phase of the clearance kinetics is faster than the later stage. Among these15-mer peptides, positive TC was observed with gp41(532–546), gp41(535–549), gp41(538–552), gp41(541–555), gp41(544–558) and gp41(547–561), while gp41(550–564), gp41(553–567) and gp41(555–560) displayed little effect on the T20 suspension.



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Fig. 3. Kinetic profiles showing the reduction in turbidity, monitored at 400 nm, upon introduction of various peptides to the T20 suspension at pH 7.0 and room temperature. The added peptides were (•) gp41(516–566), ({circ}) gp41(545–587) and (x) fusion peptide gp41(512–534). The T20 concentration was 42 µM and the molar ratio of added peptides to T20 was 1:1.

 
To ensure that the observed TC of T20 suspension is due to the direct reaction between the 15-mer peptide and T20, and not to the 15-mer peptide alone, we examined the UV absorbance behavior of one of the peptides which is TC-active toward T20, gp41(541–555). Curve c in Figure 2AGo (measured at 400 nm) and curve d in Figure 2BGo show the time and wavelength scan experiments for the peptide at pH 4.0 and room temperature. These data illustrate that the peptide in 10% DMSO aqueous solution is transparent to and shows little scattering on the 400 nm UV light and is stable over the period of several hours. Thus the TC phenomenon displayed in Figure 4Go cannot be attributed to the HR1-derived peptides.

To address further the specificity of the interaction of HR1-derived peptide with T20, a scrambled sequence of a T20-active gp41(541–555), gp41(541–555)sc, was mixed with T20. As demonstrated in Figure 4AGo, the sequence-scrambled peptide was unable to solubilize T20 despite the high TC activity of the parent peptide. The result indicates a specific binding between T20 and HR1-derived peptides. Binding specificity was also evidenced by titration of T20 suspension with a 15-mer peptide. Figure 4BGo illustrates the TC change of the T20 dispersion with increasing amounts of gp41(535–549). The stoichiometry of 1:1 for the gp41(535–549):T20 complex can be discerned from the molar ratio at which the TC reduction reaches a plateau.

Strikingly, when we examined the secondary structure elements of these 15-mer peptides with CD measurements (Figure 5AGo), a correlation was observed between their TC property and content of helix and ß-sheet. Thus gp41(532–546), which has highest reactivity toward T20 dispersion, exhibits the highest helix/ß-sheet population in aqueous medium. Figure 5BGo summarizes TC index (TCI) values and secondary structure elements for the HR1-derived peptides. The helix content is clearly more critical in determining the TC reactivity.



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Fig. 5. (A) Far-UV CD spectra of 15-mer peptides derived from HR1 and neighboring regions of gp41 ectodomain. Note that the peptides with little helix content (as judged from the ellipticity at 222 nm) are those display no effect on the turbidity of T20 suspension, suggesting secondary structure elements may play a role in the complex formation. (B) Summary of the TCI against T20 suspension and secondary structure element of the 15-mer HR1 peptides in aqueous buffer illustrating the correlation of helix plus ß-sheet content with TCI for these peptides. The analysis of the secondary structure is based on CD data shown in (A) using the Varselec program.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
T20 is a potent inhibitor for the HIV-1 mediated fusion with an EC50 of 50 ± 22 nM and as such its mode of inhibition has received wide attention. It was also employed to capture a putative intermediate of the fusogenic state of gp41 ectodomain (Furuta et al., 1998Go). Various non-natural elements, which were covalently attached to a peptide fragment from the outer helix, have been explored for inhibition of gp41 mediated cell fusion (Ferrer et al., 1999Go). Other HR2 peptides with a sequence overlapping a large portion of T20 have also been found to inhibit the viral fusion at low concentrations (Jiang et al, 1993bGo; Wild et al., 1994Go; Ferrer et al., 1999Go). These endeavors pointed to an essential role of the outer helix in the formation of a stable helix hairpin complex. Although the HR2 region of the gp41 ectodomain was predicted to be helical (Gallaher et al., 1989Go), peptides from this region lack discernible secondary structures in solution (Wild et al., 1994Go; Judice et al., 1997Go). The formation of a stable complex between this peptide and the peptides from the HR1 domain has been studied in detail (Chen et al., 1995Go; Lu et al., 1995Go, 1999Go; Chan and Kim, 1998Go). The helical core structure formed by the two heptad repeat regions shares common features with influenza hemagglutinin HA2 and other viral fusion proteins (Chan and Kim, 1998Go; Weissenhorn et al., 1999Go).

With largely disordered structure and limited solubility, T20 was used in this study to locate the critical binding region within the HR1 region. Figure 2Go demonstrates that, upon addition of a selected peptide from the HR1 region, decreased scattering intensity (turbidity) resulting from an interaction with T20 can be employed effectively. Advantage was taken of the fact that T20 suspension is stable over a period of a few hours. The TC measurements suggested that the formation of the complex by HR1 helices with T20 is of the order of minutes (Figures 3Go and 4AGo). Figures 3Go and 4Go provide several pieces of evidence that only fragments with effective and specific interaction with T20 [e.g. gp41(532–546)] results in dissolution of T20 suspension leading to a reduction in size and thus scattered light. The crucial question of the specificity of interaction between T20 and HR1-derived peptides is addressed by Figure 4AGo (marked by an asterisk) and B. Thus scrambling the sequence of gp41(541–555) resulted in a loss of reactivity with T20 (Figure 4AGo). An initial increment in TC of T20 suspension followed by a TC saturation with increased gp41(535–549)/T20 ratio (Figure 4BGo) demonstrates a 1:1 stoichiometry for the 15-mer/T20 association. The latter result is consistent with the notion that the turbidity clearance observed in the mixing of gp41(535–549) with T20 suspension does not arise from precipitation by non-specific association of the two peptides, since the latter property would lead to a continuous increase in A0Af with added 15-mer peptide. A similar TC approach has been utilized to estimate the protease concentrations using the turbid and stable suspension of a misfolded lens protein (Trivedi et al., 1999Go).

Analysis of the TC data in Figure 4AGo reveals that the 15-mer peptides not containing the sequence LLSGIV (residues 544–549) are inactive to T20. In contrast, the partially overlapping 15-mer peptides encompassing the LLSGIV sequence or part of it exhibit a positive TC property, hence this segment constitutes a critical T20 docking site. The peptides reactive to T20 also displayed a significant helix content in aqueous solution, in addition to the ß-sheet form. For these two conformations, helix is more important than the sheet conformation, as demonstrated by the highly T20-active gp41(532–546), which has only LLS triad at its carboxy terminus. The GIV triad is not as critical as LLS in that gp41(544–558) through gp41(550–564) exhibit gradual decreased T20 activity. That the region 535–549 of gp41 is rich in helix and ß-sheet forms (Figure 5AGo) has been suggested by the coupling constant evaluation from gp41(516–566) in aqueous and SDS micellar solutions (S.F.Cheng and D.K.Chang, unpublished data). It is of interest that this stretch is close to the gp120 association site (Cao et al., 1993Go). A recent investigation on the mechanism of gp41-mediated fusion suggested that the formation of a six-helix bundle provided free energy needed for the membrane merging (Melikyan et al., 2000Go). Based on these results, we propose the following model illustrated in Figure 6Go. The region near residues LLSGIV is in ß-sheet conformation in association with gp120, but converts into helix, acquiring a stabilized triple-stranded (oligomeric) structure of gp41 ectodomain after trimeric gp120 molecules disengaged from each other and dislodged from gp41 triggered by CD4–gp120 and coreceptor–gp120 interactions. The HR1 coiled coil forms an inner core and, along with the (newly formed) helix of residues 535–549, creates a long helix rod spanning residues 535–593. The rigid rod helps the fusion peptide to project toward and insert into the target membrane. These processes are faster than the refolding of the HR2 domains and their packing against the grooves between the monomers of HR1 helices, because this property would allow the freely diffusing T20 molecules to bind to the coiled coil core and block formation of six-helix bundle. In the absence of T20, the adjacent helix bundles promote membrane fusion by releasing the free energy of helix hairpin formation to provide, at least in part, the energy needed to surmount the barrier of dehydration of the fusing membrane surfaces prior to their merger (Melikyan et al., 2000Go). It is noted that the inhibitory action of T20 is dominant negative because attachment of a single T20 molecule to one helix bundle in the cluster of homo-trimeric subunits would greatly compromise the function of fusion pore, which requires cooperation of all constitutive subunits.



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Fig. 6. Model for the folding of gp41 ectodomain to form helix bundle core. (A) The fusion peptide (red) and the linker (light blue, residues 535–549) regions are sequestered (denoted by dotted form) by bound gp120 molecules before cellular receptor CD4 binding. Residues 535–549 are in non-helix conformation in this metastable state. (B) The trimeric gp120 becomes loose and begins to dissociate from gp41, resulting in a more exposed fusion peptide. The linker region undergoes a conformational transition into helix. Also, the tightly packed HR1 coiled coil is formed to which the HR2 segments will later relocate. (C) Although the six-helix bundle is thermodynamically stable, the refolding of HR2 toward HR1 inner core may be slow, allowing a freely diffusing T20 molecule, when present, to attach to the HR1 coiled coil, thereby disrupting the formation of the gp41 core structure. It is noteworthy that a defective six-helix bundle caused by engagement of one T20 molecule to the inner coiled coil is sufficient to abrogate the function of fusion pore, which requires coordination of member subunits to overcome the hurdle of membrane dehydration and destabilization. (D) Assisted by the helix rod of linker and HR1 coiled coil regions and driven by its hydrophobicity, the exposed fusion peptide reorients toward the cell membrane and inserts into it. (E) In the absence of T20, the attachment of HR2 domains to the outer surface of HR1 coiled coil reorients the six-helix core parallel to the cellular and viral membranes. The helix hairpin core is thus poised to promote the membrane fusion by bringing the two apposing membranes into proximity and the cooperation of adjacent subunits to form fusion pore (F). Free energy provided by the hairpin complex formation would help the process to proceed to complete fusion (or content mixing).

 
A recent genetic study implicating the GIV motif in the HIV-1 resistance to T20 (Rimsky et al., 1998Go) can be rationalized by the idea that the LLSGIV stretch is crucial to interaction of the HR1 region with T20 in the context of the present model. It is proposed that, after the release of gp120 that suppresses the hairpin formation, refolding of HR2 to pack against HR1 coiled coil is relatively slow so that T20 peptide is able to interfere with the HR1:HR2 complex formation. It is plausible that the mutation in the GIV motif retards the association of the N-terminal helix with the HR2 domain and with the exogenous T20 peptide. T20 then loses the advantage of attaching to the HR1 domain before HR2 folding to the proximity of HR1. In other words, the delay in binding between the HR1 domain of the G547S/V549M mutant and C-helix sequence renders relocation of HR2 segment toward the N-terminal helix a non-rate limiting step. The ability of T20 to block the hairpin formation is then greatly reduced (Rimsky et al., 1998Go). Our model also explains a difference of three orders of magnitude in inhibition efficacy of HIV-1 replication between T20 and DP107 [gp41(553–590)] because the affinity of the HR2 segment for the HR1 coiled coil is higher than for DP107 which lacks the LLSGIV sequence.

Previous reports proposing possible participation of the fusion peptide in binding to the inhibitory molecule (Jiang et al., 1993aGo; Neurath et al., 1995Go) are not substantiated by the data presented here. Another distinct region near L568 and W571 forming a cavity in the coiled coil has been proposed as a drug target and shown to contribute to the stability of HR1:HR2 complex (Chan et al., 1997Go, 1998Go). The use of 15-mer peptides encompassing residues 526–569 enabled us to pinpoint the T20 binding site within HR1. TC experiments, performed at a minimal concentration of DMSO to overcome the solubility problem without interfering with the absorbance measurements, have provided us with a simple means to assess the ability of these peptides to associate with T20 and probe the kinetic aspect of the specific interaction. The method of scanning the sequence in a triad or tetrad step in the turbidity experiment to pinpoint a critical site for interaction between peptides and proteins can also be extended to other systems.

From the crystal data for HIV-1 gp41 ectodomain (Tan et al., 1997Go; Lu et al., 1999Go) and crystal and solution structures for the homologous SIV gp41 (Caffrey et al., 1998Go; Yang et al., 1999Go), the following interacting pairs can be observed: (L544, L663), (S546, L660), (G547, N656) and (V549, Q653). These residues are conserved in various HIV and SIV strains, suggesting their functional importance. The latter notion is in line with the study of the anti-viral effect of T20 variants, which indicated that truncation of the sequence near the region Q653–L663 resulted in an increase of more than four orders of magnitude in EC50 (Lawless et al., 1996Go). Similar results on the inhibition of cell–cell fusion have also been documented (Ferrer et al., 1999Go).

Identification of the LLSGIV sequence as an essential segment for HR1:HR2 interaction may be important in antiviral drug and vaccine design because the sequence may be accessible to drugs and antibodies after gp120 shedding from gp41 and the HR1 domain is the most conserved region in the HIV and SIV envelope glycoproteins (Chan and Kim, 1998Go; Ferrer et al., 1999Go).


    Acknowledgments
 
This work was supported by the National Science Council, Taiwan (NSC 89-2113-M-001-016) and Academia Sinica.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received May 2, 2002; revised February 11, 2003; accepted February 11, 2003.





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