Influence of peptide conformation on oligosaccharide binding characteristics—a study using apamin-based chimeric peptide

Peptides 24 (2003) 1853–1861

Influence of peptide conformation on oligosaccharide binding characteristics—a study using apamin-based chimeric peptide Cheng Wei Wu, Gurunathan Jayaraman∗ , Kun Yi Chien, Yaw Jen Liu, Ping Chiang Lyu1 Department of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan Received 31 July 2003; received in revised form 16 October 2003; accepted 16 October 2003

Abstract Interactions between proteins and heparin play a crucial role in most of the cellular process. Unraveling the forces that govern the formation of these complexes is vital for understanding the specificities involved in these biomolecular events. In the present study, a detailed analysis has been undertaken to evaluate the effect(s) of peptide conformation on heparin-binding, using a chimeric peptide, apaK6—a chimera of a highly stable neurotoxic peptide from honey-bee venom and a de novo designed lysine-rich peptide. The dissociation constants of these peptide–heparin complexes were found to be in the submicromolar range. Comparison of the results obtained from the titration of the disulfide-reduced and disulfide-intact chimeric peptide with various sulfated oligosaccharides, derived from heparin, suggest that the initial structure of the peptide has pronounced effect on the binding affinity, binding modes and also on binding preferences. The results of this study indicate that the heparin-binding specificity of an isolated peptide and that exhibited by the same peptide when present in a globular protein could be significantly different, especially if the isolated peptide undergoes conformational change(s) upon binding to the sulfated oligosaccharides. In addition, such dependency of the binding specificity on the preformed structures could be utilized for the design of high-affinity and sequence-specific heparin-binding polypeptides. © 2003 Elsevier Inc. All rights reserved. Keywords: Binding specificity; Chimeric peptide; Heparin; Peptide conformation; Peptide design

1. Introduction The chemical basis underlying the interaction of heparin/heparan sulfate and proteins, belonging to certain classes, are being unraveled in recent years [7,8]. Heparin and heparan sulfate are linear polymers that are composed of disaccharide repeating units, consisting of glucuronic acid and glucosamine linked by (1 → 4) glycosidic linkage [21,36]. Non-uniformity of the sulfation pattern in the disaccharide repeating units forms the basis for the complex structure of these glycosaminoglycans. Such diverse structural features seem to be responsible for the observed specificity in the protein–glycosaminoglycan binding reaction [2,7,12,19,35,36]. It is common that the heparin-binding region(s) of proteins contain high density of positively charged residues (lysine, arginine and occasionally histidine). Analysis of the arrangement of these ∗ Corresponding author. Present address: Centre for Protein Engineering and Biomedical Research, The Voluntary Health Services, Adyar, Chennai 600 113, India. Tel.: +91-44-2254-2252; fax: +91-44-2254-2018. E-mail address: [email protected] (G. Jayaraman). 1 Co-corresponding author.

0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.10.004

basic residues in the heparin-binding domain of proteins led to the proposal of diverse heparin-binding consensus motifs: XBBXBX, XBBBXXBX, XBBBXXBBBXXBBX and TXXBXXTBXXXTBB [6,18,32], where B is the positively charged amino acid residue, X is any hydropathic residue and T signify a turn. The above motifs are dependent on the backbone conformation of the protein. Recently, it has been proposed that a distance of about 20 Å between two positively charged residues in peptides/proteins and their relative orientation form a very important topology for heparin/heparan sulfate binding [24]—a model that is independent of the protein conformation. In either case, the electrostatic interaction(s) of the positively charged residues in proteins and the negatively charged functional groups (sulfates and carboxylates) on the heparin/heparan sulfate chain is presumed to be the dominant force responsible for the binding reaction. This led to the general belief that the recognition of heparin by proteins could be non-specific [33]. Such a view was further strengthened by several facts, viz., arginines were preferred over lysines for better binding [13]; decrease in the dissociation constant of the protein–heparin complexes with increasing chain length of the oligosaccharides (derived from heparin); and drastic

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decrease in the binding affinity upon increasing the ionic strength of the medium [26]. However, studies with model peptides [11,13,14,34] have proved that the interaction of proteins with heparin is largely dependent on the spacing between the positively charged amino acid residues and the availability of unique sulfation patterns on the oligosaccharide chain. Moreover, in a few cases, the amino acids between these positively charged residues were shown to play an important role in the binding process [9,18]. Thus, it was clarified that these biomolecular interactions need not arise solely due to electrostatic interactions and not all the positively charged residues in the heparin-binding region contribute to the formation of the complex. In addition to the electrostatic interactions, hydrogen bonding, van der Waals forces and hydrophobic interactions are shown to occur in a few complexes [9]. Therefore, at present, two aspects are presumed to be critical in dictating the binding specificity of these protein–heparin interactions: (a) spacing between the positively charged residues rather than the density of positive charges [14,18,24] and (b) most importantly, the type of the intervening amino acid residues. While these aspects are studied in detail (using model peptides), less attention is being paid with regard to the role of peptide conformation in dictating the binding affinity [15]. Interestingly, in most cases, conformational changes have been observed in the polypeptide backbone [1,10,17,25] upon binding to these glycosaminoglycans. This process is more pronounced in studies involving short peptides. These short peptides when severed from the native protein are mostly unstructured in water, even though they fold into ␣-helix or ␤-sheet in the intact protein. It is unclear whether these unstructured peptides could recognize the same sulfated oligosaccharide as it would when present in the native protein. With this view, we embarked upon understanding the effect of peptide conformation on its heparin-binding characteristics using a chimeric peptide termed apaK6—a chimera of a neurotoxic peptide from honey-bee venom [5] and the de novo designed lysine-rich peptide, K6.

2. Materials and methods

aliquots of the reaction mixture were pipetted out, at regular intervals and quenched using 300 mM acetic acid. The mixture was then analyzed using reverse phase HPLC column. It was found that the oxidation was essentially complete by 60 min. In order to avoid the formation of disulfides during the heparin-binding studies using the disulfide-reduced peptide, we preferred to acetamidate the thiols. The reaction was carried out by incubating apaK6 in a solution containing 10 M excess of iodoacetamide, dissolved in 10 mM Tris, 6 M guanidinium hydrochloride (pH 8.2). After incubation in dark at 25 ◦ C for 30 min, the reaction was quenched by the addition of 30 M excess ␤-mercaptoethanol with subsequent acidification. Both the oxidized and modified apaK6 was purified by reverse phase (C18 ) HPLC column. The authenticity of the peptide was confirmed by mass spectrometry. The peptide concentration was determined from its extinction coefficient value (280 nm) of 5930 and 5690 M−1 cm−1 [16]. 2.2. Preparation of homogeneous oligosaccharide fragments Enzymatic digestion of high molecular weight heparin was performed according to the procedure of Linker and Hovingh [22]. Briefly, 200 mg of heparin was dissolved in 5 ml of a solution containing 25 mM Tris, 1 mM CaCl2 , 1% BSA and 25 units of heparinase I (pH 7.4) and incubated at 37 ◦ C. After 24 h, the depolymerization reaction was quenched by heating the reaction mixture at 100 ◦ C, for 5 min. The oligosaccharide fragments thus obtained were fractionated based on their size using Bio-Gel P10 resin (Pharmacia), with 0.2 M (NH4 )2 CO3 at a flow rate of 4 ml/h. The elution of the saccharides was monitored based on the changes in the absorbance at 232 nm. Further purification of the octasaccharide fragments were performed using Q-Sepharose HP resin (Pharmacia). A linear gradient of 0–3 M NaCl was applied (flow rate, 0.75 ml/min) to elute the bound octasaccharides. The pure fragments thus obtained were desalted using Supherdex 10 HR (Pharmacia) column. The concentration of the oligosaccharides was determined based by the uronic acid assay [3].

2.1. Preparation of peptides 2.3. Circular dichroism spectroscopy The chimeric peptide, apaK6, was synthesized by SynPep Corp. (Dublin, CA, USA) and was further purified using reverse phase (C18 ) HPLC column, using appropriate acetonitrile–water gradient. Heparin, heparinase I, glucoronic acid were obtained from Sigma. All other chemicals were of high quality analytical grade. Oxidation of apaK6 was effected by dissolving the peptide in a solution containing 100 mM Tris, 0.1 mM EDTA, 1 mM reduced glutathione and 0.3 mM oxidized glutathione. The pH of the mixture was maintained at 8.0 and the final concentration of the peptide used in the reaction was 1 mg/ml. In order to confirm the complete oxidation of the peptide,

All CD measurements, in the far UV region of the spectrum (195–260 nm) were made using a 1 cm path length cell on a AVIV 62A DS CD spectrometer, equipped with a programmable heating unit. The spectropolarimeter was periodically calibrated using d10 -camphor sulphonic acid. All titrations were performed (at 25 ◦ C, unless otherwise mentioned) by the addition of heparin/oligosaccharides to a constant concentration of the peptide. Appropriate baseline and buffer corrections were made. Changes in the ellipticity values of the peptides, upon titrating with heparin/oligosaccharides, were also monitored at a fixed

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wavelength (222 nm). All the CD spectra used for conformational analysis were an average of three scans and that each data point used for quantification represents an average of 100 data points.

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zero-filled once before Fourier transformation. Data processing was performed by XWIN-NMR software. Proton chemical shifts were referenced to 3-(trimethylsilyl)propionate (sodium salt).

2.4. Data analysis 3. Results The apparent dissociation constant (Kd ) values of the peptide–oligosaccharide complex were estimated from the changes in the ellipticity values of the peptides at 222 nm, upon titrating with heparin or its enzymatically digested oligosaccharide fragments, using the non-linear relation
  (ΘS − ΘO ) Θ = ΘO + [(Kd + nLt + Pt ) 2Pt  (1) − ((Kd + nLt + Pt )2 − 4nLt Pt )0.5 ]

3.1. Design of the chimeric peptide

where [Na+ ] is the total NaCl concentration, Kd(non-ionic) is the contribution of the interactions arising other than pure electrostatics to the observed dissociation constant (Kd(obs) ), Z is the number of ionic interaction and Ψ represents the fractional charge density parameter (0.8 for heparin).

Apamin is a small neurotoxic peptide (aa 18) present in the honeybee (Apis mellifera) venom [5]. The presence of two disulfide bridges in the molecule confers greater thermodynamic stability to the peptide. The architecture of apamin is such that the covalent crosslinks are located at the N-terminal half of the molecule whereas the C-terminal residues enjoy greater degree of conformational freedom [29]. Owing to these interesting structural features, this short peptide has been often used as a template for the design of chimeric peptides. In the present design, we replaced most of the amino acid residues at the C-terminal half of the native apamin with the de novo designed lysine-rich peptide, K6 (Fig. 1). K6 differs from our previous de novo designed K8 [20] both in chain length and in the total number of lysine residues. K8 contains eight lysines in its amino acid sequence whereas K6 consists of only six lysines. The arrangement of the lysines (at alternate positions of three and four) is similar in both the peptides (K6 and K8). Detailed description of the design of the lysine-rich K8 is described elsewhere [20]. The position of the cystine residues is the same as that found in native apamin (Fig. 1). Of the six lysines in K6, two of them are locked within the disulfide framework of apamin. The resultant chimeric peptide, apaK6, contains the disulfide patterns that are identical to the native apamin molecule and a flexible, highly charged C-terminal.

2.5. NMR spectroscopy

3.2. Conformational analysis of the chimeric peptide

NMR experiments were carried out (at 20 ◦ C) using 1 mM of apaK6 in 50 mM d3 -sodium acetate (95% H2 O/5% D2 O; pH 5.0) on a 500 MHz (Bruker) NMR spectrometer. Two-dimensional TOCSY and NOESY [30] experiments were carried out with a spectral width of 6000 Hz in both dimensions. Mixing periods of 90 and 300 ms were used for TOCSY and NOESY experiments, respectively. In these experiments 2048 and 512 K complex data points were collected in F1 and F2 dimensions, respectively. The data were

The intrinsic conformation of the peptides, used in the present study, was examined by circular dichroism spectroscopy. Far UV CD spectrum of the de novo designed lysine-rich peptide, K6, indicates that the conformation of the peptide is highly disordered (Fig. 2). Careful examination of amino acid residues in the peptide would suggest that all the residues exhibit high propensity to adopt helical conformation. But the presence of excess positively charged residues has obviously destabilized the preferred

where ΘO and ΘS represent the ellipticity values of the peptides in the absence and presence of saturating concentrations of a particular oligosaccharide, Pt and Lt are the total concentration of the peptide and oligosaccharide, respectively. The ionic and non-ionic contributions involved in the complex formation of the chimeric peptides with the oligosaccharide fragment were inferred from the dependence of the dissociation constant values on the ionic strength of the medium, which could be described as: log Kd(obs) = log Kd(non-ionic) + ZΨ log[Na+ ]

Apamin K6 ApaK6

(2)

CNCKA PETAL CARRC QQH SK QAKQA QKAQK AQAKQ AKQW CNCKA PETAK CAKQC QKAQK AQAKQ AKQW

Fig. 1. Amino acid sequence of the chimeric peptide, apaK6. Portion of the peptide that correspond to the primary sequence of the bee venom peptide, apamin, is indicated in bold. The positions of the disulfide bridges (Cys1–Cys11 and Cys3–Cys15) are identical to the native apamin molecule.

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Fig. 2. Far UV CD spectra of K6 (A), apaK6 (B) and apaK6R (C) in the presence (dotted line) and absence (solid line) of heparin. An increase in the helical conformation is observed for apaK6, whereas a structural transition from a random coil to helical conformation is seen in K6 and apaK6R.

helical conformation. However, K6 adopts helical conformation in the presence of helix inducing solvents like TFE (data not shown). This indicates that the backbone of this lysine-rich peptide could transform into ␣-helical structure, under favorable condition(s). Helical wheel representation (not shown) of the amino acid sequence of K6, indicates that the peptide exhibits amphipathic nature, with the positively charged residues sticking out on one side of the helical axis. We have recently shown that peptide of this type preferentially binds to iduronic acid containing glycosaminoglycans [20]. The CD spectrum of the chimeric peptide, apaK6, exhibits a double minimum at 208 and 222 nm, implying that the peptide backbone adopts helical conformation (Fig. 2). The conformation of apaK6 was probed, in detail, by NMR experiments. Proton chemicals shifts of apaK6 were traced by the sequential assignment strategy. Signals originating from the N-terminal half (Cys1–Cys15) of the chimeric peptide

are well dispersed compared with those from the C-terminal (Qln16–Trp29) residues (Fig. 3). As the amino acid residues, in apaK6, from Cys1 to Ala9 are identical to that in the parent apamin molecule [16], the conformation adopted by this segment should be similar. This is authenticated by the presence of characteristic NOE patterns (Fig. 3) and by the similarity of the amide as well as the alpha proton chemical shifts. Continuous HN–HN and H␤i –NHi+1 NOES observed for the amino acid residues from Lys10 to Gln16 imply that this region is also ordered. Presence of (i, i + 3) NOEs such as Ala12H␣–Cys15HN, Ala12H␣–Gln16HN and Lys13H␣–Cys15HN indicate that the peptide backbone spanning these residues adopts helical conformation. Proton chemical shifts for the residues Lys20–Trp29 could not be assigned unambiguously due to severe overlap of the resonances. The amide chemical shifts of these protons lie within a narrow range of 8.1–8.4 ppm, indicating that this region of apaK6 could possibly be unstructured. Clearly,

Fig. 3. NOESY spectrum of apaK6. Crosspeaks in the HN–H␣ (A) and HN–HN (B) regions are shown. Residue numbers are used in the annotations. Medium range interactions involving the backbone atoms are also indicated in (A).

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the N-terminal region (Cys1–Cys15) of apaK6 is structured whereas the C-terminal (Qln16–Trp29) is less ordered, conformationally. CD spectrum of disulfide-reduced and carboxyamidated apaK6 (termed as apaK6R) depicts a single minimum at 200 nm (Fig. 2). This indicates that in the absence of the disulfide bonds, the peptide does not possess any well-defined secondary structure. Thus, conformational analysis of the designed peptides reveals that apaK6 is partly helical whereas apaK6R is unstructured. Given the same environmental conditions the two peptides, apaK6 and apaK6R, possessing identical amino acid sequence exhibit two different conformations. Therefore, these two peptides could form an ideal system to explore the influence of the peptide conformation on heparin-binding. Fig. 2 also depicts the conformation of the peptides in the presence of heparin. Far UV CD spectra of all these three peptides (K6, apaK6 and apaK6R) display a double minimum at 208 and 222 nm in the presence of heparin, implying that they tend to adopt ␣-helical conformation. ApaK6 exhibits increased ␣-helical content whereas K6 and apaK6R undergo a conformational transition from random coil to ␣-helix, upon binding to heparin. The ratio of the mean residue ellipticity values of 222–208 nm would reflect on the quality of the helical structure. A value of 0.85 and 0.62 for apaK6 and apaK6R indicates that the quantity of heparin-induced secondary structure is greater in apaK6 than apaK6R. As heparin is highly non-homogeneous in terms of both sulfation and length, we decided to carry out extensive studies using the oligosaccharides obtained from the enzymatic digestion of heparin in order to draw meaningful comparison on the effect(s) of preformed structures of the peptides (apaK6 and apaK6R) on its binding to sulfated oligosaccharides.

disulfide-reduced apaK6, titrations were performed with hexa-, octa- and decasaccharides. As observed in the titrations with low molecular weight heparin, all the three oligosaccharides induced ␣-helical conformation in the peptide backbone, irrespective of the presence or absence of the disulfide bridges. The titration curves represented as the changes in the mean residue ellipticity values at 222 nm as a function of oligosaccharide concentration is given in Fig. 4. Several interesting observations could be made upon careful inspection of the titrations curves (Fig. 4): (a) the percentage of helix induced in the peptide backbone is dependent on the oligosaccharide chain length for apaK6, whereas no drastic effects were observed for apaK6R; (b) the apparent dissociation constant values and the stoichiometry of binding is dependent on the oligosaccharide length for apaK6 but the dependency is less for apaK6R. The non-linear fits represented in Fig. 4 results in the apparent dissociation constant values of 7.02 × 10−7 M, 2.47 × 10−7 M and 5.42 × 10−7 M for apaK6 complexed with hexa-, octaand decasaccharides, respectively. On the other hand, the same oligosaccharides upon binding with apaK6R exhibited the Kd values of 2.73 × 10−7 M, 2.84 × 10−7 M and 2.84 × 10−7 M, respectively; (c) for a given oligosaccharide, the molar ratio of binding (peptide:oligosaccharide) is always greater for apaK6 than required for the neutralization of apaK6R. 3.4. Effect of sulfation pattern/density Oligosaccharide fragments that are derived from heparin are composed of repeating units of hexuronic acid (either iduronic acid or glucoronic acid) and glucosamine [21]. The diversity that originates from the differences in the sulfation patterns in the oligosaccharide chain, is a critical factor in deciding the binding specificity [7]. As the binding affinity of the octasaccharide fragment (Fig. 4) is similar for both apaK6 and apaK6R, this fraction was chosen for further analysis. In order to evaluate the differences in the binding behavior of apaK6 and apaK6R on the different

3.3. Titrations with oligosaccharides of different lengths

Relative Mean Residue Ellipticity

In order to judge the effect of the oligosaccharide chain length on the binding affinity of both the native and the

4

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(A)

(B)

6 3 4 2 2

1

0

0 0

5

10

15

20

0

5

10

15

20

Oligosaccharide ( µM)

Fig. 4. Titration of apaK6 and apaK6R with oligosaccharides of different length. The data points are fit using Eq. (1) (see Section 2). Hexasaccharides (circles), octasaccharides (triangles) and decasaccharides (squares) are used for the titrations. The binding affinity as well as the binding stoichiometry of these two peptides are different, indicating that all these parameters are dependent on the initial conformation of the peptide.

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1.4

1.3

8 1.2 6 1.1

NaCl (M)

Absorbance at 232 nm

10

4 1.0

2

0

0.9 100

150

200

250

300

Elution time (min) Fig. 5. Ion exchange chromatographic profile of the octasaccharide fragment. Q-Sepharose HP resin was used for the purification of the octasaccharide fragment using a linear gradient of 0–3 M NaCl (flow rate, 0.75 ml/min; pH 2.50). Fractions that were used for the titrations are numbered.

sulfation patterns of oligosaccharide of a particular chain length, we purified the octasaccharide fragment using the anion exchange chromatography (see Section 2). The chromatographic profile is given in Fig. 5. Order of elution is dictated by the sulfation density and by the number of sulfates on the octasaccharide. Accordingly, octaVIII is the octasaccharide fragment possessing the highest sulfation number whereas octaI is the saccharide fragment containing the lowest possible sulfation number/density. Comparison of the best non-linear fit of the titration curves indicates that the binding reaction exhibits highly complex patterns for both apaK6 and apaK6R. Analyses of these titration curves indicate that the oligosaccharides of same size but with different sulfation pattern need not exhibit similar molar ratio of binding to the peptides (data not shown). For apaK6, the stoichiometry of interaction is 1:1 for oligosaccharides with low and medium sulfation, whereas binding of two peptides to a single oligosaccharide chain is preferred if the sulfation density is increased. This trend is not evident in the titrations of the same oligosaccharides with apaK6R. A molar ratio of 1:1 is preferred for all the binding reactions, except with octaVI, involving apaK6R. On the basis of pure electrostatic interactions alone, one would predict that increase in the sulfation density of the octasaccharide would increase the binding strength of the peptide. This would indicate that the octaVIII fraction would exhibit the lowest apparent dissociation constant value. Interestingly, this was not observed for both apaK6 and apaK6R (Fig. 6). Therefore, it appears that the octasaccharide containing the largest sulfation number need not necessarily be the oligosaccharide fragment that could exhibit the highest binding affinity. Moreover, no apparent relation could be drawn between the concentration of the salt needed to elute a particular octasaccharide fraction from the anion exchange column and its binding strength to the chimeric peptides (both oxidized and reduced). Highest binding affinity is observed with octaVI for apaK6 whereas

the disulfide-reduced form of the peptide prefers the octaV fraction (Fig. 6). OctaVI fraction binds twice more strongly to apaK6 than to apaK6R. On the other hand, comparison of the binding affinity of the octaV fraction to the chimeric peptides implies that the binding is weaker by four times for apaK6 than apaK6R. On the whole, the results of these titrations not only indicate the importance of initial conformation of the peptide with respect to its binding affinity to a particular oligosaccharide but also in dictating the binding specificities. Three of the total seven positively charged residues, namely Lys4, Lys10 and Lys13, adopt drastically different conformation in apaK6 and apaK6R. On the other hand, the amino acid residues in the C-terminal half of these two peptides are likely to exhibit very similar conformation. It is quite possible that the differences in the binding behavior observed for these two peptides could result from the differences in the orientation of the residues (before complex formation) at the N-terminal of both apaK6 and apaK6R. Variations in the binding behavior could also arise from the

Fig. 6. Changes in the dissociation constant values of octasaccharide fragments complexed with either apaK6 (filled bars) or apaK6R (hatched bars).

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Fig. 7. Dependence of Kd on ionic strength. The changes in the Kd values of the complex formed by the octaVIII fragment with apaK6 (open circle) and apaK6R (filled circle) as a function of NaCl concentration are depicted. The number of ionic contacts (Z) between the apaK6 peptides and the octaVIII fragment appears to be dependent on the initial conformation of the peptide.

fact that the lysines Lys10 and Lys13 in apaK6 act as template or ‘binding nucleus’ for the initial binding process, whereas such structural ‘motif’ is not present in apaK6R.

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in the values of 4 and 5 for apaK6 and apaK6R, respectively (Fig. 7). Assuming that in the presence of 1 M salt concentration, the complex is no more governed by intermolecular charge–charge interaction, extrapolation of the linear fits shown in Fig. 7 would yield the apparent dissociation constant of 8.57 × 10−3 M and 6.46 × 10−2 M for apaK6 and apaK6R, respectively. At physiological salt concentration (150 mM), non-ionic forces contribute about 46 and 28% to the total apparent dissociation constant of the complex involving apaK6 and apaK6R, respectively. It should be noted that though the apparent dissociation constant value of apaK6R–octaVIII is lower than apaK6–octaVIII, the contribution of the non-ionic forces in the latter complex is larger. This might indicate that the apparent dissociation constant value does not directly signify the total strength of the complex. Such trends have also been observed in studies involving globular proteins [7]. Thus, the results of the ionic strength dependence of the dissociation constants do indicate that the mode of interaction of the peptides with sulfated oligosaccharides could be dependent on the preformed conformation of the peptide.

3.5. Influence of ionic strength Protein/peptide sulfated oligosaccharide interactions have been shown, in most cases, to be dominated by intermolecular charge–charge interactions. Therefore, it is expected that the binding affinity would substantially decrease upon increasing the ionic strength of the medium. The extent of decrease has been observed to be dependent on the amino acid composition of the heparin-binding domain that could also in turn dictate the type of interaction involved in the complex formation. The effect(s) of salt concentration on the apparent dissociation constant values of the complex, is a powerful tool to estimate the contribution of the non-ionic interactions involved. Therefore, we performed the titration of a purified oligosaccharide fragment with both apaK6 and apaK6R. OctaVIII has been chosen for this purpose owing to its high abundance. The dissociation constant values for the complexes formed by octaVIII with the two peptides (apaK6 and apaK6R) differ by four times (Fig. 6). The reduced form of the chimeric peptide exhibits stronger binding than the oxidized form. Also, two molecules of apaK6 are involved in complex formation with one molecule of octaVIII whereas the molar ratio of binding is 1:1 for apaK6R. Titration, at constant temperature, in the presence of various salt concentrations indicate that the dependence of the overall apparent dissociation constant is more pronounced for apaK6R–octaVIII complex than for apaK6–octaVIII (Fig. 7). Increasing the NaCl concentration from 17 to 150 mM exhibits an increase of 59 times in the dissociation constant values with apaK6 whereas an increase of about 630 times is observed for apaK6R (Fig. 7). This indicates that the arrangement of the charges prior to complex formation could be an important criterion for binding affinity. Estimation of the number of ionic contacts involved in both these complexes (apaK6 and apaK6R with octaVIII) results

4. Discussion Protein–heparin interaction plays a pivotal role in many of the cell regulatory processes, especially involving intercellular interactions. The structure of heparin, though composed of the repeating disaccharide units of glucoronic acid and glucosamine, exhibits quite complex and disordered sulfation patterns. Such diversity in the placement of the charged functional groups are indeed responsible for the observed specificity in this type of biomacromolecular interaction(s). Proteins from a wide variety of classes have been shown to exhibit high-affinity for heparin and its analogs [4,7,9,12,27,28,31]. Although studies are being carried out with intact proteins or with the peptide segments that has been proposed to exhibit heparin-binding activity, the requirements that are vital for displaying sequence-specific binding is not yet understood in detail. The primary amino acid sequence (forming the consensus motif) certainly plays an important role in dictating the heparin-binding behavior, but the initial conformation of the protein/peptide could play a significant role in terms of the binding strength and also the specificity involved in the interaction. It has been shown previously that a linear peptide mimicking the heparin-binding domain of FGF bound less strongly than the corresponding cyclic peptide [15]. Also, heparin-binding studies of a peptide derived from superoxide dismutase indicate that the peptide exhibit affinity in par with the native protein only in the presence of a structure inducing co-solvent like TFE [23]. The results of these studies corroborate well with those obtained in the present study. Therefore, it is clear that heparin-binding strength is dependent on the peptide conformation. It has to be noted that most of the heparin-binding studies with model peptides indicate that the peptides are

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unstructured in water but adopt ␣-helix/␤-sheet upon binding. Owing to the highly heterogeneous composition of heparin/heparan sulfate chains, the binding specificities of these peptides cannot be overlooked. It is likely that these peptides might recognize a different heparin sequence when compared with that of the native protein, inspite of the fact that the peptide backbone could adopt a similar conformation as that in the native protein, upon binding to sulfated oligosaccharides. The case study presented in this report unequivocally supports this view. Both the peptides (apaK6 and apaK6R) adopt helical conformation upon binding to different sulfated oligosaccharides. Though both apaK6 and apaK6R have identical amino acid sequence, the initial conformation of these peptides plays a significant role in dictating the binding specificity. Both the peptides do not exhibit optimal binding to the same sulfated oligosaccharide chain. This clearly implies that the initial conformation and the presence of localized charges in the preformed conformational state of the peptide plays significant role in deciding the binding specificity. Results from the binding experiments, performed in the presence of varied salt concentrations, indicate that the difference in the binding modes is not only governed by electrostatic interactions but also significantly contributed by the non-ionic forces. Such dramatic difference(s) in the binding modes reflects the importance of the initial interactions between the peptide and the oligosaccharide, which in turn brings out the significance of the conformation of the peptide prior to complex formation. Therefore, the results obtained in the present study implies that the characterization of the oligosaccharide binding capabilities of peptides, derived from heparin-binding domain(s) of globular proteins, should be performed with due caution in order to draw meaningful conclusions, especially when a conformational disparity (between the free peptide and that adopted by the same peptide when present in the native protein) exists. The information gained from this report could serve as an useful tool for peptide design strategies. In addition to the contribution of the amino acid sequence to the binding preferences, alteration in the peptide conformation could also be considered to modulate the binding modes and sequence preferences, in the design of sequence-specific high-affinity oligosaccharide binding polypeptides.

Acknowledgments This work is supported by research grants from the National Science Council, Taiwan and also by the grant from the National Health Research Institutes, Taiwan to G.J. References [1] Aoyama H, Naka D, Yoshiyama Y, Ishii T, Kondo J, Mitsuka M, et al. Isolation and conformational analysis of fragment peptide corresponding to the heparin-binding site of a hepatocyte growth factor. Biochemistry 1997;36:10286–91.

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