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Interaction of Synthetic HPV-16 Capsid Peptides with Heparin: Thermodynamic Parameters and Binding Mechanism Jian Sun,† Ji-Sheng Yu,‡ Shi Jin,† Xiao Zha,§ Yuqing Wu,*,† and Zhiwu Yu*,‡ State Key Laboratory for Supramolecular Structure and Materials, Jilin UniVersity, No. 2699, Qianjin Street, Changchun 130012, China; Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua UniVersity, Beijing 100084, China; and Sichuan Tumor Hospital and Institute, Chengdu 610041, China ReceiVed: February 1, 2010; ReVised Manuscript ReceiVed: July 2, 2010
Capsid proteins binding cell surface proteoglycans is a key early event in human papillomavirus (HPV) infection. The positively charged sequences at the C-terminus of the L1 protein and the N- and C-termini of the L2 protein of HPV-16 can efficiently bind to heparin receptors, which were characterized in the present study by quantitative isothermal titration calorimetry experiments primarily, fluorescence spectroscopy, and static right-angle light scattering. The binding constant, K, was at an order of magnitude of 107 M-1 for the two peptides at the N- and C-termini of HPV-16 L2 and segment b at the C-terminus of HPV-16 L1, while that for other L1 analogues were of a smaller order, illustrating that the heparin binding is a typical sequencespecific and -dependent phenomenon. These results suggest that, in addition to L1, the L2 protein may participate in cell surface attachment during HPV infection. Furthermore, the calorimetry results demonstrated that hydrophobic interactions and hydrogen bonding are involved in peptide binding to heparin in addition to the essential electrostatic interactions. Meanwhile, circular dichroism spectroscopy revealed that binding to heparin does not induce obvious secondary structural changes in the peptides. Introduction Human papillomaviruses (HPVs) are nonenveloped epitheliotropic DNA viruses, and more than 100 different types have been identified to date. Infection with low-risk HPVs as HPV-6 or HPV-11 generally causes benign genital warts; while invasion by high-risk types as HPV-16 will produce anogenital malignancies, particularly cervical cancer, the second most common cause of cancer-related death in women worldwide.1,2 HPV viral capsids are 55-60 nm in diameter and each contains 72 pentamers of the major structural L1 protein (capsomers).3 The capsids also include 12 copies of L2, the minor structural protein possibly associated with the 12 pentavalent capsomers.4 L1 proteins can self-assemble into virus-like particles (VLPs) in the presence or absence of L2 proteins, either in vivo or in vitro.5 The life cycle of a HPV virus starts with its adsorption and attachment to host cells by binding to receptor molecules on the surface, being followed by amplification of the viral genome and production of progeny virus. Therefore, the prerequisite for exploring the viral life cycle should be the investigation on how the viruses adsorb to cells. Because efficiently propagating HPVs is nearly impossible to obtain in vitro, VLPs generated by synthesizing L1 and L2 proteins have been used as a mode in exploring interaction between the cell surface and the viral capsid. These studies implicated cell surface heparan sulfate (HS) proteoglycans (HSPGs) as the primary attachment factors for most HPV types: surface modifications by sulfate groups have been shown to be essential for HPV-11, HPV-16, and * To whom correspondence should be addressed. Tel: +86-431-85168730 (Y.W.), +86-10-62792492 (Z.Y.). Fax: +86-431-85193421 (Y.W.), +86-1062771149 (Z.Y.). E-mail:
[email protected] (Y.W.),
[email protected] (Z.Y.). † Jilin University. ‡ Tsinghua University. § Sichuan Tumor Hospital and Institute.
HPV-33 capsid interactions with cells,6,7 suggesting an involvement of electrostatic interactions between the basic amino acid residues of the capsid protein with the negatively charged sulfate groups. The C-terminal portion of HPV-11 L1 interacts with heparin and has been shown to be crucial for interaction with the cell surface.7 Similarly, HPV-16 L1 contains two basic amino acid segments at the C-terminus, comprising residues 473-488 (GLKAKPKFTLGKRKAT) and 492-505 (SSTSTTAKRKKRKL), respectively. The L2 protein also contains two similar amino acid segments at the N-terminus (residues 1-13, MRHKRSAKRTKRA) and C-terminus (residues 454-462, MLRKRRKRL). These short segments have been shown to be the nuclear localization signals (NLS) and sufficient for binding DNA due to the high proportion of positively charged residues (K and R).8,9 Using these synthetic Arg- and Lys-rich peptides and a competition assay measuring the binding of HPV-31 VLPs to heparin-coated plates, Bousarghin et al. showed that these peptides can bind HS or heparin.10 In addition, the surfaceexposed basic residues of the HPV-16 L1 protein have been identified as attaching to cell surface proteoglycans by electrostatic interactions.11 Despite a number of studies demonstrating that sulfated cell surface sugars are the general receptors for HPVs capsid protein and the positively charged sequences may play an important role in cell binding, both the molecular mechanism and thermal dynamics of the interaction remain poorly understood to date. With the goal of characterizing the interactions involved in peptide binding to the HS portion of cell surface in detail at the molecular or submolecular level, we studied the binding of synthetic peptides using heparin as a substitute for cell surface HS. Heparin and HS are closely related glycosaminoglycans (GAGs) and are composed of the same monosaccharide building blocks.12-17 Despite heparin having a higher degree of sulfation,
10.1021/jp1009719 2010 American Chemical Society Published on Web 07/15/2010
Interaction of HPV-16 Capsid Peptides with Heparin
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TABLE 1: Thermodynamic Data for the Interaction of Heparin with Five Peptides at 28 °C peptide L2NtW L2CtW L1CtaW L1CtbW Set 1 L1CtbW Set 2 L1CtbScW
amino acid sequence WMRHKRSAKRTKRA WMLRKRRKRL WLKAKPKFTLGKRKAT WSTSTTAKRKKRKL WKSRSRTKTAKSTRL
position (aa) in protein 1-13 of L2 454-462 of L2 474-488 of L1 493-505 of L1
n
K (M-1)
∆H (kcal/mol)
∆G (kcal/mol)
T∆S (kcal/mol)
11.8 ( 0.3 15.2 ( 1.6 23.7 ( 3.0 15.0 ( 0.2 3.56 ( 0.44 22.3 ( 1.4
(2.02 ( 0.40) × 107 (6.61 ( 1.70) × 107 (1.53 ( 0.51) × 105 (4.74 ( 1.62) × 107 (6.04 ( 2.76) × 105 (2.52 ( 1.45) × 105
-78.93 ( 1.26 -70.82 ( 1.36 -36.94 ( 6.29 -29.28 ( 0.61 -1.91 ( 0.39 -95.81 ( 6.78
-10.06 ( 0.08 -10.69 ( 0.23 -7.14 ( 0.20 -10.57 ( 0.22 -7.90 ( 0.29 -7.44 ( 0.12
-68.87 ( 1.33 -60.13 ( 1.13 -29.80 ( 6.49 -18.71 ( 0.39 5.99 ( 0.58 -88.37 ( 6.90
both GAGs generally have similar peptide binding properties, and most studies of the interactions with proteins have used heparin instead of HS because of its easier commercial availability.18-20 In this study, we report on the interaction of heparin with five synthetic peptides of HPV-16 (Table 1), comparing the different sequential peptides of HPV-16 capsid proteins. Using highly sensitive isothermal titration calorimetry (ITC), as well as fluorescence spectra, and static right-angle light scattering (SLS) experiments, we determine the binding constant K, reaction enthalpy ∆H, and the number of peptide binding sites on each heparin molecule in the interaction. In addition, the peptide structural changes induced by heparin binding are followed by circular dichroism (CD) spectra in solution. Materials and Methods Materials. The analogues of HPV-16 peptides L2NtW, L2CtW, L1CtaW, L1CtbW, L1CtbScW, and AVL1CtbW were purchased from GL Biochem Ltd. (Shanghai, China) (purity >98% by reverse phase HPLC). As shown in Table 1, the L2NtW and L2CtW peptides are the N-terminus (residues 1-13) and C-terminus (residues 454-462), respectively, of the HPV16 L2 protein with the addition of a Trp in their N-terminus; L1CtaW and L1CtbW are the positively charged sequences (residues 474-488, 493-505) present at the C-terminus of the HPV-16 L1 protein after the first residue in the N-terminus is replaced with Trp; and L1CtbScW is a scramble peptide of L1Ctb with an additional Trp in the N-terminus, which was used to investigate the effect of the specific sequence by comparing it to L1CtbW. Trp was added to the N-terminus of the synthetic peptides or substituted the first residue in the N-terminus to use fluorescence spectra, facilitating the interaction between the peptides and heparin. AVL1CtbW is a control peptide of L1CtbW, where the segment of STSTT in L1CtbW was changed to AVAVV. Porcine intestinal mucosa heparin (sodium salt, H4784) was obtained from Sigma-Aldrich (Shanghai, China). All other chemicals were spectrum pure or reagent grade. Isothermal Titration Calorimetry. The ITC experiments were performed at a number of selected temperatures using a MicroCal VP-ITC calorimeter (Northampton, MA). Heparin and peptide solutions were prepared in buffer A (50 mM sodium phosphate, pH 7.4) containing different concentrations of NaCl. The concentration of heparin ranged from 0.1 to 0.3 mM as evaluated with the average molecular mass of 12 kDa, and the concentration of the peptide solutions was 0.1 mM. The titrations involved the injection of 5 µL aliquots of the heparin solution into the calorimeter cell (Vcell ) 1.4616 mL) containing peptide solution at a stirring speed of 307 rpm, with a 240 s delay between injections. The heat released by reactions with dilutions was determined in control titrations by injecting heparin solution into buffer A or buffer A into peptide solution, which showed almost zero heat release by the reaction and subtraction of it is thus unnecessary. The reversed titration of 1 mM peptide into 5 µM heparin was performed under identical conditions. Raw
data were obtained as a plot of power (µcal/s) against time (min) and integrated to obtain a plot of observed enthalpy change per mole of injected heparin (∆H, kcal/mol) against molar ratio (heparin-peptide). Binding stoichiometries (n) and binding constant (K) were determined by fitting the data to a one-set or two-set binding model, respectively, using the Origin 7 nonlinear least-squares program supplied with the MicroCal VP-ITC instrument. The binding isotherm for heparin to each peptide is the representative of two repeat experiments, and the reported experimental data is an average of them where the error bars represent the standard deviations of mean values from the experimental results. Fluorescence Spectra and Static Right-Angle Light Scattering. Fluorescence emission spectra of peptides were recorded on a computer-controlled Shimadzu RF-5301PC fluorescence spectrophotometer (Tokyo, Japan). Samples consisted of 10 µM peptides titrated with heparin in buffer A at 25 °C. A fixed excitation wavelength of 280 nm and collecting emission spectra of 300-500 nm were used. Static light scatter (SLS) at a right angle was measured at a wavelength of 350 nm with a Shimadzu RF-5301PC fluorescence spectrophotometer under constant stirring and 25 °C. Samples consisted of 10 µM peptides titrated with heparin in buffer A. As a control, heparin was injected into pure buffer A without peptide. Circular Dichroism. The CD spectra were recorded for heparin, peptides, and peptides with heparin in buffer A using a Jasco J-810 spectrophotometer (Tokyo, Japan). A quartz cuvette with a path length of 0.5 cm was used. All spectra were corrected by subtracting the buffer baseline. In the mixing solutions, the peptide concentration was 50 µM and heparin concentrations were 2.5, 5.0, and 25 µM, respectively. Results are reported as the mean residue ellipticity (deg cm2/dmol). Results Interaction of the Synthetic Peptides and Heparin Measured by ITC. We first investigated the interaction of the five peptides with heparin in buffer A (50 mM sodium phosphate, pH 7.4) using ITC at 28 °C, which enabled us to explore the different binding properties of heparin for the segments originating from HPV-16 L1 and L2. Depending on the structural aspect of the peptides, we obtained three different types of titration curves, as illustrated in Figure 1A-C. Figure 1A shows a calorimetric heat flow trace and the corresponding titration curve obtained by the titration of L2NtW with heparin at 28 °C. The calorimeter cell contained a 100 µM peptide solution, and 5 µL aliquots of a 300 µM heparin solution were injected at 4 min intervals. In Figure 1A, the heat measured for the first few injections was rather constant with hi ∼ -76.4 kcal/mol heparin. With more heparin injection, the concentration of free L2NtW decreased and the heat of the reaction decreased suddenly. The titration plot for L2NtW with heparin can be fitted well using a single-set binding model, where heparin is suggested to have n independent and equal
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Figure 1. Binding isotherm for the interaction of heparin with (A) L2NtW, (B) L1CtaW, and (C) L1CtbW: (top pane) calorimetric trace, the peptide (100 µM in the cell) was titrated with 300 µM heparin (in the syringe) at 28 °C; (bottom pane) integrated heat (squares) and fitted results (lines) of the reaction.
binding sites for peptides. This model has been widely used for the binding of other peptides and heparin.19,21,22 The solid line in the bottom pane is the best least-squares fit of the data using this model, which gives the standard thermodynamic parameters ∆H ) -78.93 kcal/mol, K ) 2.02 × 107 M-1, and n ) 11.8 (Table 1). The Gibbs free energy change (∆G) and entropy change (∆S) were then calculated by using the thermodynamic equations: ∆G ) -RT ln K and ∆G ) ∆H T∆S and the data are summarized in Table 1. The results of L2CtW (see Supporting Information, Figure S1A) are similar to those of L2NtW. Therefore, an identical analysis was performed for L2CtW. The complete thermodynamic parameters are also listed in Table 1. The binding constants of L2CtW and L2NtW are of the same order of magnitude (107 M-1), which is obviously (1 or 2 orders) higher than those reported for heparin-peptide or heparin-protein interactions.20,23-27 These results unambiguously show a sufficiently strong binding affinity between heparin and the two L2 peptides. The binding constant of L2CtW was roughly 3 times that of L2NtW, which may be due to the cluster of basic sequences (K, R) in L2CtW, which is known to have high compatibility with and binding affinity to heparin. To validate this conclusion, we further compare the results of L2CtW and L1CtaW in detail. Although each has six basic residues, the separated ones in L1CtaW resulted in 2 orders lower binding constant with heparin. In addition, the ITC flow trace of L2NtW demonstrated an unusual shape, where the heat of the reaction was initially exothermic, but then dropped to zero and became slightly endothermic before approaching zero again (Figure 1A). This endothermic process was not observed when the titration was performed at high NaCl concentration (200 mM, data not shown). The addition of heparin to peptide probably leads to an aggregation of several heparin molecules via the peptide. This aggregate would be disassociated with the addition of excessive heparin, which may be endothermic. Both the formation and dissociation processes of the aggregate are clarified clearly by the SLS experiment in which the maximum fluorescence scattering intensity is shown at a peptide/heparin
Figure 2. SLS of the peptides (10 µM) or pure buffer A titrated with heparin at 25 °C. Except L2NtW and L2CtW, the fluorescence scattering intensity of other peptides as well as buffer A in titrated by heparin leads to a flat baseline. The scattering curves showed the maximum at a peptide/heparin molar ratio of n ) 12.5 and 14.3 for the L2NtW and L2CtW, respectively.
molar ratio of 12.5 and 14.3 for the L2NtW and L2CtW, respectively (Figure 2). These ratios are close to the n ) 11.8 and n ) 15.2 obtained by the ITC experiment. In addition, at the inverse peptide-to-heparin titration, the SLS plots showed that the addition of L2 peptide to heparin also lead to aggregation and a scattering maximum, but it was less intense and showed an exactly reciprocal molar ratio (data not shown). In addition, in the inverse peptide-to-heparin ITC experiment, there was an excess exothermic process during the initial stage of the titration and an endothermic process in the final stage, which reveals the formation and dissociation of the aggregated heparin-L2 complexes (data not shown). Therefore, these results indicate that, upon further addition of either reactant beyond the stoichiometric maximum, the aggregates can dissolve into solution. A similar phenomenon was observed in a previous report of HS/TAT-PTD binding, and it was also attributed to the aggregates of the complexes.19 Figure 1B shows the binding isotherm for the interaction between L1CtaW and heparin at 28 °C. Although the shape of
Interaction of HPV-16 Capsid Peptides with Heparin the titration curve is close to that of L1CtbScW (Figure S1B, Supporting Information), they will not be discussed together here because of their different physical origination. Different from L2 peptides, once heparin was injected into the solution containing L1CtaW, the heat of the reaction progressively decreased, and after a few injections the heat nearly reached zero. The titration plot for L1CtaW with heparin was also well fitted using the single-set binding model, which supplies the following set of thermal dynamic parameters: ∆H ) -36.94 kcal/mol, K ) 1.53 × 105 M-1, and n ) 23.7 (Table 1). Notably, the binding constants of L1CtaW was close to other reported heparin-peptide interactions,19,21 which are all 2 orders of magnitude lower than those of L2 peptides and show a moderate binding ability. In addition, in possessing six basic residues (K and R) as L2CtW, the value of n obtained for L1CtaW here is unexpectedly large compared to that of L2CtW, which may relate to the peptide sequence and, in particular, the distance between the basic amino acids in the peptide. We speculated that the discontinuous basic residues in L1CtaW create unmatched binding sites with heparin and, consequently, some of them cannot bind with heparin and result in relatively larger n. Figure 1C shows the calorimetric heat flow trace and corresponding titration plot obtained for L1CtbW with heparin. The theoretical curve calculated with a single-set binding model indicated that, after 12 injections, almost all L1CtbW in the calorimeter cell was bound to heparin. After this point, an additional exothermic reaction takes place, and the titration is an obvious two-stage interaction. Similar phenomena have been observed for the titration of DNA with multivalent cations28 and the titration of Mel-SH with HS,29 which were interpreted in terms of DNA condensation and aggregation or a reorganization of the complex into a more compact aggregate, respectively. We propose here two possibilities for the second-stage interaction between L1CtbW and heparin. The first possibility corresponds to the aggregates of L1CtbW-heparin complexes via further hydrogen bonding of the unbound polar cluster (abundant S and T) in L1CtbW, which will show aggregate or colloidal suspensions in the solution and would be detected by the SLS measurement. The second possibility may correspond to the direct interaction between the polar cluster (S and T) of L1CtbW and the specific chain of hydrophilic heparin after the heparin concentration excess the saturated limit, where amorphous precipitates are not expected. Because neither precipitation nor obvious scattering intensity in the SLS measurement was observed for L1CtbW (Figure 2), we denied the first possibility. Such conclusion was further approved by using a control peptide of L1CtbW, AVL1CtbW, where the segment of STSTT in L1CtbW was changed to AVAVV (Figures S4 and S5, Supporting Information). Therefore, the formation of a complex between L1CtbW and heparin can only be assessed using a twoset binding model in which the binding sites are divided into two nonequivalent groups (Table 1). A similar model was presented in the ITC curve fitting for the complexation between BSA and nanoparticles; a strong interaction for set 1 and a weak interaction for set 2 was proposed.30 Because the interaction for set 2 was weaker and minor, only the interaction for set 1 of L1CtbW with heparin will be discussed and contrasted to other peptides in this study. The shape of the titration curve of L1CtbScW with heparin was distinct from that of L1CtbW but similar to that of L1CtaW, and it was fitted well using the single-set binding model. The thermodynamic data for L1CtbScW are also listed and compared in Table 1. The binding constant of L1CtbScW was close to that of L1CtaW, 2 orders of magnitude lower than the constants
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Figure 3. Temperature dependence of the ∆H for five HPV-16 peptides in binding to heparin. The linear regression analysis of the experimental data (solid line) yields ∆H ) -74.01 - 0.156T (°C) for L2NtW (9); ∆H ) -55.40 - 0.511T (°C) for L2CtW (O); ∆H ) -13.02 - 0.845T (°C) for L1CtaW (b); ∆H ) -25.12 - 0.140T (°C) for the first binding set of L1CtbW (4) and ∆H ) -15.54 - 0.658T (°C) for the second binding set of L1CtbW (2); and ∆H ) -32.69 - 2.305T (°C) for L1CtbScW (0), respectively.
for L1CtbW and L2 peptides, showing a much weaker heparin binding ability compared to L1CtbW. Effect of Temperature on the Heparin-Peptide Interaction. In order to investigate the effect of temperature on the biding interactions, calorimetric titrations were performed at several temperatures (15-37 °C) for each peptide. For all five peptides, the binding constants decrease with temperature as a result of the exothermic character of the heparin-peptide interaction (see Supporting Information, Table S1). With the exception of the second binding set of L1CtbW at 15 and 28 °C, all reactions were completely enthalpy driven at the measured temperature range (∆H < 0 and ∆S < 0) under standard conditions. In addition, ∆H decreased in magnitude with increasing temperature (Figure 3). The slope of the straight line shown in Figure 3 describes the constant pressure heat capacity change (∆Cp) of the heparin-peptide interaction. As an example, the reaction enthalpy of L2NtW decreased linearly with increasing temperature and yielded a ∆Cp of -156 cal mol-1 K-1 (Figure 3). Similarly, the ∆Cp for the first binding set of L1CtbW (-140 cal mol-1 K-1) was achieved. However, the values are more negative for L2CtW (∆Cp ) -511 cal mol-1 K-1) and L1CtaW (∆Cp ) -845 cal mol-1 K-1), and very negative for L1CtbScW (∆Cp ) -2305 cal mol-1 K-1). These results are quite different from those obtained in previous heparin-binding studies of brain natriuretic peptide (BNP), which revealed large positive values for ∆Cp (1 kcal mol-1 K-1),31 or HS binding studies using cellpenetrating peptides (CPPs) TAT and R9, which also revealed positive values (∆Cp,TAT ) 135 cal mol-1 K-1 and ∆Cp,R9 ) 155 cal mol-1 K-1).19,22 However, similar negative values of ∆Cp were reported in two other studies on the temperature dependence of melittin with heparin or HS (∆Cp ) -285.1 cal mol-1 K-1 and ∆Cp ) -227 cal mol-1 K-1),20 and it was more negative (∆Cp ) -322 cal mol-1 K-1) for the melittin analogue with HS.29 Generally, ∆Cp has been shown to correlate with the burial of surface area induced by binding; thus, the positive ∆Cp confirmed a reduction in the number of polar surfaceexposed residues and negative ∆Cp values confirmed a reduction in the number of nonpolar surface-exposed residues,32-36 whereas the magnitude of ∆Cp per unit surface area accompanying the exposure of nonpolar surface is larger.37,38 On the basis of the differences in ∆Cp, the value has demonstrated that electrostatic interactions and hydrogen bonding play a major
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Figure 4. Salt concentration dependence of the interaction between heparin and five peptides. The K values are plotted as a function of the Na+ concentration on a log/log scale. The linear regression analysis of the experimental data (solid line) yield log K ) 4.41 - 2.79 log [Na+] for L2NtW (9), log K ) 3.07 - 4.55 log [Na+] for L2CtW (O), log K ) 2.99 - 2.07 log [Na+] for L1CtaW (b), log K1 ) 2.74 - 4.74 log [Na+] for the first set of L1CtbW (4), log K2 ) 3.02 - 2.69 log [Na+] for the second set of L1CtbW (2), and log K ) 3.05 - 2.25 log [Na+] for L1CtbScW (0), respectively.
role in the binding of TAT, R9, and BNP with GAGs,19,22,31 while hydrophobic interactions contribute to the binding of melittin or its analogue with GAGs.20,29 Consequently, the negative ∆Cp values for the HPV-16 peptides indicate that hydrophobic and electrostatic interactions must be involved in peptide binding to heparin. The overall contribution of ionic and nonionic components in the interaction between peptide and heparin was clarified quantitatively by NaCl affected titration in the following. Effect of NaCl on the Heparin-Peptide Interaction. The thermodynamic parameters of the five peptides (Table S2, Supporting Information) provide information about the effect of NaCl on the heparin-peptide interaction. As anticipated on the basis of the electrostatic interaction, the binding affinity of heparin for each peptide decreased with increased salt content in the buffer solution. We analyzed the salt concentration dependence of the binding constant using a popular model for protein-polyelectrolyte interactions.21,39 The model predicted that the extent of peptide binding would decrease with increasing Na+ concentration (Table S2, Supporting Information), and the relationship between them was calculated by eq 1.31,40
log K ) log Knonionic - zψ log [Na+]
(1)
where K is the measured binding constant and Knonionic is the contribution of nonionic interactions. A plot of the experimental log K as a function of log[Na+] gives a straight line (Figure 4) with a slope that provides the number of purely ionic interactions involved in the association reaction, and the y-intercept at 1 M
NaCl gives an estimate of Knonionic, which is equivalent to K minus electrostatic effects.40,41 Regression analysis of the experimental data yielded a straight line with a slope of zψ ) -2.79 for L2NtW. By using ψ ) 0.8 for heparin,21 z ) 3.49 is obtained, suggesting that approximately three to four Na+ are released when one L2 peptide binds to heparin. The y-intercept (log Knonionic) of the regressed line was 4.41 for L2NtW. For a comparison, the relationship between log K and log [Na+] for other four peptides are also presented (Figure 4). The linear relationship between the binding constant and Na+ concentration suggests a predominantly electrostatic association between heparin and each peptide. Regression analysis of the experimental data yielded the parameters and percentage of ionic contribution listed in Table 2 for each peptide. The comparison of the binding parameters between Tables 1 and 2 evaluated the different energy contributions from electrostatic and other interactions. These values suggest that a high fraction, 60.7% and 64.3%, of the total free energy of heparin binding to L2CtW and L1CtbW originated from electrostatic interactions under the present conditions. However, the relatively low values were obtained for L1CtaW (42.5%) and L1CtbScW (43.5%). Surprisingly, the value for L2NtW was unexpectedly low (39.7%) based on the polyelectrolyte theory, whereas its total binding constant, K, was similar to that of L2CtW and L1CtbW. No explanation can be made for this result. Interaction of HPV-16 Peptides and Heparin Assayed by Fluorescence Spectra. Tryptophan fluorescence is very sensitive to microenvironmental changes and is commonly used as probe in exploring protein unfolding or peptide binding.42,43 Exposure to UV light will produce an emission peak of Trp residues in proteins or peptides at approximately 350 nm, which sensitively changes along the condition switches of residue. The fluorescence spectra of one representative peptide, L2NtW, titrated with heparin at 25 °C (Figure S2, Supporting Information) showed a slight blue shift and a quenching of the fluorescence emission band with the addition of heparin, which remained constant to a certain point (Figure 5). At this point, the molar ratio of heparin to peptide was 0.1, which is close to the n ) 0.0849 obtained by ITC measurements. The other four peptides had similar blue-shifted and quenched fluorescence spectra as that of L2NtW, which reflected the strong interaction between the peptides and heparin. However, differences between peptides in the degree of quenching were observed (Figure 5). The plots of I/I0 versus the molar ratio of heparin to peptide showed relatively greater quenching for L2NtW and L2CtW than the other three L1 peptides, which should be ascribed to the stronger interaction between the L2 peptides and heparin as revealed by ITC (K ) ∼107 M-1). In addition, such strong fluorescence quenching of L2 peptides may also relate to the aggregation of the heparin-peptide complex as revealed by SLS measurement (Figure 2). Reasonably, the lesser quenching of L1CtaW and L1CtbScW reflects the relatively weak interaction between the peptides and heparin (K ) ∼105 M-1). Surprisingly, similar
TABLE 2: Thermodynamic Parameters for the Interaction of Heparin with Five Peptides at 28 °C peptide L2NtW L2CtW L1CtaW L1CtbW L1CtbScW
z
K (M-1)
Knonionic (M-1)
Kionic (M-1)
the percentage of ionic contribution (%)
3.49 5.56 2.59 5.93 3.36 2.81
(2.02 ( 0.40) × 107 (6.61 ( 1.70) × 107 (1.53 ( 0.51) × 105 (4.74 ( 1.62) × 107 (6.04 ( 2.76) × 105 (2.52 ( 1.45) × 105
2.57 × 104 1.17 × 103 9.77 × 102 5.50 × 102 1.05 × 103 1.12 × 103
7.86 × 102 5.65 × 104 1.57 × 102 8.62 × 104 5.75 × 102 2.25 × 102
39.7 60.7 42.5 64.3 47.8 43.5
Interaction of HPV-16 Capsid Peptides with Heparin
Figure 5. Change of fluorescence relative intensity at the fluorescence emission peak of five peptides with heparin as a function of heparin/ peptide molar ratio. I0 and I are the fluorescence emission maximum of the peptide in the absence and presence of heparin, respectively. Each sample consisted of 10 µM peptides titrated with heparin in buffer A at 25 °C.
lesser quenching was observed for L1CtbW, though it was shown to strongly bind with heparin in ITC as L2NtW and L2CtW (K ) ∼107 M-1). The reason for this difference between L1CtbW and two L2 peptide may be that the long distance between Trp and the basic cluster (KRKKRK) in L1CtbW reduced the fluorescent response of Trp to the peptide-heparin interaction, while the close distance between Trp and the basic residues in L2 peptides lead to a readily fluorescence response and more extensive quenches. In addition, the fluorescence intensity of L1CtaW or L1CtbScW continued to decrease even after excessive heparin beyond the binding stoichiometry was added (Figure 5), which may also relate to the weaker interaction between L1CtaW and heparin; a low binding affinity leads to an unstable equilibrium between the peptide and heparin, needing an excess of heparin. As a control, we also investigated the fluorescence titration of heparin into L-tryptophan under identical conditions. We observed neither the quenching nor the band-shift of the fluorescence emission (data not shown), excluding observed fluorescence changes of the peptide being due to the direct interaction of Trp with heparin. CD Spectra. To the best of our knowledge, no crystal structural data are available for the HPV-16 L1 and L2 peptides used in this paper.44 We followed any structural changes in the synthetic peptides induced by heparin binding using CD spectroscopy (Figure S3, Supporting Information). These experiments provided additional information about the conformation of free and heparin-binding L1CtbW. There was no significant change of CD spectra with the addition of heparin and they did not exhibit the features of the regular secondary structure,21,45 illustrating that L1CtbW always adopts an essential random coil conformation. The CD spectra of the other peptides, either in the absence or in the presence of heparin (data now shown), were qualitatively similar to those obtained for L1CtbW. Therefore, identical conclusion as L1CtbW could be addressed as that there are no conformational changes induced by heparin binding to the peptides. Discussion Binding Equilibrium of the HPV-16 Peptide-Heparin Interaction. The ITC results showed that heparin can rapidly bind to the five HPV-16 peptide analogues at 28 °C with intrinsic binding constants in the range of ∼105-107 M-1. These results demonstrate that heparin has one set of multiple
J. Phys. Chem. B, Vol. 114, No. 30, 2010 9859 independent binding sites for L2NtW, L2CtW, L1CtbScW, and L1CtaW, but two sets of independent binding sites for L1CtbW. The thermodynamic parameters also show that, with the exception of stage 2 of L1CtbW, binding is overwhelmingly enthalpy driven and entropy-unfavorable at 28 °C because both ∆H and ∆S are negative. These results are in agreement with previous reports on the interaction of heparin with other peptides31 and several protein-small ligand associations36 in which both ∆H and ∆S are negative. In general, in the process of peptide-heparin complex formation, there are several different driving forces, of which the ionic interaction contributes positive ∆S and near zero ∆H, the hydrophobic association contributes positive ∆S and ∆H, and the only contributions to negative ∆S and ∆H are from hydrogen bonding and van der Waals interactions.36 Thus, our results of both negative ∆H and ∆S indicate that hydrogen bonding and van der Waals interactions may also participate in the peptide-heparin interaction. In addition, the entropy decrease generally originates from a reduction in side chain mobility at the binding site,46 which could be due to the large negative entropy contribution associated with more degrees of freedom lost in heparin upon binding.18 The binding stoichiometry of L2CtW/heparin was 15.2, which corresponds to approximately 15 L2CtW bound to each heparin. For the binding of L1CtbW to heparin in the first binding set, there are a similar number of positive charge binding sites, owing to the similar n of peptide/heparin and the same number of basic sequences. The n value for L2NtW/heparin was 11.8 and different from that of L2CtW, which is not surprising because each L2NtW has seven basic amino acids. For the other two peptides, L1CtaW and L1CtbScW, the n values were larger than that of L2CtW, even though they all have six basic amino acids. For L1CtaW or L1CtbScW, approximately 23 molecules are bound to each molecule of heparin. We speculate that there are some basic amino acids in the peptide that do not bind because they are far from each other and do not match the distance of the negative charges on heparin. Therefore, only some of the six basic residues can interact with the heparin, subsequently generating relatively higher binding stoichiometries. Ionic and Nonionic HPV-16 Peptide-Heparin Interaction. Although molecular interpretations of thermodynamic results must be viewed with caution, the results obtained with ITC do suggest general differences between the binding of L1 and L2 peptides or the different clusters within L1 and L2 with heparin. The binding constants are of the same order of magnitude (∼107 M-1) for L2CtW, L2NtW, and the first set of L1CtbW which are obviously greater than the constants of the other two peptides (∼105 M-1). These results are reasonable because the competition assay performed by Bousarghin et al.10 indicated that the peptides of HPV-16 L1Ctb, L2Ct, and L2Nt are more efficient at mediating gene transfer into target cells via the HS receptor. However, heparin binding to L1Cta or L1CtbSc is much weaker than to the other three peptides. This conclusion is also supported by the results that the L1CtbScW had a much weaker interaction with heparin compared to L1CtbW, though both have exactly the same residues. The reduced binding ability of L1CtbScW compared to L1CtbW suggests that heparin binding is a sequence-specific and -dependent phenomenon. The interaction between peptides and heparin consists of both ionic and nonionic components.20,29,31 Table 2 shows that there are conspicuous distinctions in the electrostatic interactions between the anionic heparin and the different cationic peptides, despite the similar number of basic amino acids they possess. The result that the five synthetic peptides are not equal in binding to heparin suggests that it is the pattern and spatial arrangement
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of the basic, polar, and/or hydrophobic residues that is important for their interaction with heparin and not the nonspecific interaction of charges. Notably, the cluster of six basic residues in the structures of L2CtW and L1CtbW contributed greatly to the ionic interaction between the peptides and heparin. The Kionic were smaller for the other three peptides, which do not have the basic residue cluster. The finding that the spatial arrangement of the six basic residues is critical and that L1CtbW is as effective as L2CtW and L2NtW in binding to heparin should aid in the exploration of other HPV analogue peptides as potential agents for cell surface attachment. Considering the size difference, and based on the predominance of electrostatic interactions to the free energy of binding, the HPV-16 capsid peptides presumably maximize their electrostatic interactions by changing their tertiary structure. Biological Aspects of HPV-16 Peptide-Heparin Interaction in Vitro. Early studies have already identified HSPGs as the primary attachment receptors for papillomaviruses,47 and this interaction was shown to be essential for productive infection.6,7 Despite the importance of HS-protein binding, there were relatively few in vitro studies at the molecular level being reported until the appearance of high-sensitivity methods, such as ITC. Basically, heparin is more easily available and has the same basic structure as HS, repeating disaccharides IdoA/GlcUA and GlcNAc, in spite of heparin having approximately twice the sulfate residues per disaccharide unit compared to HS.19,48 Several studies on HS-protein binding have used the more abundant heparin or heparin-related oligosaccharides as models of HS due to the similar chemical composition.18,19 Moreover, heparin was demonstrated to have similar thermodynamic parameters and binding mechanism as HS in the interaction with melittin.20 The basic stretch of amino acids at the C-terminus of L1 was previously suggested to mediate HS-VLPs binding.7,10 The majority of the C-terminal arm is surface-exposed despite being located within the intercapsomeric cleft and may provide surfaces for receptor binding and the induction of neutralizing antibodies.49,50 The L1 protein can self-assemble into icosahedral VLPs in the absence of L2, which is nonetheless necessary for establishing infection,51 especially the N-terminal sequence of L2. An N-terminally truncated BPV L2 was previously reported to be incapable of producing infectious virions,52 and a 23 amino acid peptide at the C-terminus of HPV-33 L2 is necessary for membrane-penetrating activity, which is absolutely essential for efficient HPV infection.53 The L2 density was probably located at the central internal cavity of each capsomer, as shown by cryoelectron microscopy; however, the central cavity of capsomers is not large enough to allow the passage of polypeptide chains. Thus, the L2 N-terminus likely extends to the capsid surface between neighboring capsomers.54 In addition, interaction with receptors at the cell surface, most likely HSPG, induces conformational changes that affect both capsid proteins, particularly the exposure of the L2 N-terminus.55 Therefore, studying the binding of heparin to these lysine- and argininerich segments originating from the C-terminus of L1 and the N- and C-termini of L2 is necessary and crucially important. In this study, the binding constants were ∼107 M-1 for L2NtW, L2CtW, and L1CtbW, meaning that the binding of HPV-16 capsid peptides to heparin is very strong relative to other peptides or proteins binding heparin or HS (e.g., 105 M-1 for TAT-PTD,19 105 M-1 for BNP,31 106 M-1 for melittin,20 and 106 M-1 for ECSOD18). The high binding affinity of these peptides for heparin further demonstrated that cell surface HSPGs and the capsid protein peptides rich in basic amino acids
Sun et al. were the primary attachment factors in the adsorption and attachment of HPV-16 to host cells. The in vitro results presented here reveal that the employed HPV-16 peptides of L1Ctb, L2Ct, and L2Nt have the ability to participate in cell binding. These peptides have similar properties and heparinbinding affinity in vitro for the cell-penetrating peptides (CPPs), such as HIV-1 TAT-PTD19 or R9.23 Thus, the interaction between HPV-16 capsid peptides and heparin is of general biological relevance for revealing the binding mechanism between HPV capsid proteins and cells at the molecular or submolecular level. Similar research on the binding mechanism between these peptides with DNA is currently in process and may quantitatively explain both the peptide-DNA binding and effects of peptide-DNA interaction observed in cells. Conclusions For the first time, ITC was used to provide detailed quantitative insight into the interaction between the cationic peptides of HPV-16 capsid protein and anionic heparin. Our study supplied solid evidence of strong interactions between several basic-sequence-rich L1 and L2 peptides and the negatively charged polyelectrolyte. The binding constants of L2NtW, L2CtW, and the first set of L1CtbW are at the level of ∼107 M-1 and roughly 1 or 2 orders of magnitude higher than the binding constants observed for L1CtaW, L1CtbScW, and other reported CPPs. Moreover, several driving forces, including ionic interaction, hydrophobic forces, and hydrogen bonding, are involved in the interaction between the positively charged peptides and heparin. In addition, the distinction between the bindings of different peptides to heparin was attributed to the species and the arrangement of the residues in the peptides. The tight interaction between HPV-16 peptides and heparin provide a solid physical-chemical foundation for the involvement of proteoglycans in viral infection. Acknowledgment. This work was supported by the Natural Science Foundation of China (No. 20934002 and 20973073), the National Basic Research Program (2007CB808006), Natural Science Foundation of Jilin Province (No. 20070926-01), the Programs for New Century Excellent Talents in University (NCET) and the 111 Project (B06009) (to J.S., S.J., and Y.W.), and the State Key Laboratory for Supramolecular Structure and Materials, Jilin University (to X.Z.). Supporting Information Available: The thermodynamic data at different temperatures, effect of NaCl on the thermodynamic data, fluorescence emission spectra of L2NtW in titrated with heparin, CD spectra of L1CtbW upon the addition of heparin, and the binding isotherm for the interaction of heparin with L2CtW, L1CtbScW, and AVL1CtbW. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Salzman, N. P., Howley, P. M., Eds.; The PapoVaViridae. Vol. 2. The PapillomaViruses; Plenum Press: New York, 1987. (2) zur Hausen, H. Biochim. Biophys. Acta 1996, 1288, F55. (3) Baker, T. S.; Newcomb, W. W.; Olson, N. H.; Cowsert, L. M.; Olson, C.; Brown, J. C. Biophys. J. 1991, 60, 1445. (4) Sapp, M.; Volpers, C.; Muller, M.; Streeck, R. E. J. Gen. Virol. 1995, 76, 2407. (5) Kirnbauer, R.; Taub, J.; Greenstone, H.; Roden, R.; Durst, M.; Gissmann, L.; Lowy, D. R.; Schiller, J. T. J. Virol. 1993, 67, 6929. (6) Giroglou, T.; Florin, L.; Schafer, F.; Streeck, R. E.; Sapp, M. J. Virol. 2001, 75, 1565. (7) Joyce, J. G.; Tung, J. S.; Przysiecki, C. T.; Cook, J. C.; Lehman, E. D.; Sands, J. A.; Jansen, K. U.; Keller, P. M. J. Biol. Chem. 1999, 274, 5810.
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