Báo cáo khoa học: Vibrio cholerae hemolysin Implication of amphiphilicity and lipid-induced conformational change for its pore-forming activity ppt

Resistance of the HlyA to proteases together with the tryp-tophan fluorescence emission spectrum suggested a compact structure for the toxin.Fluorescence energy transfer from the HlyA to

Vibrio cholerae hemolysin

Implication of amphiphilicity and lipid-induced conformational change

for its pore-forming activity

Kausik Chattopadhyay1, Debasish Bhattacharyya2and Kalyan K Banerjee1

1 National Institute of Cholera and Enteric Diseases, Kolkata 700 010, India; 2 Indian Institute of Chemical Biology,

Kolkata 700 032, India

Vibrio choleraehemolysin (HlyA), a water-soluble protein

with a native monomeric relative molecular mass of 65 000,

forms transmembrane pentameric channels in target

bio-membranes.The HlyA binds to lipid vesicles nonspecifically

and without saturation; however, self-assembly is triggered

specifically by cholesterol.Here we show that the HlyA

partitioned quantitatively to amphiphilic media irrespective

of their compositions, indicating that the toxin had an

amphiphilic surface.Asialofetuin, a

b1-galactosyl-termin-ated glycoprotein, which binds specifically to the HlyA in a

lectin-glycoprotein type of interaction and inhibits

carbo-hydrate-independent interaction of the toxin with lipid,

reduced effective amphiphilicity of the toxin significantly

Resistance of the HlyA to proteases together with the

tryp-tophan fluorescence emission spectrum suggested a compact

structure for the toxin.Fluorescence energy transfer from the

HlyA to dansyl-phosphatidylethanolamine required the

presence of cholesterol in the lipid bilayer and was synchronous with oligomerization.Phospholipid bilayer without cholesterol caused a partial unfolding of the HlyA monomer as indicated by the transfer of tryptophan residues from the nonpolar core of the protein to a more polar region These observations suggested: (a) partitioning of the HlyA to lipid vesicles is driven by the tendency of the amphiphilic toxin to reduce energetically unfavorable contacts with water and is not affected significantly by the composition of the vesicles; and (b) partial unfolding of the HlyA at the lipid– water interface precedes and promotes cholesterol-induced oligomerization to an insertion-competent configuration Keywords: pore-forming toxin; amphiphilicity; lipid-induced conformational change; oligomerization; protein fluorescence

Vibrio choleraehemolysin (HlyA), a water-soluble cytolytic

protein expressed by many V cholerae El Tor O1 and

non-O1 strains [1,2], belongs to a large, heterologous family of

pore-forming toxins (PFT) [3,4] that are ubiquitous in

prokaryotic and eukaryotic organisms.The toxin has been

cloned and sequenced [5,6].The HlyA permeabilizes a wide

spectrum of eukaryotic cells including human and rabbit

erythrocytes [2] and synthetic lipid vesicles [7,8] by forming

transmembrane pentameric [9] diffusion channels with a

diameter of approximately 1.5 nm In addition to binding

specifically to cholesterol [9], the toxin shows a lectin-like

property in interacting with b1-galactosyl-terminated

gly-coconjugates such as asialofetuin and asialothyroglobulin

[10].The purified toxin evokes secretion of fluid in a rabbit

ligated ileal loop, suggesting its involvement in pathogenesis

of cholera [11]

A consensus on the pathway of induction of membrane

damage by PFTs postulates a sequence of at least three

discrete biochemical events: binding of the toxin monomer

to a cell surface receptor, self-assembly to an amphiphilic

prepore oligomer and insertion in the lipid bilayer gener-ating a functional pore that mediates passive flux of molecules across the membrane [12–15].Extensive studies

of the interaction of V cholerae HlyA with synthetic lipid vesicles suggest that the binding is nonspecific and nonsa-turable [8,9].However, permeabilization of the target lipid vesicle and more precisely oligomerization of the toxin monomer to a pentameric channel shows a specific requirement for cholesterol [9,16] and is augmented dramatically by inclusion of ceramides in the lipid bilayer [9].Recent studies indicate that sphingolipids and glycero-lipids with cone-shaped structures modify the energetic state of membrane cholesterol, which in turn promotes functionally productive interaction of the sterol with the toxin [17].Earlier, we reported inhibition of hemolysis of rabbit erythrocytes by glycoproteins with b1-galactosyl moieties [10].However, the sensitivity of erythrocytes to the HlyA is not correlated with the surface density of galactose Even more intriguing was the observation that these glycoproteins inhibit the carbohydrate-independent interac-tion of the toxin with immobilized phospholipid and phospholipid-cholesterol.As there is no information on the structure of the toxin and its physicochemical charac-teristics in solution and lipid bilayer, it is difficult to speculate a molecular interpretation of the toxin–membrane interaction and subsequent events

In this communication, we show that the HlyA is a compact protein with an amphiphilic surface.Partitioning

of such a molecule to a lipid bilayer seems to be driven solely

Correspondence to K.K.Banerjee, National Institute of Cholera

and Enteric Diseases, Kolkata-700 010.

Fax: + 91 33 350 5066, Tel.: + 91 33 350 1176,

E-mail: banerjeekalyan@hotmail.com

Abbreviations: ANS, 8-anilino-1-naphthalene sulphonic acid;

HlyA, hemolysin; PFT, pore-forming toxins

(Received 10 May 2002, revised 20 June 2002, accepted 25 July 2002)

by its tendency to avoid energetically unfavorable contact

with water and is relatively insensitive to the bilayer

composition.In spite of its intrinsic amphiphilicity, the

toxin monomer does not possess an insertion-competent

configuration.Secondly, we demonstrate that self-assembly

of the toxin monomer is a spontaneous event that could

occur in water as well, but at a very slow rate compared to

that observed in a membrane bilayer.Finally, we provide

evidence for partial unfolding of the toxin at the lipid–water

interface, a conformational change that precedes and is

likely to facilitate cholesterol-induced self-assembly to a

transmembrane channel

E X P E R I M E N T A L P R O C E D U R E S

Purification ofV cholerae HlyA and assay of hemolytic

activity

The HlyA was purified to homogeneity as described

previously [10] with some modification V cholerae

non-O1 strain V2, a clinical isolate kindly supplied by

R.Sakazaki, Tokyo, Japan, was grown to early stationary

phase (
6 h) in brain heart infusion (BHI,

Becton-Dickinson) broth at 37C with shaking.Bacteria were

removed by centrifugation at 30 000 g for 10 min at 4C

and the culture supernatant (6 L) was concentrated

approximately 100-fold by ultrafiltration through PM-10

(Amicon) membrane.Lipid-protein vesicles released by

bacteria during growth were removed from the ultrafiltrate

by size-exclusion chromatography on Sepharose CL-4B

(Pharmacia, 50· 2.5 cm) equilibrated with 25 mMsodium

phosphate buffer containing 1 mM EDTA and 3 mM

NaN3, pH 7.2 (Buffer A) The hemolytic activity eluted

as a totally included fraction and was subjected to

hydrophobic interaction chromatography on

phenyl-Sepharose CL-4B (Pharmacia, 50· 1.5 cm) equilibrated

with buffer A.The HlyA bound tightly to the matrix and

desorbed from the column at 46% ethylene glycol on

application of a linear 0–80% ethylene glycol gradient,

with the mixing chamber containing 200 mL of buffer A

Finally, the HlyA was separated from traces of proteases

and low relative molecular mass contaminants by

chro-matofocusing on PBE-94 (Pharmacia; 20· 1.5 cm) The

toxin bound weakly to the matrix and eluted as a slightly

retarded symmetrical peak during washing of the column

with buffer A.The purified toxin migrated in

SDS-polyacrylamide gel electrophoresis (SDS/PAGE) [18]

fol-lowing incubation in 1% SDS at 50C for 15 min, as a

single 65 kDa polypeptide and was homogeneous in

PAGE under nondenaturing condition at pH 8.3 The

HlyA was stable indefinitely in 50% ethylene glycol at

4C.The protein concentration was determined

spectro-photometrically at 280 nm based on an absorbance of 1.4

for a 1 mgÆmL)1 solution, determined by a modified

Folin-Ciocalteu method [19].The hemolytic activity was

assayed by monitoring spectrophotometrically the release

of hemoglobin at 541 nm or by measuring turbidity of the

erythrocyte suspension at 600 nm.The purified HlyA

caused 50% lysis of a 1% (v/v) suspension of rabbit

erythrocytes in 10 mM sodium phosphate buffer

contain-ing 150 mMNaCl, pH 7.2 (NaCl/Pi) for 1 h at 25C at a

concentration of 4 ngÆmL)1, corresponding to a specific

activity of 60 pM

Demonstration of protein amphiphilicity Amphiphilicity was operationally defined as preference of a protein for an amphiphilic phase relative to water as measured by its equilibrium concentrations in the two phases.Because the chemical interaction of the HlyA with phase constituents might affect the distribution of the toxin

in the two phases, a system consisting of water and an amphiphilic phase was constituted in two ways: (a) by using Triton X-114, a nonionic detergent that forms a single phase mixture with water at 4C and separates into a water-rich and detergent-rich phase at 23C [20], and (b) by using the interface of water and a low polarity organic solvent such as chloroform as an amphiphilic surface [21].To monitor partitioning of the HlyA to Triton X-114, the toxin was dissolved in a 2% (v/v) solution of the detergent at 4C, the aqueous and detergent phases collected separately at 25C, precipitated with nine volumes of cold acetone and subjec-ted to SDS/PAGE for visualization of the toxin content.In the second system, the HlyA in the concentration range 25–100 lgÆmL)1 was mixed in duplicate with an equal volume of chloroform at 25C, vortexed for 10 min to maximize the area of water–chloroform interface and allowed to separate.The HlyA concentration in the aqueous phase was determined in triplicate by assay of the hemolytic activity as well as by the enzyme-linked immunosorbent assay (ELISA) using rabbit anti-(HlyA) IgG as described previously [10]

Preparation of liposome Phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) or a mixture (1 : 1 by weight) of PtdCho and cholesterol in chloroform/methanol (2 : 1 by volume) was evaporated to dryness, dispersed in buffer A and sonicated for 10 min at 23 kHz.Multilamellar vesicles were removed

by centrifugation at 30 000 g.Large unilamellar liposomes were prepared from the pool of vesicles by size-exclusion chromatography on Sepharose CL-4B [22]

Protease treatment The HlyA was incubated with trypsin, chymotrypsin and Pronase (1 : 50, protease/toxin ratio, w/w) in NaCl/Pior NaCl/Pi-2Murea at 25C.The reaction was terminated by boiling in sample buffer containing 1% SDS for 5 min Proteolytic digestion was monitored by SDS/PAGE [18] Spectroscopic measurements

Fluorescence and light scattering measurements were car-ried out with a Hitachi 4500 Spectrofluorimeter.Light scattering was recorded at a right angle to the incident beam

in the wavelength range of 500–650 nm at slit widths of 2.5 nm Because the intensity of the scattered light was related to the wavelength, k as 1/k4, the sensitivity of scattering was low in the long wavelength used; however, it eliminated interference by ultraviolet absorption of the protein.The intrinsic Trp fluorescence of the HlyA was monitored in buffer A by exciting the sample at 295 nm using band widths of 2.5 nm Lipid-induced perturbation

of the conformation of the HlyA was investigated by monitoring intrinsic Trp fluorescence of the toxin at a

protein : lipid ratio of 1 : 10 (w/w), a ratio at which the

unbound toxin was not detected by gel filtration, using the

excitation wavelength and slit widths as above.The

fluorescence energy transfer from Trp in the toxin to

dansyl- PtdEtn incorporated in PtdCho and PtdCho

-cholesterol vesicles was followed kinetically by setting

excitation and emission wavelengths at 280 and 512 nm,

respectively, with 5 nm slit widths [22].The quenching of the

intrinsic Trp fluorescence by acrylamide was studied by

using excitation wavelength of 295 nm, and the data were

analyzed using the Stern–Volmer equation [23]

F0/F¼ 1 + Ksv[Q]

where F0 and F refer to the fluorescence intensities in the

absence and presence of acrylamide, respectively, Ksvis the

collisional quenching constant, and [Q] the concentration of

the quencher

The binding of the dye, 8-anilino-1-naphthalene

sul-phonic acid (ANS) to the HlyA monomer and oligomer was

monitored spectrofluorimetrically by exciting at 390 nm a

mixture of the dye and protein at concentrations of 50 lM

and 90 lgÆmL)1, respectively.The emission was recorded in

the wavelength range of 410–600 nm at a slit width of 5 nm

R E S U L T S

The HlyA is an amphiphilic protein

Previously we showed that V cholerae HlyA interacted

strongly with phenyl-Sepharose CL-4B and desorbed from

the hydrophobic matrix at an ethylene glycol concentration

of 8.25M [10] (see Experimental Procedure).Because

surface hydrophobicity of a protein might drive it to a lipid

bilayer, we explored the physicochemical nature of the

HlyA surface in more details.The purified toxin was

dissolved in 2% Triton X-114 at 4C.On separation of the

solution into detergent- and water-rich phases by raising the

temperature to 25C [20], the HlyA was found by SDS/

PAGE to be concentrated exclusively in the detergent phase

(Fig.1), a behavior that is commonly considered

charac-teristic of an integral membrane protein and somewhat

unusual for a water-soluble globular protein [24]

Water-soluble amphiphiles like detergents prefer the

interface of water and air or a nonpolar organic solvent

that enables them to attain an energetically favorable

arrangement by orienting their polar and nonpolar ends to

water and the less polar phase, respectively.In order to see if

the partitioning of the HlyA to the detergent phase was

dictated by intrinsic amphiphilicity of the protein and not by its affinity for Triton X-114 or a conformational change induced by the detergent, we monitored accumulation of the toxin at the interface of water and chloroform.The ratio of the aqueous phase concentration of the HlyA following partitioning with chloroform, c to the initial value, co, estimated by the ELISA and also by the assay of hemolytic activity, was: 5.2· 10)3 (co¼ 100 lgÆmL)1), 7.6· 10)3 (co¼ 50 lgÆmL)1) and 1 2· 10)2 (co¼ 25 lgÆmL)1) implying that approximately 99% of the toxin was actually present at the interface.For comparison, bovine serum albumin showed a c/co value of 0.58 in an identical experiment at a protein concentration of 100 lgÆmL)1 Interestingly, asialofetuin, a b1-galactosyl-terminated gly-coprotein [25] that binds to the HlyA with an affinity constant of 9.4· 107

M )1and inhibits its interaction with phospholipid vesicles [10] caused an approximately 10-fold increase in the bulk aqueous phase concentration of the toxin, as measured by a c/covalue of 8· 10)2at HlyA and asialofetuin concentrations of 50 and 200 lgÆmL)1, respect-ively.These observations demonstrate: (a) the HlyA was a surface-active molecule that tended to be squeezed out of an aqueous phase; and (b) a carbohydrate ligand reduced significantly the effective amphiphilicity of the toxin, thereby rendering it energetically more compatible with an aqueous environment

Because amphiphiles form micellar aggregates to avoid exposure of the nonpolar surface to water, we examined the HlyA for self-aggregation by light scattering.The intensity

of light scattered by 1 lMHlyA in water was significantly higher than that by a solution at the same concentration in 50% ethylene glycol (Fig.2) indicating promotion of self-aggregation of the toxin by an aqueous environment.Size-exclusion chromatography of the HlyA on Biogel A-0.5 m,

an agarose-based matrix with a fractionation range of 10–500 kDa led to partial exclusion of the toxin with the bulk of the material being spread out over the entire bed volume (data not shown).Use of dextran or polyacryl-amide-based matrices led to similar profiles.Such profiles

Fig 1 Distribution of the HlyA in water-Triton X-114 system The

purified toxin, dissolved in 2% (v/v) Triton X-114 in buffer A at 4 C

at a protein concentration of 100 lgÆmL)1, partitioned into water- and

detergent-rich phases at 25 C.Aliquots of 100 lL were withdrawn

from each phase, precipitated with nine volumes of cold acetone and

subjected to SDS/PAGE Lane 1, detergent-rich phase; lane 2,

water-rich phase.

Fig 2 Dependence of the intensity of light scattered by HlyA on solvent polarity Light scattering by the HlyA (65 lgÆmL)1) in buffer A (—) and 50% ethylene glycol in buffer A (– -) were recorded at slit widths

of 2.5 nm.

could arise from a combination of self-aggregation of the

toxin and nonspecific low affinity interaction with the

matrix, both of which reflected the tendency of the protein

to avoid exposure to water

The HlyA is a compact protein

Having established the amphiphilic nature of the HlyA

monomer, we investigated if the toxin possessed a compact

solution structure characteristic of a native folded protein

The fluorescence emission spectrum of the HlyA excited at

295 nm to eliminate contribution by Tyr showed a

maxi-mum at 330 nm (Fig.3A).Exposure of the protein to 4M

guanidinium hydrochloride caused a red shift of the

emission maximum to 345 nm suggesting transfer of Trp

residues from a nonpolar environment in the native protein,

to water in the unfolded state (data not shown).As there are

11 Trp residues at positions 31, 33, 113, 186, 210, 230, 318,

400, 402, 570 and 574 in the HlyA polypeptide [26] with

possible differences in solvent exposure, we examined if the

heterogeneity in microenvironments of the indolyl residues

could be resolved by quenching of fluorescence by

acryl-amide.Although acrylamide induced a large decrease in

fluorescence intensity, there was no shift in emission

maximum (Fig.3B) The Stern–Volmer plot [23] of the

relative fluorescence intensity vs.acrylamide concentration

was perfectly linear over a fairly wide range of the quencher

concentrations (Fig.3C) with a KSVvalue of 2.2 indicating

that the multiple indolyl groups behaved in essence as a

single emitting center with restricted accessibility to water

These data, along with the large blue shift of the emission

maximum of the native protein indicated that Trp residues

were located in a nonpolar region that could be provided by

the core of a compactly folded protein

Limited proteolysis of the HlyA supported a compact

solution structure with restricted accessibility of peptide

linkages to a water-soluble probe.There was no degradation

of the toxin on incubation of the protein with trypsin

(Fig.4A) or chymotrypsin (Fig.4B) at an enzyme :

sub-strate ratio of 1 : 50.Repetition of the enzyme digestion in

2Murea led to partial degradation of the HlyA to a 50-kDa

fragment.The native toxin was, however, fairly susceptible

to digestion by Pronase, a nonspecific protease (data not

shown)

Self-assembly of the HlyA and association with lipid

bilayer

Oligomerization of the HlyA, as distinct from

amphiphili-city-driven nonstoichiometric self-aggregation in water

described above, was monitored by stability of the pentamer

in SDS at 60C, a characteristic it shares with several other

PFTs, e.g Staphylococcus aureus a-toxin [27].In agreement

with previous reports [9,16], self-assembly of the HlyA was

essentially complete within 1 min of incubation with

PtdCho-cholesterol vesicles (Fig.5A) at 25C and very

slow in pure PtdCho vesicles (Fig.5B).However,

incuba-tion of the HlyA in a homogeneous dispersion of cholesterol

in water at a toxin : sterol molar ratio of 1 : 60 for 20 min

failed to induce detectable oligomerization (data not

shown).Self-assembly of the HlyA was detected in water

on storage of the toxin for several weeks but remained

incomplete even after a year suggesting that oligomerization

was essentially a spontaneous event that was accelerated dramatically by cholesterol in a lipid bilayer matrix.The near identity of the fluorescence emission spectra of the

Fig 3 Fluorescence emission spectra of the HlyA monomer andoligo-mers (A) The HlyA monomer (– -), the oligomer formed during storage of the toxin in water for 30 days (— – —), and the oligomer generated in PtdCho-cholesterol vesicles and delipidated by treatment with sodium deoxycholate and size-exclusion chromatography on Sepharose CL-4B (15) (—) were dispersed in buffer A at a protein concentration of 90 lgÆmL)1.Samples were excited at 295 nm and spectra recorded at slit widths of 2.5 nm (B) Acrylamide-induced quenching of fluorescence of the HlyA monomer was monitored at a protein concentration of 65 lgÆmL)1 in buffer A (—) and 1 M

acrylamide in buffer A (– -).Excitation wavelength and slit widths were set at 295 and 2.5 nm, respectively (C) Stern–Volmer plot for quenching of intrinsic fluorescence of the HlyA monomer (d) and oligomer (D) by acrylamide.The protein concentration was kept constant at 65 lgÆmL)1.Excitation and emission wavelengths were set

at 295 and 330 nm, respectively, with slit widths of 2.5 nm.

HlyA oligomer formed in water and the one generated in

PtdCho-cholesterol vesicles indicated a similarity in

confor-mations of the two pentamers (Fig.3A).Notably,

oligo-merization led to no significant change in either Trp

fluorescence spectrum or quenching of fluorescence by

acrylamide, suggesting Trp residues sensed similar

microen-vironments in the monomer and pentamer.However, the

monomer and oligomer differed in binding to ANS, a

fluorescent probe used widely to detect surface-exposed

hydrophobic patches on proteins [28].While there was no

significant change in the ANS emission spectrum in the

presence of the monomer, the oligomer induced a small twofold increase in intensity but a significant blue shift in the emission wavelength maximum by 21 nm (Fig.6)

As reported previously [10], incubation of the HlyA with PtdCho-cholesterol led to a total loss in hemolytic activity

In contrast, the HlyA bound to PtdCho remained hemo-lytically as potent as the free toxin, indicating that oligo-merization of the toxin signalled its irreversible association with the lipid bilayer.The difference in modes of association

of the HlyA monomer and the oligomer with a lipid bilayer was reflected in the fluorescence energy transfer from Trp in the toxin to dansyl- PtdEtn incorporated in lipid bilayers

No increment in fluorescence intensity was observed during

a 15 min incubation of pure PtdCho vesicles with the HlyA monomer (Fig.7).In contrast, the increment in fluorescence intensity was almost immediate with PtdCho-cholesterol vesicles and essentially complete within 1 min.It appears therefore that the increment in fluorescence intensity

Fig 4 Protease-cleavage pattern of the HlyA The toxin was incubated

with the protease at an enzyme : substrate ratio of 1 : 50 (w/w) at

25 C.(A) Lane1, digested with trypsin for 2 h; lane 2, digested with

trypsin for 15 min in 2 M urea.(B) Lane 1, digested with chymotrypsin

for 2 h; lane 2, digested with chymotrypsin for 15 min in 2 M urea.

Fig 5 Lipid-induced oligomerization of the HlyA detected by SDS/

PAGE The protein : lipid weight ratio was maintained at 1 : 10 to

ensure absence of the unbound toxin.Samples were treated with 1%

SDS at 50 C for 15 min, a temperature at which the oligomer does not

dissociate into monomer.(A) Incubation with PtdCho-cholesterol for

1 min (lane 2) and 2 min (lane 3).Lane 1 shows HlyA in buffer A.(B)

Incubation with PtdCho for 5 min (lane 2), 10 min (lane 3), 15 min

(lane 4) and 20 min (lane 5).The HlyA in buffer A (lane 1) was

included for comparison.

Fig 6 The binding of ANS to the HlyA monomer and oligomer The incubation mixtures contained the monomer (– -) and oligomer (—) at

a protein concentration of 90 lgÆmL)1and ANS at 50 l M Excitation was performed at 390 nm and slit widths were set at 5 nm (— – —) indicates ANS in buffer A.

Fig 7 Fluorescence energy transfer from the HlyA to dansyl-PtdEtn incorporatedin Ptd Cho-cholesterol (—) andPtd Cho (– -) liposomes The toxin was incubated with liposome at a protein : lipid ratio of 2 : 1 (w/w), a ratio at which the sensitivity of the assay was found to be maximum.The incubation mixture was excited at 280 nm and fluor-escence emission was recorded at 512 nm approximately 10 s after mixing the toxin with liposome.Slit widths were 5 nm.Dansylated liposome suspensions without the HlyA served as the control.

followed exactly the time-course of self-assembly of the

HlyA.Because the efficiency of the fluorescence energy

transfer depends, among other things, on the proximity of

the energy donor and acceptor groups [23], we interpret

these observations as implying a tight association of the

HlyA oligomer with the core of the PtdCho-cholesterol

bilayer

Interaction with lipid induces conformational change

in the HlyA monomer

As incorporation of the HlyA in a lipid bilayer seemed to

require cholesterol induced self-assembly of the toxin

monomer, we thought it interesting to see if interaction of

the toxin with the amphiphilic lipid matrix by itself induced

a conformational change in the monomeric protein.To

avoid complexity introduced by oligomerization of the toxin

in PtdCho-cholesterol bilayer, we examined the fluorescence

emission spectra of the toxin incubated with PtdCho and

PtdEtn liposomes at an excitation wavelength of 295 nm

(Fig.8) Surprisingly, there was a drastic change in the

spectrum, with the emission maximum showing a red shift

of approximately 10 nm from 330 to 340 nm and a decrease

of intensity by approximately twofold, indicating transfer of

Trp residues from the nonpolar core of the protein to more

polar surroundings, presumably at the lipid–water interface

These data suggest that although the HlyA monomer and

oligomer had similar conformations (Fig.3), the

self-assembly involved a partially unfolded state of the protein

as an intermediate that survived in the absence of cholesterol

for a fairly long time without undergoing oligomerization

D I S C U S S I O N

Structure–function analysis of several PFTs, e.g S aureus

a-toxin [13,27], aerolysin [12], pneumolysin [29], and

perfringolysin [14] have established that oligomerization of

the toxin is a critical cell surface event that precedes insertion

of the protein in the target membrane.It is unusual for a water-soluble monomeric PFT to possess surface-exposed, uninterrupted hydrophobic stretches, and integration of such proteins with the nonpolar core of a lipid bilayer is energetically expensive.As an alternative, PFT binds to specific cell surface receptors and an increase in surface concentration by several orders of magnitude compared to that in water provokes self-assembly of the monomer to an amphiphilic oligomer.In this communication, we show that

V cholerae HlyA does not adhere to the details of this mechanistic framework of membrane permeabilization by PFTs

Despite solubility in water, the HlyA monomer is amphiphilic and the tendency of the toxin molecule to decrease its area of contact with water seems to dominate much of its functional and hydrodynamic properties.The HlyA, which was dissolved initially in 2% Triton X-114 at

4C, partitioned quantitatively to the detergent-rich phase

on raising the temperature to 25C [20].Because seques-tration of the HlyA by Triton X-114 could possibly be caused by affinity of the toxin for the detergent or by a conformational change in the native protein, we used an alternative strategy to monitor intrinsic amphiphilicity of the toxin.Amphiphilic molecules like detergents, which have high Gibbs free energy in water, adopt an energetically favorable orientation at the interface of water with air or a nonpolar organic solvent and tend to accumulate at the surface.Increase in the surface concentration of a protein, estimated indirectly by the decrease in the bulk aqueous phase concentration would therefore provide a measure of amphiphilicity.As chloroform does not affect the property

of the aqueous phase due to its insolubility and lacks a functional group that can interact with a protein, we chose it

as the organic solvent.The aqueous phase concentration of the HlyA was found to be 100-fold less in a water– chloroform system than in water in comparison to a twofold decrease observed with bovine serum albumin demonstra-ting that amphiphilicity was an intrinsic characteristic of the native HlyA.Amphiphilicity of the toxin was reflected in nonstoichiometric association of the protein in water, as indicated by light-scattering (Fig.2) and gel filtration experiments.Furthermore, the complex of the HlyA with asialofetuin, a glycoprotein inhibitor of the interaction of the HlyA with synthetic lipid vesicles and biomembranes [10], was considerably more hydrophilic than the unbound toxin.On the basis of this positive correlation between effective amphiphilicity and affinity of the toxin for lipid vesicles and erythrocytes, we conclude that amphiphilicity drives the HlyA to phospholipid vesicles and is a major determinant of the interaction of the toxin with erythro-cytes.Such an interpretation is consistent with the nonspe-cific and nonsaturable interaction of the HlyA with lipid vesicles [8] and might resolve the controversy in the identity and role of the erythrocyte surface receptor in initiating the action of the toxin [10,30]

The preceding observations would suggest that the amphiphilic HlyA monomer might itself posses an inser-tion-competent configuration.We addressed the question

by delinking lipid-binding from oligomerization by using PtdCho and PtdCho-cholesterol vesicles.On excitation of the HlyA incubated with such liposomes incorporating dansyl-PtdEtn as a fluorescent probe at 280 nm, transfer of Trp fluorescence energy to the dansyl moiety occurred only

Fig 8 Lipid-induced perturbation of the fluorescence emission spectrum

of the HlyA The HlyA was incubated with PtdCho (—) and PtdEtn

liposomes (– -) for 10 min at a protein concentration of 50 lgÆmL)1

and a protein-lipid weight ratio of 1 : 10 and excited at 295 nm (—

– —) shows the spectrum of the HlyA in buffer A at the same

con-centration.Slit widths were 2.5 nm.Liposome suspensions without the

HlyA served as the blank.

in PtdCho-cholesterol vesicles and was synchronous with

the self-assembly of the toxin to the SDS-stable pentamer

(Fig.5A).In addition, the hemolytic activity of the

lipid-bound HlyA correlated exactly with the quantity of the

toxin present in the monomeric form, implying that the

monomer and not the oligomer could exchange rapidly

between the amphiphilic matrices of the synthetic lipid

vesicle and erythrocyte membrane.Requirement of

oligo-merization for integration of the HlyA with the nonpolar

core of the lipid bilayer implies that the monomer does not

adopt an insertion-competent configuration in spite of its

intrinsic amphiphilicity

As a folded protein has nonpolar amino acids buried in a

hydrophobic core insulated from water, it seems doubtful if

the amphiphilic HlyA possesses a compact structure

Although no information is available for the HlyA

struc-ture, a blue shift of 15 nm of the wavelength maximum of

Trp fluorescence emission spectrum of the HlyA monomer

(Fig.3A) together with the Stern–Volmer analysis of the

quenching of fluorescence by acrylamide (Fig.3C) indicated

that the multiple indolyl residues were located in a nonpolar

region with limited accessibility to water.Because Trp

residues are scattered along the length of the 65 kDa

polypeptide chain [26], such an arrangement would imply a

compact folded structure for the HlyA monomer.The

compactness of the native HlyA in water was corroborated

by resistance of the toxin to proteolytic degradation by

trypsin and chymotrypsin (Fig.4).Furthermore, the

iden-tity in shape of the spectra of the HlyA monomer and the

oligomer (Fig.3A) and the similarity in quenching

charac-teristics of the two proteins indicated that the two toxin

forms shared similar global conformations.Nevertheless,

the HlyA monomer and the oligomer could be distinguished

by ANS.Although the increase in fluorescence intensity was

relatively small in comparison to the large enhancements

caused by proteins in molten globule states [31] the data

suggested the presence of exposed hydrophobic patches on

the oligomer that might be instrumental in conferring it the

insertion-competent configuration.It may be recalled that

self-assembly of monomer to insertion-competent oligomer

without major changes in secondary structure and tertiary

foldings have been documented for S aureus a-toxin [13]

and aerolysin [12]

Although amphiphilicity-driven partitioning of the HlyA

in phospholipid vesicles devoid of cholesterol failed to

trigger rapid oligomerization of the toxin, the interaction

caused a profound change in the conformation of the

protein (Fig.8).A red shift of the wavelength maximum of

intrinsic Trp fluorescence of the monomer by 10 nm

induced by phospholipids irrespective of head groups

indicated that a significant fraction of the multiple indolyl

emitting centers were transferred from an apolar region in

the protein core to a more polar environment, presumably

at the lipid–water interface.A partial collapse of the folded

HlyA, as suggested by the spatial redistribution of Trp

residues, might be similar to the lipid-induced transition of

the native conformation to a molten globule state observed

with a number of proteins, e.g colicin A [32], cytochrome c

[33], and human apolipoprotein [34].Cholesterol had a

dramatic effect on the kinetics of self-assembly of the HlyA,

an irreversible process that seemed to be guided by the

higher thermodynamic stability of the oligomer; however, it

exerted its effect in a lipid bilayer matrix only.Although the

nature and extent of the disruption in structure of the HlyA induced by phospholipids is unclear, self-assembly of the toxin appears to involve a partially unfolded state as a fairly stable intermediate, followed by cholesterol-assisted recon-stitution to the oligomer with recovery of much of the folding patterns of the monomer.In recent years, there has been wide acceptance of chaperone-like roles of specific lipids in the correct folding of unfolded integral membrane proteins [35].Such examples include the roles of PtdEtn and PtdCho in the folding of Escherichia coli lactose permease [36] and OmpA [37], respectively.Cholesterol might play a similar role in conversion of the HlyA monomer to the oligomer.Adoption of such a mechanism for generating functional channels has enabled the HlyA to select eukary-otic cells as targets in consonance with its survival and multiplication in animal guts and spare prokaryotic cells that lack cholesterol in their membrane

A C K N O W L E D G E M E N T

We thank the Council of Scientific and Industrial Research, New Delhi, India for a grant (Scheme no.37/0955/97-EMR-II) and a senior research fellowship to K.C.We thank S.K.Bhattacharyya, Director, National Institute of Cholera and Enteric Diseases, Kolkata, for support.

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