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Toxicon 58 (2011) 8–17

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Toxicon

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Cys mutants in functional regions of Sticholysin I clarify the participationof these residues in pore formation

A. Valle a, A. López-Castilla a, L. Pedrera a, D. Martínez a, M. Tejuca a, J. Campos b, R. Fando b,E. Lissi c, C. Álvarez a, M.E. Lanio a, F. Pazos a,*, S. Schreier d

aCenter for Protein Studies, University of Havana, Vedado, Ciudad de la Habana 2400, CubabNational Center for Scientific Research, CubacChemistry and Biology Faculty, Santiago de Chile University, Chiled Institute of Chemistry, University of São Paulo, Brazil

a r t i c l e i n f o

Article history:Received 23 December 2010Received in revised form 1 April 2011Accepted 5 April 2011Available online 13 April 2011

Keywords:Pore-forming toxinActinoporinSticholysinsProtein–membrane interaction CDFluorescenceMembrane permeability

* Corresponding author. Tel./fax: þ5378321321.E-mail address: fpazos@fbio.uh.cu (F. Pazos).

0041-0101/$ – see front matter � 2011 Elsevier Ltddoi:10.1016/j.toxicon.2011.04.005

a b s t r a c t

Experimental evidence shows that the mechanism of pore formation by actinoporins isa multistep process, involving binding of the water-soluble monomer to the membrane andsubsequent oligomerization on the membrane surface, leading to the formation of a func-tional pore. However, as for other eukaryotic pore-forming toxins, themolecular details of themechanism of membrane insertion and oligomerization are not clear. In order to obtainfurther insightwith regard to the structure-function relationship in sticholysins, we designedandproduced three cysteinemutants of recombinant sticholysin I (rStI) in relevant functionalregions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StIR52C (in the membrane binding site). The conformational characterization derived fromfluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests thatreplacementof these residues byCys in rStI did not noticeably change the conformation of theprotein. The substitution by Cys of Arg52 in the phosphocholine-binding site, provokednoticeable changes in rStI permeabilizing activity; however, the substitutions in theN-terminal region (Glu2, Phe15) did not modify the toxin’s permeabilizing ability. The pres-ence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showedhigher pore-forming activity thanwhen theprotein is in themonomeric state, suggesting thatsticholysins pre-ensembled at the N-terminal region could facilitate pore formation.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Thepore-forming actinoporin Sticholysin I (St I) is a 20kDacysteineless basic protein purified from the sea anemoneStichodactyla helianthus which displays hemolytic activity atrather low concentrations (Lanio et al., 2001). The toxinincreases ion membrane permeability by forming a channelresulting from the association of three or four toxin mono-mers (Tejuca et al.,1996). The three-dimensional structures ofthree actinoporins, equinatoxin II (Eqt II) from Actinia equina(Athanasiadis et al., 2001; Hinds et al., 2002), St I (Castrillo

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et al., 2009) and Sticholysin II (St II) (Mancheño et al., 2003),an isoform of St I (Lanio et al., 2001), have been solved. Allthese actinporins display a very similar b-sandwich foldflanked on each side by two short a-helices.

Actinoporins are characterized by a high affinity forsphingomyelin (SM), a sphingophospholipid containing thephosphorylcholine moiety, also present in phosphatidylcho-line (PC) (Bakrac et al., 2008). The structure of the complexbetween monomeric St II and phosphocholine revealed theexistence of a putative phospholipid-binding site (Mancheñoet al., 2003). As for other eukaryotic pore-forming toxins, themolecular details of the mechanism of pore formation byactinoporins are not clear. At present, experimental evidenceshows that the mechanism of pore formation is a multistep

Table 1Relative accessible molecular surface of the rStI amino acid selected formutation.

rStI Residues ASAa (%) in the rStIstructural model

Glu 2 85.5Phe 15 31.2Arg 52 50.7

ASA < 20%: buried; 20% � ASA �50%: partially buried and ASA > 50%:exposed (Gromiha et al., 1999).

a ASA: Accessible Surface Area calculated as the accessible molecularsurface of the amino acid (X) in rStI structural model relative to a Gly-X-Gly tri-peptide in vacuum (WATHIF: http://swift.cmbi.ru.nl/servers/html/index.html)

Table 2rStI sense and antisense oligonucleotide primers.

Mutantes oligos Sense oligonucleotides (s)

Antisense oligonucleotides (as)

StI-E2C NdeI-E2C s (50-GGG CAT ATG TCC TGC CTC GCT GGCACC ATT ATT GAT-30)

Ct-BamHI as (50-GGG GGA TCC TTA GCG TGA AAT CTTAAT TTG CAT-30)

StI-F15C T7s (50-GTA ATA GGA CTC ACT ATA GGG-30)F15C as (50-TTT GTC AAG GAC TTG GCA GGT TAG -30)F15C s (50-CTA ACC TGC CAA GTC CTT GAC AAA -30)Ct-BamHI as (50-GGG GGA TCC TTA GCG TGA AAT CTT

AAT TTG CAT-30)StI-R52C T7 s (50-GTA ATA GGA CTC ACT ATA GGG-30)

R52C as (50-GTC CGT AGT ACC AGA GCA GAA ATA-30)R52C s (50-TAT TTC TGC TCT GGT ACT ACG GAC -30)Ct-BamHI as (50-GGG GGA TCC TTA GCG TGA AAT CTT

AAT TTG CAT-30)

The codons in bold only or in bold and underlined indicate the restrictionsites by BamHI and NdeI and the mutation sites, respectively.

A. Valle et al. / Toxicon 58 (2011) 8–17 9

process, involving binding of the water-soluble monomer tothe membrane and subsequent oligomerization on themembrane–water interface, leading to the functional pore(Hong et al., 2002). The phosphocholine-binding site anda cluster of exposed aromatic residues, together with a basicregion, would be involved in the initial interaction with themembrane (Malovrhet al., 2000;Honget al., 2002;Mancheñoet al., 2003; Bakrac et al., 2008), whereas the N-terminalregion, containing hydrophobic and amphipathic sequencesas part of a more flexible segment, would be essential foroligomerization and pore formation (Belmonte et al., 1993;Tejuca et al., 1996; Malovrh et al., 2000; Hong et al., 2002;Mancheño et al., 2003; Gutiérrez-Aguirre et al., 2004;Kristan et al., 2004; Alegre-Cebollada et al., 2007).

In order to obtain further information concerning thestructure-function relationship in sticholysins and deter-mine in greater detail both the structural importance andthe position of specific amino acid residues involved ininteraction and insertion in the membrane, we designedand produced three mutants of recombinant St I (rStI)containing Cys in relevant functional regions of the protein:StI E2C and StI F15C (in the N-terminal region), and StI R52C(in the phosphocholine-binding site). CD and fluorescencedata suggested that replacement of Glu2, Phe15, andArg52 byCys residues in rStI did not noticeably change the confor-mation of the protein. In addition, the substitution of Arg52

by Cys in the binding region caused more pronouncedchanges in rStI pore-forming activity than substitution ofGlu2 and Phe15 in the N-terminal region. StI E2C exhibitedthe highest permeabilization capacity in non-reducingconditions, when part of the –SH groups are oxidized,suggesting that the presence of pre-existing dimers stabi-lized by disulfide bonds in this region could favor poreformation in the membrane.

2. Materials and methods

2.1. Design of mutants

Mutants were designed taking into account the solventexposure of each residue and their location in regions relevantfor function. In order to estimate the degree of exposure ofresidues in the rStI three-dimensional model (Pazos et al.,2006), we employed the WhatIf server (http://swift.cmbi.kun.nl/whatif/) to calculate the accessible surface area (ASA)as the accessiblemolecular surface of the amino acid (X) in therStI structural model relative to a Gly-X-Gly tri-peptide invacuum. Mutated residues are exposed in the order:Glu2 > Arg52 > Phe15 (Table 1). Since these residues arelocated in relevant functional regions (Glu2 and Phe15 in theN-terminal region, and Arg52 in the membrane binding site),they were replaced by Cys in order to allow for studies of theeffect of dimer formation, or, in further work, chemicalmodification such as the introduction of spin labels or fluo-rescent labels for EPR or fluorescence studies, respectively.

2.2. Expression and purification of mutants

StI E2C, StI F15C, and StI R52C were obtained byreplacing the corresponding wild-type amino acid by Cys.Mutations were introduced by the PCR technique using as

a template the vector pET3a-rStI (Pazos et al., 2006). Senseand antisense oligonucleotide primers employed to intro-duce the mutations are shown in Table 2. PCR was per-formed as previously described (Pazos et al., 2006). The PCRproducts were restricted with NdeI and BamHI and clonedinto pET3a, where the nucleotide sequence of putativepositive clones was verified using a T7 Sequencing TM Kitas previously described (Pazos et al., 2006). The con-structed vectors pET3a-StI E2C, pET3a-StI F15C and pET3a-StI R52C were used to transform the E.coli BL21 (DE3) pLysSstrain, and expression of mutants was induced with 0.2%lactose. Purification of the mutants and rStI was carried outfrom supernatants of lysed bacteria using ion-exchangechromatography on carboxymethyl cellulose (CM-52) aspreviously described (Pazos et al., 2006). For the mutants,an intermediate washing step with 100 mM b-mercaptoe-thanol was included before the elution gradient in order toeliminate anymolecule eventually linked to the Cys residue(Kim and Levine, 2005). The homogeneity of the proteinswas verified by SDS-PAGE (Laemmli, 1970) and HPLC ona Vydac reverse phase column RP-C4. Protein concentrationwas determined by measuring the absorbance at 280 nmand using the extinction coefficient of St I (Lanio et al.,2001). Protein molecular mass was determined by ESI-MSin a JEOL JMS-HX110/110A spectrometer (JEOL, Peabody,MA, USA) equipped with an ESI double focusing interface,as described by Lanio et al. (2001).

A. Valle et al. / Toxicon 58 (2011) 8–1710

2.3. Hemolytic activity (HA) assays

HA was determined turbidimetrically at 650 nm witha microplate reader (Multiskan EX, Labsystems) as previ-ously described (Martínez et al., 2001). Human red bloodcells (HRBC) were prepared from fresh citrated blood bywashing three times (700� g for 10min) and resuspendingin saline buffer: 145mMNaCl,10mMTris–HCl, pH7.4. HRBCconcentration was adjusted with buffer to an apparentabsorbance of 0.1 at 650 nm. Protein samples were two-foldserially diluted in saline buffer using flat-bottom 96-wellmicroplates, and a volume of HRBC was added to each well(200 ml final volume). From the results, we calculated HC50,the protein concentration that causes 50% lysis.

2.4. Preparation of vesicles

Large unilamellar vesicles (LUV) were prepared byextruding multilamellar liposomes of 1-palmitoyl-2-oleylphosphatidylcholine (POPC) and sphingomyelin (SM) frombovine brain (both from Avanti Polar Lipids, Alabaster, AL,USA) at 85:15 M ratio in the presence of 80 mM carboxy-fluorescein (CF) at pH 7.0, as described by Tejuca et al. (1996).Extrusion was carried out employing a two-syringe extruder(Liposofast Basic unit, from Avestin Inc., Ottawa, Canada),equippedwith two stackedpolycarbonatefilterswithpores of100 nm average diameter (Nucleopore, Plesanton, USA). Non-encapsulatedfluorescent probewas removed from the vesiclesuspension by a Sephadex G-50 gel filtration column underisosmotic conditions. Small unilamellar vesicles (SUVs) wereprepared by sonication (Branson 450) of 85:15 POPC:SMmultilamellar vesicles, as previously described (Pazos et al.,1998). The vesicles final lipid concentration was determinedaccording to Rouser et al. (1970).

2.5. Intrinsic fluorescence measurements

Fluorescence spectra were recorded in a HitachiF-4500 spectrofluorimeter using 1 cm path length quartzcuvettes. Slit widths of 5 nm nominal band pass were usedboth in the excitation and emission beams. Intrinsic fluo-rescence emission spectra of St I mutants and rStI (1 mM) insolution or in the presence of 85:15 POC:SM SUV, wererecorded from 300 to 450 nm after excitation at 295 nm toobtain fluorescence spectra derived from Trp residues.Background intensities measured in samples withoutprotein were subtracted. Changes in the intrinsic fluores-cence of rStI and mutants upon addition of increasingquantities of SUV were also measured. Selective quenchingof the fluorescence of Trp residues located in differentmicroenvironments was achieved by adding increasingacrylamide concentrations in the absence or presence ofSUV (Lakowicz, 1999).

2.6. Permeabilization assays

Permeabilization was assayed by measuring the fluo-rescence of CF (Sigma) released from LUV according toTejuca et al. (1996). After adding the toxin to the vesicles,the release of CF produced a time-dependent fluorescenceincrease. Maximal release was obtained by adding Triton X-

100, which yielded Fmax. If a single pore in a vesicle isenough to (instantaneously) lead to the total leakage of theentrapped fluorophore (Tejuca et al., 1996; De los Ríos et al.,1998), the final extent of the fluorescence increase elicitedby addition of the toxin is readily related to the fraction (f)of vesicles in which at least one pore has been formed.

f ¼ �Ff � Fo=Fmax � Fo

� � 100 (1)

where Fo and Ff represent the fluorescence values beforeand after adding the toxin. In order to characterize thepermeabilizing activity of the proteins, C50, the proteinconcentration necessary to achieve 50% leakage, wasestimated.

2.7. Circular dichroism (CD)

Far-UV and Near-UV CD spectra of St I mutants andrStIwere recorded on a CD6 Jobin Yvon spectropolarimeter(Longjumeau, France) coupled to a Multiscan Computer(D&D Technology); spectra were baseline corrected byusing control samples of solutions not containing protein.Spectra in the far-UV and near-UV ranges were obtainedfrom 190 to 260 nm and 250–350 nm in 1 and 5 mm pathlength quartz cuvettes, respectively. The reported spectraare averages of 6 and 12 scans for far-UV and near-UV,respectively.

2.8. Surface pressure measurements

Surface pressure measurements were carried out usinga Micro-Trough-S system from Kibron (Helsinki, Finland) at25 �C with constant stirring. The aqueous subphase con-sisted of 0.3 ml ultra-pure milli-Q water. An 0.2 mM (totallipid) mixture of POPC (Avanti Polar Lipids, Alabaster, AL,USA) and SM (85:15 M ratio) was prepared in chloroform,and gently spread over the water until the desired initialsurface pressure was achieved. After attaining stabilizationof the signal, the protein was injected with a Hamiltonmicrosyringe to the subphase at different concentrations.The temporal course of surface pressure increase wasrecorded until a stable signal was obtained.

3. Results and discussion

The mechanism of pore formation by actinoporins is notcompletely understood. Different regions of the proteinstructure seem to play important roles in the interactionwith membranes. A phosphocholine-binding site anda cluster of exposed aromatic residues, togetherwith a basicregion, are thought to be involved in the initial interactionwith themembrane (Malovrh et al., 2000; Hong et al., 2002;Mancheño et al., 2003; Bakrac et al., 2008), whereas theN-terminal region, containing hydrophobic and amphi-pathic sequences as part of a more flexible segment, wouldbe essential for oligomerization and pore formation(Belmonte et al., 1993; Tejuca et al., 1996; Malovrh et al.,2000; Hong et al., 2002; Gutiérrez-Aguirre et al., 2004;Kristan et al., 2004; Alegre-Cebollada et al., 2007).

In order to obtain information at the molecular levelwith respect to the mechanism of pore formation and the

Fig. 2. Assessment of expression of rStI’s mutants by SDS-PAGE (15%)(Laemmli, 1970).The constructed vectors pET3a-StI E2C, pET3a-StI F15C, andpET3a-StI R52C were used to transform the E.coli BL21 (DE3) pLysS strain.Expression of the rStI mutants was induced with 0.2% lactose for 4 h: lane 1,non-induced cells with pET3a-StI E2C; lane 2, induced cells with pET3a-StIE2C; lane 3, non-induced cells with pET3a-StI F15C; lane 4, induced cellswith pET3a-StI F15C; lane 5, rStI purified (molecular weight w20,000 Da);lane 6, non-induced cells with pET3a-StI R52C and lane 7, induced cells withpET3a-StI R52C.

A. Valle et al. / Toxicon 58 (2011) 8–17 11

structure-function relationship of sticholysins, we havedesigned and produced three Cys mutants of rStI, takingadvantage of the fact that actinoporins are cysteinelessproteins (Huerta et al., 2001). The position of Cys residueswas selected considering their solvent exposure (Table 1),as well as their location in regions relevant for function: StIE2C and StI F15C (in the N-terminal region) and StI R52C (inthe phosphocholine-binding site) (Fig. 1). The introductionof Cys residues should allow specific binding of spin labelsor fluorescent labels and their use in studies of toxin–membrane interaction.

3.1. Expression and purification of St I mutants

Putative positive clones selected by restriction analysiswere sequenced to confirm the presence of desired Cysmutations in the construction and the integrity of the rest ofthe genes. The mutants were expressed both in the cell-soluble fraction and in inclusion bodies (Fig. 2). rStI, con-taining a Gln16 instead of Glu16, was purified according toPazos et al. (2006). The rStImutantswere similarly obtainedbut with an additional washing step with 100 mM b-mer-captoethanol during the ion-exchange chromatography.Fig. 3 illustrates a typical chromatographic profile obtainedduring the purification of StI R52C. Similar results wereobtained for the othermutants. The purity of the toxins washigher than 95% as estimated by SDS/PAGE and RP-HPLC,showing a behavior similar to that of rStI (inserts A and B,Fig. 3). The amount of protein produced was 3–4 mg/L ofculture broth both for the wild-type and for the mutants.

3.2. Intrinsic fluorescence emission and UV CD spectra of rStIand the mutants

Tryptophan emission spectra of rStI, StI E2C, StI F15C,and StI R52C in solution are shown in Fig. 4. The substitu-tion of each selected residue by Cys did not modify the

Fig. 1. Three-dimensional model of rStI (Pazos et al., 2006). The three aminoacids selected for mutation by cysteine are highlighted, E2 and F15 (locatedin the N-terminal sequence), and R52 (located in the phosphocholine-binding site). The Figure was generated by DeepView–Swiss-PdbViewerprogram, v. 3.7 (http://www.expasy.org/spdbv/).

average degree of Trp exposure to solvent since the fluo-rescence maximum wavelength and intensity were verysimilar to those of rStI (Fig. 4 and Table 3). The averagelocation of the Trp residues can also be estimated fromfluorescence quenching experiments employing acryl-amide as a soluble quencher. Stern–Volmer plots wereessentially linear, suggesting that the emitting residues canbe treated as an approximately homogeneous population(data not shown). Values of the Stern–Volmer constants(Ksv) obtained assuming linearity are given in Table 3, andsuggest a similar behavior of rStI and the mutants.

Typical far and near-UV CD spectra of rStI and themutants are shown in Fig. 5. The secondary structurecontent of the toxinswas estimated byanalysis of the far-UVCD spectra. The spectra of the wild-type and the mutantsare very similar, exhibiting a positive band centered at195 nm and a minimum around 217 nm, typical of proteinscontaining mainly b-sheet structure (Venyaminov andYang, 1996). Deconvolution of these spectra according tothe CONTIN (Provencher and Glockner, 1981; Van Stokkumet al., 1990) and SELCON3 (Sreerema and Woody, 1993;Sreerema et al., 1999) programs in the DICHROWEBInternet server (Lobley et al., 2002; Whitmore andWallace,2004, 2008) provide the contents of secondary structuregiven in Fig. 5A (inset). The structure of rStI and its mutantsconsists of predominantly b-sheet structure, showing thatmutation of the selected residues by Cys did not modifysignificantly the proteins secondary structure. The resultsare in agreement with data derived from FTIR (Menestrinaet al., 1999) and far-UV CD (Martínez et al., 2001; Álvarezet al., 2003; Pazos et al., 2006) studies of sticholysin I andthe homologous protein sticholysin II, and also with thethree-dimensional structures of St I and St II, which alsorevealed a high content of b-sheet structure (Castrillo et al.,2009; Mancheño et al., 2003).

Further characterization of the protein conformation canbe obtained by analysis of near-UV CD spectra, where themain bands originate from aromatic and Cys residues(Woody andDunker,1996). The actual shape andmagnitudeof the near-UV CD spectrum of a proteinwill depend on the

Fig. 3. Profile of ion-exchange chromatography of the StI R52C mutant on a carboxymethyl cellulose (CM-52) column. Purification was carried out fromsupernatant of lysed bacteria, as previously described (Pazos et al., 2006). An intermediate washing step with 100 mM b-mercaptoethanol was included beforethe elution gradient in order to eliminate any molecule linked to the Cys (Kim and Levine, 2005). Absorbance at 280 nm (OD280nm), (- and solid line); hemolyticactivity (- and dashed line) (mg of toxin estimated by rStI standard curve) and (solid line) NaCl concentration gradient. Insert A: SDS-PAGE 15% (Laemmli, 1970)lane 1, unbound fraction; lane 2, washed step #1; lane 3, washed step with 100 mM b-mercaptoethanol; lane 4, washed step #2; lane 5, rStI purified, lane 6,elution fraction. Insert B: HPLC of the elution fraction on a Vydac reverse phase column RP-C4, as previously described (Pazos et al., 2006). The homogeneity ofthe proteins was higher than 95% and the amount of protein produced was 3–4 mg per liter of culture broth both for wild-type and mutants.

300 320 340 360 380 400 420 440

0

100

200

300

400

500

600

700

800

flu

orescen

ce in

ten

sity

wavelength (nm)

Fig. 4. Tryptophan fluorescence spectra of rStI and its mutants. Spectra of1 mM toxin in 10 mM Tris–HCl buffer, pH 7.4, were recorded between300 and 450 nm after excitation at 295 nm, at 25 �C. Slit widths of nominalband pass of 5 nm were used both in the excitation and emission beams.Background intensities measured in samples without protein were sub-tracted. rStI (---); StI E2C (- - -); StI F15C (.. .. ..); StI R52C (- .. -).

A. Valle et al. / Toxicon 58 (2011) 8–1712

number of each type of aromatic amino acid present, theirmobility, the nature of the environment where they arelocated, and their spatial disposition in the protein (Kellyet al., 2005). Therefore, the position and intensity of thenear-UV bands can be used to follow changes in proteintertiary structure. Neither St I nor rStI contain Cys, thus theresulting signals arise from the aromatic amino acid micro-environments; however, StI E2C, StI F15C, and StI R52Ccontain an additional Cys residue that might modify theshape of the spectra. Interestingly, the mutants and rStIshowed similar spectra with the same main band positions(Fig. 5B).

Altogether, the fluorescence and CD studies suggest thatreplacement of Glu2, Phe15 and Arg52 by Cys does notsignificantly change the conformation of the protein.Nevertheless, a more detailed observation of near UV-CDspectra might suggest a slight difference in the microenvi-ronment of aromatic residues of StI R52C, as suggested bythe more pronounced negative bands at 266 nm and270nmwhen compared to the spectra of the other proteins.

3.3. Hemolytic activity of rStI and the mutants

The hemolytic activity of rStI and its mutants was eval-uated by following the decay of turbidity of the HRBC

Table 3Conformational and functional properties of the mutants.

rSt I St I E2C St IF15C St I R52C

lmax (nm)in solutionb

335.4 � 0.8 333.7 � 0.4 333.7 � 0.9 334.4 � 1.3

lmax (nm) inthe presenceof SUVa,b

329.9 � 2.2 329.3 � 0.6 328.3 � 1.1 330.6 � 1.1

Ksv (M�1) insolutionc

3.25 � 0.21 2.95 � 0.18 2.80 � 0.15 2.97 � 0.14

Ksv(M�1) inthe presenceof SUVa,c

2.10 � 0.05 1.80 � 0.20 1.80 � 0.09 2.40 � 0.40

HC50d 1.72 � 0.21 1.32 � 0.006 1.34 � 0.11 3.13 � 0.45pc (mN m�1)e 47.1 47 69.3 44.6me 0.63 0.51 0.27 0.57

The values in the Table are the result of 3–4 experimental determinations.a SUV comprised of POPC:SM (85:15 M ratio).b Fluorescence spectra in solution or in the presence of POC:SM (85:15)

SUV were recorded from 300 to 450 nm after excitation at 295 nm. Thewavelength of the emission maximum (lmax) was determined by spectralintegration.

c Selective quenching of the fluorescence emitted by Trp residueslocated in different microenvironments was achieved by addingincreasing acrylamide concentrations in the absence or presence of SUV.Stern–Volmer constants (Ksv) were calculated making use of the Stern–Volmer equation (Lakowicz, 1999).

d The hemolytic activity of rStI and its mutants was evaluated followingthe decay of turbidity of HRBC suspensions as a consequence of the loss ofcellular integrity after addition of the toxin. HC50 was estimated byplotting the percentage of hemolysis at the end of each assay (10 min) asa function of toxin concentration.

e The critical pressure (pc) and slope (m), were obtained by linear fit ofthe increase in pressure (Δp) as a function of po and extrapolating the lineto zero. pc corresponds to the pressure that must be applied to avoidincorporation of the toxin to the monolayer and is directly correlated withthe affinity of the toxin for the lipids in the monolayer (Brockman, 1999).

A. Valle et al. / Toxicon 58 (2011) 8–17 13

suspension as a consequence of the loss of cellular integrityupon addition of the toxin. We estimated HC50 by plottingthe percentage of hemolysis achieved at the end of eachassay (30min) as a function of toxin concentration (Table 3).The data show that StI E2C and StI F15C exhibited slightlyhigher hemolytic activity than rStI. Studies with EqT IIrevealed that itsN-terminus extends through thepore to thetrans sideof themembraneand itwasproposed that thefirstfive residues help to anchor the amphipathic helix,contributing to the stabilization of the final transmembranepore (Kristan et al., 2007). Considering these observations, itcould be speculated that the elimination of a negativecharge in StI E2C would favor the translocation of thissequence through the membrane. On the other hand,Alegre-Cebollada et al. (2004) reported that substitution ofPhe14 of St II, which is equivalent to position 15 in St I, byanother aromatic residue like Tyr did not modify its hemo-lytic activity. Interestingly in this work, the substitution ofPhe15 of St I by a relatively more polar Cys residue seems toenhance the functional role of the N-terminus in poreformation. As expected, StI R52C showed lower hemolyticactivity, suggesting an important role for the Arg52 residuein the functional capacity of the toxin.

3.4. Dimerization ability of rStI Cys mutants

Introduction of Cys residues in relatively exposed posi-tions (Table 2) in the structure of rStI could facilitate the

spontaneous dimerization by formation of disulfide bonds.SDS-PAGE showed that, under non-reducing conditions, StIE2C at concentrations of 8,12, 24 and 50 mMdimerizes by 3,12, 34 and 46%, respectively (Fig. 6). As expected, uponincubation with b-mercaptoethanol the protein remainsmonomeric (data non shown). StI F15C and StI R52C alsodimerize by about 3 and 30% at concentrations of 25 and60 mM, respectively. Furthermore, in StI W111C, the puta-tively exposed position Trp111 led to 90% dimerization, evenat low protein concentrations (not shown). In studies withthe homologous actinoporin EqT II, only three cysteinemutants (K77C, R126C and A179C) were found to sponta-neously form dimers (Anderluh et al., 1998). The differentability of these mutants to dimerize could result from thedifferent exposure of the Cys to the solvent and/ora different reactivity. StI E2C, where the Cys residueexhibits the highest exposure to the solvent (Table 2),experiences the largest dimerization (Fig. 6).

3.5. Binding of rStI and its mutants to model membranes

In order to obtain insight into the interaction of St I withmembranes, we studied the binding of rStI and its mutantsto lipid monolayers and bilayers of POPC:SM (85:15). Insome experiments, reducing conditions were employed toensure that the protein was in the monomeric state. Theresults were similar to those obtained under non-reducingconditions.

The increase in surface pressure elicited by the associ-ation of the toxin to previously formed lipid monolayerscan be employed to characterize its ability to interact withorganized lipids (Martínez et al., 2007). The increase insurface pressure associated to the presence of the toxinwasevaluated at several initial pressures (p0) of the lipidmonolayer keeping constant the monolayer area. Theincrease in surface pressure at equilibrium caused by rStIand the mutants as a function of the initial pressure isshown in Fig. 7. Δp decreased with increasing po for allproteins, because tighter lipid packing prevents insertion.A suitable parameter for characterization of the interactionis the critical pressure (pc), obtained by extrapolating toΔp ¼ 0 the linear fit obtained by plotting the increase inpressure (Δp) as a function of po. This parameter corre-sponds to the pressure that must be applied to avoidincorporation of the toxin and is directly related to theprotein affinity for the lipids in the monolayer (Brockman,1999). Table 3 shows the values obtained. The value ofpc ¼ 35 mN m�1 corresponds to the lateral pressure ofa typical biological membrane (Brockman, 1999). It hasbeen proposed that when pc is higher than this criticallimit, the protein not only associates to the monolayer, butpenetrates into it (Caaveiro et al., 2001). If pc is lower thanthis value, the association takes place without significantpenetration of the toxin into the monolayer (Gutiérrez-Aguirre et al., 2004). rStI, StI E2C and StI R52C showcurves with similar values of pc and slope (Fig. 7, Table 3),suggesting that their affinity for the monolayer and finalinserted state are similar. Interestingly, StI F15C exhibitedthe smallest slope with the highest pc value, suggestinga different insertion of this mutant into the POPC:SMmonolayer. Phe15 is located in the St I N-terminal a-helix

Fig. 5. Far (A) and near (B) UV CD spectra of rStI (---) and the mutants StIE2C (- - -), StI F15C (.. .. ..), and StI R52C (- .. -) in water, at 25 �C. Far and NearUV-CD spectra were recorded between 190 and 260 nm and between250 and 350 nm in 1 and 5 mm path length quartz cuvettes using 4 mM and16 mM toxin, respectively. The baseline was corrected by using similarlyprepared solutions devoid of protein. The reported spectra are the average of6 and 12 scans for far-UV and near-UV, respectively. The spectra wereconverted to molar ellipticity, [q], using molecular weights determined byESI-MS (Lanio et al., 2001): rStI, 19390 Da; StI E2C, 19364 Da; StI F15C,19346 Da, and StI R52C, 19337 Da. The inset shows the secondary structurecontent estimated by deconvolution of the CD spectra according to theContin and SELCON3 algorithms.

Fig. 6. Analysis of the dimerization ability of StI E2C by SDS-PAGE (15%).SDS-PAGE was performed as previously described (Laemmli, 1970), butwithout heating and under non-reducing conditions. Lane 1, 8 mM StI E2C inwater (3% of dimeric structure); lane 2, 12 mM StI E2C in water (12% ofdimeric structure); lane 3, 24 mM StI E2C in water (34% of dimeric structure);lane 4, 50 mM StI E2C in water (46% of dimeric structure); lane 5, molecularweight markers (6500 Da, aprotinin; 14,300 Da, chicken egg lysozyme;45,000 Da, Ovalbumin; 66,000 Da, bovine serum albumin).

A. Valle et al. / Toxicon 58 (2011) 8–1714

(Castrillo et al., 2009). In the three-dimensional structure ofSt I, this residue is located close to Gln171 and Lys169 (<5 Å)(PDB: 2KS4, Castrillo el al., 2009), contributing to theassociation of the N-terminal a-helix with the mainb-sandwich core. Substitution of Phe15 by a Cys in St I couldaffect the association between the N-terminal a-helix andb-sandwich, giving rise to a different final inserted state ofthe protein in the monolayer.

To obtain further insight into the insertion mechanismof the proteins into lipid membranes, binding of rStI andthe mutants to SUVs of POPC:SM (85:15) was assessed bymeasuring the effect of lipid concentration on proteinintrinsic fluorescence. Addition of liposomes progressivelyincreased the fluorescence intensity until a plateau wasreached, indicating quantitative association of the proteins(Fig. 8). The curves in Fig. 8 and the data in the insert showa similar behavior for rStI and the mutants: StI E2C, StI F15C

and StI R52C. In the fluorescence quenching studies withthe water-soluble quencher acrylamide, the smallerdifference between Ksv values in solution and in thepresence of SUV (Ksv in solution - Ksv in SUV, Table 3)exhibited by StI R52C (DKsv ¼ 0.57) when compared to rStIand the other mutants (DKsv y 1), could be related todifferent final states of StI R52C upon binding to the lipidmembrane. This result could explain the lower hemolyticactivity of this mutant (Table 3), which would result froma less competent binding for pore formation. Interestingly,the binding assay using POPC:SM SUV slightly reflects thedifferent behavior of StI F15C observed in the monolayermodel (Fig. 7, Table 3); what could be a result of thedifferent characteristics of the lipidic monolayer andbilayer as membrane model systems.

3.6. Permeabilizing activity of rStI and its mutantsin model membrane

The pore-forming capacity of rStI and the mutants wasevaluated from their ability to release CF encapsulated in85:15 POPC:SM LUVs. The rate and extent of the process canbe characterized by the increase in fluorescence associatedto the release of the fluorescent probe (Kayalar andDuzgunes, 1986). The fraction of vesicles bearing at leastone pore (f) vs. time, assuming that the release of CF issimultaneous to pore formation, is illustrated in Fig. 9A.Since the mutants can be found as a mixture of dimeric andmonomeric structures, the permeabilization time coursewas also followed under reducing conditions to obtaina homogenous monomeric population. Maximal fluores-cence intensity measured at “infinite” time (fN) and therates of the process depend on toxin concentration.The value fN, defined by fN¼ (FN - Fo)/(Ftrit – Fo) gives the

5 10 15 20 25 30 35 40 45 50

0

5

10

15

20

25

30

mN

/m

0

Fig. 7. Increase in surface pressure of a POPC:SM (85:15) monolayer uponaddition of rStI and its mutants. The measurements were carried out at 25◦C with constant stirring. A mixture of 85:15 POPC:SM was gently spreadover water until the desired initial surface pressure was achieved.. Once thesignal stabilized, the protein was injected into the subphase at differentconcentrations. The time course of surface pressure increase was recordeduntil a stable signal was obtained. Similar results were obtained under non-reducing conditions. rStI (-); StI E2C (B); StI F15C (:) and StI R52C (6).

A. Valle et al. / Toxicon 58 (2011) 8–17 15

fraction of vesicles that have incorporated enough toxin tolead to channel formation. Values of fN vs toxin concen-tration (Fig. 9B) showed a similar cooperative behavior inalmost all cases. This behavior is compatible with theparticipation of more than one molecule in the steps of the

0 25 50 75 100

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

F/F

0

Phospholipids ( M)

Toxins Lip 50 F/Fo

rSt I 13.43 ± 0.92 1.66 ± 0.02 St I E2C 13.98 ± 0.81 1.64 ± 0.02 St I F15C 10.42 ± 0.43 1.73 ± 0.01 St I R52C 12.50 ± 0.79 1.64 ± 0.01

Fig. 8. Increase of intrinsic fluorescence intensity of rStI and its mutants asa function of lipid concentration. Changes in toxins fluorescence uponaddition of increasing quantities of POPC:SM, 85:15 SUV were recorded at334 nm after excitation at 295 nm. Experiments were carried out at 25 �Cwith 1 mM toxin in 10 mM Tris–HCl buffer pH 7.4. Slit widths of nominalband pass 5 nm were used in both excitation and emission beams. F0 and Fare the fluorescence intensities in the absence and presence of vesicles. Thevalues represent the mean of 3–4 experimental determinations and thevertical bars are the standard deviations of the mean value. The experi-mental results were fitted to the best Boltzman function (lines in graphic,c2< 0.001). The highest fluorescence intensity ratio (F/F0) and the amount oflipid necessary to bind half of the total protein (Lip50) were estimated(Inset). rStI (-); StI E2C (B); StI F15C (:) and StI R52C (6).

Fig. 9. Permeabilization of POPC:SM 85:15 LUV by rStI and its mutants.Permeabilization was assessed by measuring the fluorescence of CF releasedfrom LUV (Tejuca et al., 1996). Changes in the fluorescence intensity at520 nm were measured after excitation at 490 nm. Slit widths of nominalband pass 5 nm were used in both the excitation and emission beams.Samples were prepared in 10 mM Tris–HCl buffer, 140 mM NaCl, pH 7.4, at25 �C. Experiments were performed both under non-reducing conditionsand after pre-incubation with 15 mM b-mercaptoethanol. (A) Kinetics of CFrelease after mixing vesicles and toxins. (B) Fraction of vesicles in which atleast one pore was formed (f) as a function of initial toxin concentration. fwas calculated using Eq.(1) at 10 min after toxin addition (see Materials andMethods). Solid symbols and lines, non-reducing conditions; open symbolsand dashed lines, samples incubated with 15 mM b-mercaptoethanol rStI(-,,); StI E2C (C,B); StI F15C (:,6); StI R52C (;,7). [CF] ¼ 80 mM;[total lipid] ¼ 13.3 mM.

pore-forming process (Tejuca et al., 1996). The data alsoshow that the fraction of channel-bearing liposomes issimilar for rStI and StI F15C over the whole concentrationrange, under reducing and non-reducing conditions. Thissuggests that toxin distribution and the number of toxinmolecules needed to form pores in a vesicle is similar inboth cases. StI R52C showed the lowest permeabilizationcapacity, no significant differences being observed betweenreducing and non-reducing conditions. These results are inagreementwith those obtained in liposome binding studies(Fig. 8) and hemolytic activity assays (Table 3). Mancheñoet al. (2003) described the phosphorylcholine group

A. Valle et al. / Toxicon 58 (2011) 8–1716

binding site in St II 3D structure as a partly hydrophilic andhydrophobic cavity due to the hydroxyl groups of Tyr andSer residues, the non-polar aromatic ring of Tyr and sidechains of Val and Pro amino acids located in this region.They proposed a role for Arg51, a highly conserved residue inthe actinoporin family, arguing that its positive chargepresumably contributes to stabilize the binding of theprotein to the phosphocholine group. Therefore, theremoval of this charge upon substitution of Arg52 by Cys inSt I would decrease the protein ability to achieve a compe-tent binding for pore formation in the membrane.

StI E2C showed a behavior similar to that of rStI in itspermeabilizing activity under reducing conditions. Interest-ingly, under non-reducing conditions StI E2C exhibited thehighest permeabilizing capacity, much lower proteinconcentrations being required to achieve similar fractions ofchannel-carrying liposomes (Fig. 9B). It should be recalledthat thismutant at the protein concentration of around50 mMand under non-reducing conditions, exhibited approximately50% of dimeric structure. Therefore, this result suggests thatthe presence of oligomeric structures in solution, such asdimers, could favor pore formation in the membrane. Thepresence of pre-aggregated protein at the bilayer surfacecould favor the complex oligomerization process. Further-more, the dimeric structure formed by StI E2C could sharestructural properties with the oligomeric pore in themembrane. Ulrih et al. (2004) suggested the involvement ofEqt II N-terminal sequence in its aggregation process underdifferent experimental conditions, such as changes intemperature and ionic strength. In a discussion about thedifferent steps of the mechanism of pore formation by acti-noporins, Alegre-Cebollada et al. (2007) did not discard thepossible binding ofwater-soluble tetramers to themembrane,although there is reasonable evidence that membranebinding byactinoporins precedes oligomerization.Itmust alsobe kept in mind that the presence of multimeric forms insolution has been detected for St II (De los Ríos et al., 1999).Moreover, it is worthwhile noting that Casallanovo et al(2006) observed that a peptide containing residues 1–30 ofSt II is able to aggregate in solution as a function of concen-tration, ionic strength, and pH. Interestingly, the peptidelacking residues 1–10 did not undergo aggregation as a func-tion of these variables. These results provide evidence for therole of the N-terminus in protein aggregation/oligomeriza-tion, whether in solution or in the membrane, and alsopossibly in the process of pore formation.

In summary, the substitution by Cys of Arg52 in thephosphocholine-binding site promoted considerable changesin rStI function; however, the amino acid substitutions in theN-terminal region (Glu2, Phe15) did not modify the toxin’spermeabilizing ability. Interestingly, the presence of a dimer-ized population stabilized by a disulfide bond in StI E2CN-terminal region showeda greater pore-forming ability thanthat of the protein in the monomeric state, suggesting thatpre-ensembled sticholysins via the N-terminal region couldfacilitate pore formation in the membrane.

Conflict of interest

The author declares that there are no conflicts ofinterest.

Funding source

All sources of funding should also be acknowledged andyou should declare any involvement of study sponsors inthe study design; collection, analysis and interpretation ofdata; the writing of the manuscript; the decision to submitthemanuscript for publication. If the study sponsors had nosuch involvement, this should be stated.

Acknowledgments

A.V. and D.M. benefited from a CAPES (Brazil) - MES(Cuba) exchange program. A.V. received an IFS GrantF/4574-1. C.A. benefited from fellowships from the Brazilianagencies CNPq and FAPESP. M.E.L. benefited from fellow-ships from CAPES (Brazil) - MES (Cuba) and CNPq (Brazil) -MES (Cuba) exchange programs. SS is recipient of a CNPqresearch fellowship. Spectroscopic studies were performedin the LSB, Institute of Chemistry, University of São Paulo,Brazil. This research was supported by grants from CNPqand FAPESP.

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