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
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Abstract |
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Keywords: binding site/coiled coil/conformational transition/inhibitory peptide/inner and outer helix regions
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Introduction |
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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., 1995; Lawless et al., 1996
; Chan et al., 1997
; Chan and Kim, 1998
; Rimsky et al., 1998
), thereby impairing the folding of N- and C-terminal helices and hence the core structure (Weissenhorn et al., 1997
, 1999
; Chan and Kim, 1998
). 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, 2000
). 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 628661) had a 10-fold weaker affinity towards the membrane (Kliger et al., 2000
). 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., 2000) 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., 1993a
,b
; Neurath et al., 1995
). 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., 1994
, 1995
; Lawless et al., 1996
; Rimsky et al., 1998
). 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.
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Materials and methods |
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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 400240 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 2, 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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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, 1987).
The mean residue ellipticity, [], in degree cm2 dmol-1, was calculated using the following relationship (Woody, 1995
):
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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.
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Results |
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The peptide gp41(516566) 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., 1999). The kinetic profiles are presented in Figure 3
. It appears that the initial rate of turbidity clearance is rapid followed by a slower phase. Also shown in Figure 3
is another peptide, gp41(545587), which covers the HR1 domain and interacts with T20. In contrast, the putative fusion peptide gp41(512534) 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(526540) through gp41(555569) as indicated in Figure 1
, 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(553567), 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 4A
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(532546), gp41(535549), gp41(538552), gp41(541555), gp41(544558) and gp41(547561), while gp41(550564), gp41(553567) and gp41(555560) displayed little effect on the T20 suspension.
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To address further the specificity of the interaction of HR1-derived peptide with T20, a scrambled sequence of a T20-active gp41(541555), gp41(541555)sc, was mixed with T20. As demonstrated in Figure 4A, 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 4B
illustrates the TC change of the T20 dispersion with increasing amounts of gp41(535549). The stoichiometry of 1:1 for the gp41(535549):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 5A), a correlation was observed between their TC property and content of helix and ß-sheet. Thus gp41(532546), which has highest reactivity toward T20 dispersion, exhibits the highest helix/ß-sheet population in aqueous medium. Figure 5B
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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Discussion |
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With largely disordered structure and limited solubility, T20 was used in this study to locate the critical binding region within the HR1 region. Figure 2 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 3
and 4A
). Figures 3
and 4
provide several pieces of evidence that only fragments with effective and specific interaction with T20 [e.g. gp41(532546)] 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 4A
(marked by an asterisk) and B. Thus scrambling the sequence of gp41(541555) resulted in a loss of reactivity with T20 (Figure 4A
). An initial increment in TC of T20 suspension followed by a TC saturation with increased gp41(535549)/T20 ratio (Figure 4B
) 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(535549) 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 A0 Af 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., 1999
).
Analysis of the TC data in Figure 4A reveals that the 15-mer peptides not containing the sequence LLSGIV (residues 544549) 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(532546), which has only LLS triad at its carboxy terminus. The GIV triad is not as critical as LLS in that gp41(544558) through gp41(550564) exhibit gradual decreased T20 activity. That the region 535549 of gp41 is rich in helix and ß-sheet forms (Figure 5A
) has been suggested by the coupling constant evaluation from gp41(516566) 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., 1993
). 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., 2000
). Based on these results, we propose the following model illustrated in Figure 6
. 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 CD4gp120 and coreceptorgp120 interactions. The HR1 coiled coil forms an inner core and, along with the (newly formed) helix of residues 535549, creates a long helix rod spanning residues 535593. 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., 2000
). 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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Previous reports proposing possible participation of the fusion peptide in binding to the inhibitory molecule (Jiang et al., 1993a; Neurath et al., 1995
) 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., 1997
, 1998
). The use of 15-mer peptides encompassing residues 526569 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., 1997; Lu et al., 1999
) and crystal and solution structures for the homologous SIV gp41 (Caffrey et al., 1998
; Yang et al., 1999
), 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 Q653L663 resulted in an increase of more than four orders of magnitude in EC50 (Lawless et al., 1996
). Similar results on the inhibition of cellcell fusion have also been documented (Ferrer et al., 1999
).
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, 1998; Ferrer et al., 1999
).
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Acknowledgments |
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References |
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Received May 2, 2002; revised February 11, 2003; accepted February 11, 2003.