Journal of Molecular Structure 891 (2008) 456–462

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Journal of Molecular Structure

journal homepage: www.elsevier .com/locate /molstruc

Conformational changes of ovine a-1-proteinase inhibitor: The influenceof heparin binding

Vivek Kumar Gupta, Lalitha R. Gowda *

Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570020, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 November 2007Received in revised form 25 March 2008Accepted 15 April 2008Available online 22 April 2008

Keywords:Dynamic light scatteringHydrodynamic radiusEquilibrium dialysisFluorescence spectroscopyFourier transform infra-red spectroscopyCircular dichroism

0022-2860/$ - see front matter � 2008 Published bydoi:10.1016/j.molstruc.2008.04.025

* Corresponding author. Tel.: +91 821 2515331; faxE-mail addresses: lrg@cftri.res.in, lrgowda@yahoo.c

a-1-Proteinase inhibitor (a-1-PI), the archetypal serpin causes rapid, irreversible stoichiometric inhibi-tion of redundant circulating serine proteases and is associated with emphysema, inflammatory responseand maintenance of protease-inhibitor equilibrium in vascular and peri-vascular spaces. A homogenouspreparation of heparin octasaccharide binds to ovine and human a-1-PI and enhances their proteaseinhibitory activity phenomenally. Size-exclusion chromatography and dynamic light scattering experi-ments reveal that ovine a-1-PI undergoes a decrease in the Stokes’ radius upon heparin binding. A strongbinding; characterizes this a-1-PI–heparin interaction as revealed by the binding constant (Ka)1.98 ± 0.2 � 10�6 M and 2.1 ± 0.2 � 10�6 M determined by fluorescence spectroscopy and equilibriumdialysis, respectively. The stoichiometry of heparin binding to ovine a-1-PI was 1.1 ± 0.2:1. The Stern–Volmer constants (Ksv) for heparin activated ovine and human a-1-PI were found to be 5.13 � 10�6 Mand 5.67 � 10�6 M, respectively, significantly higher than the native inhibitors. FTIR and CD spectroscopyproject the systematic structural reorientations that a-1-PI undergoes upon heparin binding character-ized by a decrease in a-helical content and a concomitant increase in b-turn and random coil elements.It is likely that these conformational changes result in the movement of the a-1-PI reactive site loop intoan extended structure that is better poised to combat the cognate protease and accelerate the inhibition.

� 2008 Published by Elsevier B.V.

1. Introduction

a-1-Proteinase inhibitor (a-1-PI) being the key serine proteinaseinhibitor in mammalian blood stages a crucial role in maintainingthe protease-inhibitor equilibrium in the blood and extra-vascularfluids. Associated with emphysema [1], inflammatory response [2]and host-defense mechanisms [3], a-1-PI exhibits considerable se-quence homology with other major serpins like antithrombin (AT),heparin co-factor II, protein C inhibitor and antiplasmin [4]. Theprotease inhibitory prowess of AT, heparin co-factor II and proteinC inhibitor undergo a seismic transition in the presence of catalyticquantities of heparin [5], a highly negatively charged glycosamino-glycan (GAG) composed of alternating residues of D-glucosamineand a combination of L-iduronic acid and D-glucuronic acid. Hepa-rin interaction with serpins brings about significant conforma-tional changes in the protein and renders the reactive site moreaccessible to the cognate protease [6]. Thus serpins are regulatedby GAGs that help in their localized activation. Heparin acceleratesthe irrelevant rates of biological interaction to relevant rates,although the amount of protease that can be inhibited is not al-tered [7]. It is the primary choice anti-coagulant in the manage-ment and preclusion of thrombotic disorders.

Elsevier B.V.

: +91 821 2517233.om (L.R. Gowda).

The polyelectrolyte nature of heparin allows it to bind tonumerous plasma proteins like AT, fibrinogen, fibronectin-histi-dine rich glycoprotein, platelet factor IV and several serine prote-ases [8]. In addition heparin interacts with fibroblast growthfactor-10, acidic fibroblast growth factor and growth hormone[9–11]. Heparin binding proteins are known to possess clustersof basic residues that form sectors of high positive charge densityand craft electrostatic interactions with the sulfate and carboxylategroups of heparin [12]. A comparison of several heparin interactingproteins reveals either X-B-B-X or X-B-B-B-X to be the consensussequence, where B represents a basic residue [13] and thus estab-lishing specificity for binding.

We have recently demonstrated that the protease inhibitory po-tential of human and ovine a-1-PI is activated several fold in thepresence of a homogenous preparation of a heparin octasaccharide.The heparin binding site in a-1-PI is localized at the lysine rich(K154, K155 and K174) F-helix region of a-1-PI distinct from the D-he-lix binding region of AT and HC-II [14]. The heparin induced activa-tion of a-1-PI is sigmoid indicative of an allosteric activationmechanism. Coupled with this is the fact that heparin is localizedin lungs [15] and mast cells the primary sites of a-1-PI activity[16]. The heparin induced activation of AT is facilitated byconformational changes around the reactive center loop (RCL) ren-dering it more accessible to the cognate protease. Such subtle con-formational changes are observed among most serpin–heparin

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interactions; however, the course and extent of change vary to asignificant extent [17]. A detailed biophysical study of the hepa-rin–a-1-PI-interaction would provide some insights into the struc-tural changes induced by such binding and provide an explanationfor the activation mechanism.

Molecular spectroscopic techniques such as dynamic light scat-tering (DLS), fluorescence, circular dichroism (CD) and Fouriertransform infra-red (FTIR) spectroscopy are potent biophysicaltools to understand protein–ligand interactions. Using these toolsthe present investigation was aimed at analyzing the binding prop-erties of heparin to ovine a-1-PI including structural reorientationsunder physiological pH to explain the activation phenomena. Thechanges in molecular dimensions of ovine a-1-PI upon heparinbinding, binding constants, stoichiometry of binding and perturba-tions in the secondary structural features associated with thisinteraction are illustrated. The binding of the octasaccharide hepa-rin to a-1-PI brings about significant changes in the secondarystructure of a-1-PI, which increases the accessibility of the RCL tothe cognate protease enhancing the inhibitory potency.

2. Materials and methods

2.1. Materials

Ovine blood was obtained from the local abattoir. Analytical gelfiltration markers were procured from Sigma–Aldrich, St. Louis,MO, USA. Low molecular weight heparin (Mr � 2530.2) was ob-tained from Hi-Media, Mumbai, India. All the other chemicals usedwere of highest purity available commercially.

2.2. Purification of ovine a-1-PI

a-1-PI was purified to homogeneity as described earlier [18] bysequentially employing (NH4)2SO4 precipitation, blue sepharose,Sephadex-G-200 and concanavalin-A sepharose chromatography.The purified a-1-PI of Mr 58310.0 Da had a specific activity of�1100 trypsin inhibitory units/mg.

2.3. Protein estimation

The protein concentration was determined according to the dyebinding method [19] using bovine serum albumin (BSA) asstandard.

2.4. Determination of Stokes’ radii

Stokes’ radii measurements were computed by size-exclusionchromatography on a TSK gel G2000 SWXL (7.8 mm � 30 cm;5 lm) column, using a Waters HPLC system equipped with aWaters 2996-photodiode array detector. The column was equili-brated with 0.1 M NaPi buffer, pH 7.4, containing 0.9% NaCl at aflow rate of 0.5 mL min�1. Ovine a-1-PI (5 lM) equilibrated in thesame buffer with heparin (0–10 lM) was loaded and eluted inthe same buffer. The protein–ligand complex was detected at 230and 280 nm. BSA (33.9 Å), ovalbumin (30.2 Å), carbonic anhydrase(23.6 Å) and ribonuclease (19.3 Å) were used to calibrate the col-umn. Blue dextran was used to determine the void volume.

Dynamic light scattering (DLS) measurements were performedto determine the size of heparin–a-1-PI complex and confirm thesize-exclusion results. Stokes’ radii measurements of a-1-PI(1 mg/mL in 0.1 M sodium phosphate buffer, pH 7.4, containing0.9% NaCl) in different concentrations of heparin (0–10 lM) wereevaluated on a Viscotek 802 DLS instrument with a thermostatedcell holder. All solutions for the experiments were prepared usingMilli-Q water. The experiments were performed at 90� scatteringangle at 4 ± 0.1 �C. Each spectrum obtained was an average of 10

scans and analyzed by OmniSize 3.0 software. The Stokes’ radiuswas obtained from the Stokes’-Einstein equation RS = kT/(6pg0D),where k is Boltzmann’s constant, T the temperature (K), g0 the sol-vent viscosity and D the translational diffusion coefficient.

2.5. Equilibrium dialysis

Aliquots (1.5 mL) of protein solution (20 lM) in 0.05 M Tris–HClbuffer, pH 7.4, containing 0.9% NaCl was continuously dialyzed for24 h at 4 �C against 30 mL buffer containing heparin (0–100 lM).Corresponding ‘blanks’ containing only buffers were maintained.The concentration of heparin in the dialysate was estimated bythe method of Farndale et al. [20]. The presence of protein didnot permit the estimation of heparin in the retentate. From the ob-served difference in heparin concentration between ‘blank’ andexperimental values, the number of heparin molecules bound permole of protein was calculated. The experiment was carried induplicate with varying protein concentrations. The data were ana-lyzed by Scatchard plot.

2.6. Fluorescence studies

Fluorescence measurements were recorded on a Shimadzu RF5000 spectrofluorimeter using a 10 mm path length quartz cell at27 ± 0.5 �C in 0.05 M Tris–HCl, pH 7.4, equipped with a stir control.Trp perturbations both in the absence and in the presence of 0–5 lM heparin were followed at 295 nm and the emission spectrarecorded between 300 and 400 nm. Excitation and emission slitwidths were set at 5 nm each. Appropriate blanks were used forbase line correction of fluorescence intensity. Fluorescencequenching by acrylamide was analyzed using the Stern–Volmerand Lehrer equation (Fo/F = 1 + Ksv [Q]) where [Q] is the quencherconcentration and Ksv the dynamic quenching constant.

Binding of heparin to ovine a-1-PI (10 lM) was analyzed byfluorescence titrations with aliquots of heparin solution (40 lMstock) in 0.1 M Tris–HCl buffer, pH 7.4, with fluorescence detectionat kex 280 nm and kem 335 nm. The data was analyzed using theScatchard equation,

B=L � ET ¼ n=KD � B=KD � ET;

where L, B, ET, n and KD represent free heparin, bound heparin,a-1-PI concentration, number of binding sites and the dissociationconstant for heparin–a-1-PI complex, respectively. The bound hep-arin was calculated from the decrease in fluorescence induced byheparin binding

B ¼ DF=DFmax � ET

The value of Fmax was deduced by extrapolating the double re-ciprocal plot of total heparin concentration versus fluorescence de-crease [21]. All the data points plotted are an average of threeexperimental values.

2.7. Secondary structure estimation and data analysis

Ovine a-1-PI (4 mg/mL) was incubated with heparin (0.1–10 lM) in 20 mM NaPi buffer, pH 7.4, and lyophilised. Each lyophi-lised protein–ligand complex was mixed with dry finely powderedKBr and pressed into a disc. FTIR spectra for these samples wereobtained to detect conformational changes in the a-1-PI structure,on a Nicolet 5700 FTIR spectrometer, equipped with a DTGS detec-tor. During measurements, the spectrometer was continuouslypurged with dry air to eliminate water vapor absorptions fromthe spectral regions of interest. Each spectrum was a result of sig-nal averaging of 128 scans at a resolution of 2 cm�1 at 25 ± 0.5 �C.Second derivative spectra were obtained using Savitzky–Golayderivative function [22] software with a five data point window.

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Iterative curve-fitting method to achieve the best Gaussian shapedcurves was used to calculate the relative areas of various compo-nents of the fitted band. The assignment of the composite bandsof amide III region were achieved using Fourier self-deconvolutionwith an enhancement factor of K = 1.8 and a half width of30.2 cm�1. The free protein IR spectrum was obtained by subtract-ing the spectrum of the heparin-buffer control. The percentage ofeach secondary element was calculated from the integrated areasof the component bands in amide III. The values given are an aver-age of three independent experiments (error, m ± 1 cm�1;area ± 5%). BSA, lysozyme, a-chymotrypsin, concanavalin-A andhemoglobin were used as standards and the experimental second-ary structural content for these corresponded well with the re-ported values [23].

CD spectra were collected with a JASCO J-500A (Japan Spectro-scopic Co., Tokyo, Japan) automatic recording spectropolarimeterequipped with 500 N data processor, xenon lamp and a uniformtemperature cell holder. Dry nitrogen was purged continuously be-fore and during the experiment. The instrument was calibratedusing ammonium (+)-10-camphorsulfonate. Conformationalchanges in the secondary structure of the protein (3.5 lM) in50 mM Tris–HCl buffer, pH 7.4, at 27 �C were monitored in thefar-UV region between 200 and 250 nm in the cuvette with a pathlength of 1 mm. After appropriate buffer and heparin blanks wererun and subtracted, mean residue ellipticities were calculatedusing equation [h] = hobsMRW/cl, where hobs is the observed ellip-ticity in degrees, MRW is the mean residue weight, c is the concen-tration of protein (mg/mL) and l is the path length in centimeters[24]. A mean residue molecular weight of 110 and sensitivity of1 m�/cm (millidegree per centimeter) was used with a scan speedof 10 nm/min using a bandwidth of 1 nm. An average of three runswas recorded for each sample. The secondary structural changeswere analyzed by the method of Yang et al. [25].

3. Results

3.1. Stokes’ radius determinations

Size-exclusion chromatography and DLS were performed todetermine the change in compactness of ovine a-1-PI upon bindingto heparin. Size-exclusion chromatography has been used to detectconformational changes in several proteins as a function of shift in

Fig. 1. Determination of Stokes’ radius of ovine a-1-PI by size-exclusion chromatography.incubated with 0, 1, 2, 5 and 10 lM of heparin, respectively, in 0.1 M Tris–HCl, pH 7.4.carbonic anhydrase (23.6 Å), ovalbumin (30.2 Å) and BSA (33.9 Å).

Stokes’ radii of the proteins [26]. The Stokes’ radius of native andheparin complexed a-1-PI were determined by the method ofUversky [27]. A set of proteins of known Mr and Stokes’ radii (RS)were used to construct the calibration curve (Fig. 1, inset). Theeffect of different heparin concentrations on the moleculardimensions of ovine a-1-PI as evaluated by size-exclusion chroma-tography is shown in Fig. 1. Native a-1-PI eluted as a single sym-metrical peak with a retention time of 13.31 min. In contrast, theheparin activated forms eluted much later. This increase in theretention time of the heparin complexed a-1-PIs as compared withnative a-1-PI could be due to formation of a more compact confor-mation and a decline in the hydrodynamic radii. The hydrody-namic radius of native ovine a-1-PI (33.5 ± 0.5 Å), decreased inthe presence of 10 lM heparin (30.7 ± 0.5 Å). Consistent with thesize-exclusion chromatography results, DLS measurements ofa-1-PI upon titration with heparin showed a steep decline in RS

over the concentration, 0.2–4 lM heparin, beyond which itappeared to remain constant (Fig. 2). This observed decrease inRS could be due to a compaction of a-1-PI induced by the bindingof the charged oligosaccharide chain of heparin. Light scatteringmeasurements carried out at 325 and 360 nm revealed a marginaldecrease in the absorbance as heparin concentration increasedindicating that no aggregation occurred.

3.2. Equilibrium dialysis

The heparin binding constant was determined by incubating a-1-PI (10 lM) with various concentrations of heparin for 24 h at4 �C. To obtain the number of heparin binding sites, saturation ofthese sites on a-1-PI is required. The number of heparin moleculesbound per mole of protein (m) was plotted against free heparin con-centrations [L]. Saturation of ovine a-1-PI occurred at 15 lM hepa-rin (Fig. 3). A Scatchard plot of the data reveals a single highaffinity-binding site for heparin. The binding constant (Ka) deter-mined from the slope of the least square regression line of the Scat-chard plot is 2.1 ± 0.2 � 10�6 M (Fig. 3).

3.3. Intrinsic fluorescence studies

To characterize the mechanism by which heparin accelerates a-1-PI–protease reaction, binding of the heparin–octasaccharide toa-1-PI was monitored by fluorescence measurements. The binding

1 (33.5 Å), 2 (33.3 Å), 3 (32.8 Å), 4 (31.4 Å) and 5 (30.7 Å) represent ovine a-1-PI pre-(Inset) Calibration curve for Stokes’ radius constructed using ribonuclease (19.3 ÅA

0

),

Fig. 2. Effect of heparin concentration on the apparent Stokes’ radii of a-1-PI me-asured by dynamic light scattering.

Fig. 3. Langmuir isotherm for determining Ka of heparin-a-1-PI interaction by eq-uilibrium dialysis. A plot of m (moles of heparin bound to a-1-PI) vs [L] free heparinconcentration. (Inset) Scatchard plot showing the relationship between m/[L] vs m.

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of heparin is accompanied by fluorescence quenching (Fig. 4A).Fig. 4A shows a relative quenching of fluorescence intensity, whichincreased with increasing heparin concentration. Heparin inducedquenching did not lead to any changes in band shape or emissionwavelength (Fig. 4A). The decrease in the fluorescence inducedby heparin enabled us to titrate a-1-PI with heparin. A maximumquench of 45% was observed at 5 lM of heparin. A saturable79 ± 3% quenching in the protein fluorescence reaction was de-duced from the double reciprocal plot (Fig. 4B, inset). Assumingthat heparin has ‘n’ independent binding sites on ovine a-1-PI,the binding constant and ‘n’ were determined from the non-linearregression curve (Fig. 4C). The dissociation constant (KD) for hepa-rin was 4.7 ± 1 � 10�6 M. The number of binding sites calculatedfrom the Scatchard analysis is 1.1 ± 0.2: 1 at lower concentrationsof heparin. The binding mode may be more complex and non-spe-cific at concentration beyond 1.8 ± 0.2 lM of heparin as seen fromtwo different slopes (Fig. 4C). The binding constant (Ka) given bythe slope of mass action plot is 1.98 ± 0.2 � 10�6 M (Fig. 4D).

The florescence spectrum of ovine a-1-PI shows maximal emis-sion at 336 nm suggesting that the Trp residues are buried in a pre-dominantly hydrophobic milieu shielded from the solvent.Collisional quenching experiments with acrylamide were per-formed to assess the accessibility of Trp residues and correspond-ing Stern–Volmer constants (Ksv) calculated. A higher Ksv of5.13 ± 0.2 � 10�6 M and 6.67 ± 0.2 � 10�6 M for heparin activatedovine and human a-1-PI were observed compared to that of3.25 ± 0.2 � 10�6 M and 3.76 ± 0.2 � 10�6 M for native inhibitors,respectively. These results indicate that the milieu around Trp ina-1-PI is altered upon heparin binding.

3.4. FTIR spectroscopy and data analysis

FTIR spectroscopy is a useful tool for analysis of the secondarystructures of the proteins and is particularly suitable for monitor-ing subtle conformational changes occurring within proteins [28].The amide III spectral region was used to examine the effect ofheparin binding on ovine a-1-PI secondary structure. The spectralbands corresponding to 1330–1295 cm�1 are assigned to a-helix,1295–1270 cm�1 to b-turns, 1270–1250 cm�1 to random coilsand 1250–1220 cm�1 to b-sheets, respectively [23,29,30]. Theamide I spectral region (1700–1600 cm�1) although commonlyused in secondary structural analysis suffers from several limita-tions. Cai and Singh [23] using the relatively weak signals of theamide III spectral region (1350–1200 cm�1) demonstrate that theamide III bands are more suitable for quantitative analysis of pro-tein secondary structure. Therefore, the FTIR spectra of the amideIII region were chosen to study changes in the secondary structureof ovine a-1-PI. The amide III spectral analysis used to estimatesecondary structural elements of the protein standards correlatedwell with the reported CD and X-ray data indicating the validityof the method [31,32]. Fig. 5 shows the FTIR spectra in the amideIII region of free a-1-PI and a-1-PI complexed with heparin. Inthe presence of heparin the area under bands (band strength)above 1295 cm�1 decreased and shifted to the lower wave number(Fig. 5B). Second derivative and Gaussian curve-fitting techniqueswere used to identify the overlapping sub-bands of the variouscomponents of protein secondary structures in the amide III region(1350–1200 cm�1) (Fig. 5A and B). The strength of bands near1270–1250 cm�1 increased consistent with an increase in randomcoil. The decrease in the band strength above 1295 cm�1 (Fig. 5)was dramatic suggesting of a-helix being very sensitive to heparinbinding. The percentages of each secondary structural element cal-culated from the integrated areas of the component bands in amideIII of ovine a-1-PI revealed 38.8%, 27.6%, 22.1% and 11.4% of a-helix,b-sheet, random coil and b-turn, respectively. FTIR analysis ofovine a-1-PI–heparin complex revealed a sharp decline in the a-helical content from 38.8% to 21.8% while b-sheet content de-creased marginally from 27.6% to 23.6% (Fig. 5C). A correspondingincrease in the random coil from 22.1% to 36.8% and b-turn from11.4% to 17.8% was observed. At saturating heparin concentrationsthere appears to be a general perturbation of protein structurewith no specific co-relation between various secondary structuralelements.

3.5. Circular dichroism (CD) studies

CD spectroscopy was also used to analyze and confirm the FTIRchanges in secondary structure upon heparin binding. The far-UVCD spectrum of proteins is particularly sensitive to secondarystructure and therefore useful in studying conformational changes.The far-UV CD spectra of ovine a-1-PI and complexed with heparinare shown (Fig. 6A). The spectra show strong negative ellipticitiesat 208, 215 and 222 nm suggesting that a-1-PI in the native state iscomposed of both a-helix and b-sheet rich regions. An increase in

Fig. 4. (A) Fluorescence emission spectra of ovine a-1-PI in the presence of 0, 0.2, 0.4, 0.6, 1, 1.5, 2, 2.5, 3, 4 and 5 lM from top to bottom. (B) Titrations of ovine a-1-PI withheparin. (Inset) Double reciprocal plot of this data. (C) Scatchard plot of the data in (B). (D) Mass action plot of data in (B) to calculate Ka for heparin binding [51].

Fig. 5. Curve-fitting analysis of FTIR spectra of ovine a-1-PI in amide III region. (A) Native ovine a-1-PI. (B) Ovine a-1-PI in presence of 2 lM heparin. (C) Plot depictingsecondary structural variations of ovine a-1-PI in response to different concentrations of heparin; (-d-) a-helix, (-h-) b-sheet, (-O-) random coil and (-j-) b-turns.

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heparin concentration results in a decline in the negative elliptici-ties of the ordered structures and an increase in random disorderedstructure (Fig. 6B). A quantitative analysis of the protein secondary

structure for free a-1-PI showed a-helix (48%), b-sheet (25%), b-turn (9%) and aperiodic (18%). Upon heparin binding the a-helicalstructures were reduced from 48% to 34%, b-sheet from 25% to

Fig. 6. (A) Far-UV CD spectra depicting heparin dependent conformational changesof ovine a-1-PI. 1, 2, 3, 4 and 5 correspond to spectra for native a-1-PI , a-1-PI + 1 lMheparin, a-1-PI + 2 lM heparin, buffer and heparin blank, respectively. (B) Plot de-picting secondary structural variations of ovine a-1-PI in response to different co-ncentrations of heparin; (-d-) a-helix, (-h-) b-sheet, (-O-) random coil and (-j-) b-turns.

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22%. Concomitantly the b-turn and random structures increased(Fig. 6B). These results are consistent with a similar decrease ina-helical content observed by FTIR (Fig. 5B). The reduction of a-helices and b-sheet in favor of random structure indicative of par-tial unfolding of protein in the presence of heparin.

4. Discussion

Heparin has an astounding ability to bind to an array of pro-teins, most prominent of them being serpins, especially AT. Hepa-rin binding to AT, heparin co-factor-II (HC-II), protease nexin-1,plasminogen activator inhibitor-1 (PAI-1) and protein C inhibitor(PC-1) enhances their biological activity several fold [33–37]. HC-II, PAI-1 and protease nexin-1 interact with heparin through theD-helix, while AT–heparin interaction additionally involves therole of A-helix and certain other basic residues [38]. Heparin bindsPC-1 at helices A and H [39] indicating that heparin binding sitesdiffer from serpin to serpin. Further heparin binds to a variety ofproteases like thrombin, trypsin, acrosin, activated protein C andfactor Xa [17]. This binding is mediated primarily by ionic interac-tions between the carboxylate and sulfate groups of heparin andbasic side chains of surface Lys and Arg residues [6]. Heparin

binding proteins exhibit a positively charged domain or clusteron the surface irrespective of the overall charge distribution. It isthrough such domains that the protein interacts with the GAGs.Heparin binding to proteins is correlated with directing proteinsto their sites of activity, regulating their inhibitory specificity andthe activation [40].

a-1-PI is the major circulating serpin responsible for mainte-nance of protease-inhibitor equilibrium in the physiological sys-tem and the regulation of several intra and extra-cellularpathways [17]. The interaction of a-1-PI and various physiologicalligands, has not been investigated in detail. To our knowledge theonly other study on a-1-PI–ligand interaction is the increased con-formational stability of a-1-PI in the presence of citrate [41]. Wehave recently demonstrated that a homogenous heparin octasac-charide binds to human and ovine a-1-PI at helix-F and activatesit several fold. Using chemical modifications and peptide mappingin conjunction with molecular modeling of human a-1-PI boundheparin, the heparin binding site has been identified as a clusterof Lys residues on helix-F which lies on the protein surface [14].The helix-F lies across the front of b-sheet A and plays an impor-tant role in regulating the opening of the sheet and in stabilizingthe five-strand conformation [42]. The reactive site of serpins is lo-cated on a mobile peptide loop, which has the flexibility necessaryfor complex formation. This flexibility is responsible for the modu-lation of activity necessary for the optimal locked conformationthat stabilizes the final serpin–protease complex, which might beresponsible for the activation mechanism. This increased accessi-bility of the RCL might explain the observed 45-fold activation ofa-1-PI inhibitory activity upon heparin binding [14]. The data pre-sented here illustrates that heparin binding induces secondarystructural changes leading to a compact conformation of a-1-PIrendering the RCL more accessible to the cognate protease. Manyof the questions related to biological macromolecules are con-nected with the shapes of molecules and how they interact witheach other. Heparin binding leads to an 8.4% decrease in the RS ofa-1-PI (Fig. 2). Additionally, the increased elution volume of ovinea-1-PI in the presence of heparin supports the decrease in RS

(Fig. 1). The declining RS with increasing heparin concentrationsindicates that heparin binding leads to a molecular compactiondue to structural changes. The light scattering measurements at325 and 360 nm indicate that these changes were not due toaggregation.

Using fluorescence spectroscopy an extremely sensitive tech-nique that is used to follow both protein folding and bindingevents we have studied the structural changes induced upon hep-arin binding to explain the activation phenomenon. The structuralchanges induced in a-1-PI was followed both at 295 and 282 nmwith respect to general perturbations in protein structure. Thestrong quenching of intrinsic fluorescence (Fig. 4A) clearly indi-cated that the binding of heparin to a-1-PI changed the micro-envi-ronment of a Trp residue and the secondary structure of a-1-PI.This conclusion is confirmed by both FTIR and CD studies (Figs. 5and 6). This quenching is in direct contrast to the enhancementof intrinsic fluorescence in the case of heparin–AT interaction[43]. Therefore, the structural changes upon heparin binding differbetween the two serpins. The binding constants as revealed byboth fluorescence measurements and equilibrium dialysis were1.98 ± 0.2 � 10�6 M and 2.1 ± 0.2 � 10�6 M, respectively, and aresimilar to heparin–AT binding [43].

Stern–Volmer constants for heparin activated a-1-PI increasewith increasing heparin concentrations, suggesting that the milieuaround Trp in a-1-PI is altered upon heparin binding either due to aconformational change or spatial perturbation of the surface Trpresidues. Trp192 (Trp194 in human a-1-PI) is one of the most con-served residues within the serpin superfamily being present in94% of all known serpins [44]. Trp192 is situated at the top of strand

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5A and is oriented towards the hydrophobic core of a-1-PI, a regionknown as the ‘‘breach” which plays a vital role in the initial eventsof RCL insertion. Therefore it is most probable that the milieuaround Trp192 is altered. Although quenching reflects changes inthe Trp environment it need not necessarily be so. It is possiblethat Lys residues of the F-helix (binding site of heparin) influencefluorescence intensity without affecting the emission maxima(Fig. 4A). In case of AT, heparin binding to D-helix results in expo-sure of buried Trp residues and hence fluorescence enhancement[43].

FTIR spectra analyses of the amide III region of native and hep-arin bound a-1-PI unambiguously indicate that heparin bindingcauses a global alteration in protein secondary structural parame-ters. Despite the weaker signals of amide III region when comparedto amide I and II, it has emerged as a promising and routinely usedband to estimate protein secondary structure [30,45,46]. It lacksdisturbance by the absorbance of H2O vapor and is highly sensitiveto secondary structure changes [47,48]. Both the a-helix and b-sheet content undergo a gradual decline with a concomitant in-crease in random coil and b-turn content when heparin binds toa-1-PI (Fig. 5). Heparin binding results in a decrease of a-helixand b-sheet content by 17% and 4%, respectively. This decrease ina-helix and b-sheet is concurrent with an increase of b-turn andrandom coil content of the molecule by 6.4% and 14.7%, respec-tively, as the heparin concentration is increased from 0 to 2 lM.At higher concentrations of heparin, a-1-PI shows a general pertur-bation in the secondary structural content and this may be attrib-uted to a weaker binding of heparin as revealed by the secondslope of the Scatchard plot (Fig. 4C). These results are consistentwith the CD data (Fig. 6) which also shows a decrease in the helicalcontent. The differences in the a-helical content between FTIR andCD spectroscopy can be explained by the differences in samplepreparation. The decrease in a-helical content can be extrapolatedto the presumption that the RCL of a-1-PI which is a protruded coilstructure becomes more exposed compared to the native formsand hence the activation. This conformational change may belinked to and implicated in the heparin activation of physiologi-cally important serpins.

It is possible that the interaction of heparin with a-1-PI is func-tionally similar to that of AT, yet different in the structural changescaused. The solvent accessible surface area for a cluster of basicresidues is quite high for both AT and a-1-PI [14]. In addition toincreasing the inhibitory properties of a-1-PI and AT, heparin alsointeracts with other proteins and regulates their physiologicalactivities and stability. The physical stability of human fibroblastgrowth factor 1 (FGF1), unstable at physiological temperatures ismarkedly increased in the presence of sucrose octasulfate [9].The binding of heparin sulfate to both FGF and its receptor (FGFR)facilitates the formation of a FGF/FGFR/heparin complex essentialfor cell signaling [49]. Heparan sulfate proteoglycans interactselectively with chemokines and modulate receptor binding andcellular responses [50]. Dextran sulfate and heparin markedly inhi-bit the aggregation of bovine and porcine somatotropin [9], thusincreasing protein stability.

5. Conclusions

In this study we have examined the conformational changes ina-1-PI that take place on heparin binding. The experimental ap-proach employing FTIR clearly shows that the binding of heparinto the a-1-PI induces a series of secondary structural changes thatlead to an increased random coil content of the protein and a de-crease in a-helix and b-sheet content. Stokes’ radius measurementsand fluorescence spectra analysis further confirm the structuralreorientations. It is likely that these conformational changes result

in the movement of the RCL into an extended structure that ispoised to combat the cognate protease. a-1-PI like AT becomes amore potent inhibitor when the RCL is expelled from the proteinand assumes a conformation more suitable for interaction withthe target protease. The crystal structure of a-1-PI–heparin com-plex when resolved will help ascertain the above observations.

Acknowledgements

We thank Dr. V. Prakash, Director, CFTRI, Mysore, and Dr. A.G.Appu Rao, Head, PCT, for their advice and useful discussions. Wethank Ms. Asha M. for technical assistance. Vivek Kumar Guptaacknowledges a Senior Research Fellowship from CSIR, India. Thiswork was carried out under CSIR-Networking Project CMM-004.

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