ABSTRACT The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membranebound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of a-toxin.
INTRODUCTION
alpha-Toxin is a soluble hemolytic protein exotoxin secreted by Staphylococcus aureus that is thought to be a major factor contributing to the pathogenicity of S. aureus. The amino acid sequence of a-toxin has been deduced from its gene sequence (Kehoe et al., 1983; Gray and Kehoe, 1984). The toxin is composed of 293 amino acids and corresponds to a calculated molecular weight of 33,400, which is in fair agreement with the SDS-PAGE-based determinations. alpha-Toxin damages membranes by the formation of nonspecific oligomeric pores in the target membranes, which lead to cell lysis (Fussle et al., 1981; Bhakdi et al., 1981). These pores are large in size, and diameters of 1-2 nm have been reported (Fussle et al., 1981; Menestrina, 1986). An insight into the mechanism of membrane insertion and channel formation by S. aureus alpha-toxin and the amino acids involved in channel formation have been emerging from the extensive investigations on alpha-toxin, based on mutational (Walker et al., 1993, 1994; Panchal and Bayley, 1995), biochemical (Tobkes et al., 1985; Walker et al., 1995), and biophysical approaches (Ward et al., 1994; Valeva et al., 1996), culminating in the model confirmed by the recently solved x-ray structure of the detergent-solubilized heptamer (Song et al., 1996). The developments in the understanding of the transformation of this water-soluble toxin monomer to a channel-forming membrane-bound oligomer has been described in excellent reviews (Bhakdi and Tranum-Jensen, 1991; Thelestam and Blomqvist, 1988; Bhakdi et al., 1996; Gouaux, 1998).
According to the recently described structure of the heptameric form of a-toxin (Song et al., 1996), the transmembrane pore complex is composed of three regions, the cap, the stem, and the rim domains. The large protrusions in electron microscopic images of the toxin (Ward and Leonard, 1992) have been identified as the cap and portions of the rim domain. The stem domain, which defines the 28-A wide transmembrane channel, is described as a 14-strand antiparallel beta-barrel composed of two 65-A-long beta-strands contributed by each monomer. The rim domains protrude from the underside of the heptamer and are in close proximity to the bilayer. However, the structure of the soluble form is yet unknown, and the formation of the heptameric pore requires structural transition from a water-soluble monomeric form into the oligomeric form and is believed to be catalyzed by binding to an unidentified receptor on the membrane or by binding to the membrane surface. Comparative studies of the soluble and membrane-bound forms therefore should give important information on the structural transitions that result in the functional heptameric pore.
Fluorescence spectroscopy is one of the spectroscopic techniques that provides structural information (although at a lower resolution) about structural and dynamic changes in proteins and is very useful in studying the interaction of soluble proteins with membranes. Aqueous soluble quenchers like iodide and acrylamide have been used to provide information on the gross location of tryptophan residues in the complex three-dimensional structure of soluble and membrane-bound proteins (Eftink, 1991). On the other hand, membrane-associated quenchers such as bromine atoms (East and Lee, 1982; Markello et al., 1985) or nitroxide (London and Feigenson, 1981; Blatt et al., 1984), groups covalently linked to fatty acids or phospholipids derived from these fatty acids, have been effectively used to evaluate involvement of tryptophan-containing regions of membrane-interacting proteins. They have also been used to determine the location of tryptophan residues of membranebound proteins in the bilayer (Meers, 1990; Jiang et al., 1991; Chattopadhyay and McNamee, 1991; Chung et al., 1992) and to follow the insertion of soluble proteins in membranes (Gonzalez-Manas et al., 1992). Another useful method to study the environment and organization of tryptophans is red edge excitation shift (REES) (Demchenko, 1988; Mukherjee and Chattopadhyay, 1994, 1995), which is a shift in the wavelength of emission maxima toward a higher wavelength caused by a shift in the excitation wavelength toward the red edge of the absorption band. This effect is observed when a polar fluorophore is present in a motionally restricted environment and arises from the slow rates of solvent relaxation around the excited state of the fluorophore caused by motional restriction on the solvent molecule in the immediate vicinity of the fluorophore.
In this study we have utilized the intrinsic fluorescence of tryptophan to evaluate structural changes in a-toxin on transition from a water-soluble native form to a membranebound oligomeric form. The gene sequence of a-toxin predicts 8 tryptophan residues in the toxin at positions 80, 167, 179, 187, 260, 265, 274, and 286 (Kehoe et al., 1983, Gray and kehoe, 1984). Based on the recent structure of the detergent-solubilized heptamer, 6 tryptophans at positions 80, 179, 187, 260, 265, and 274 appear to be present in the rim domain, whereas Trp-167 and Trp-286 seem to be present in the cap domain (Song et al., 1996). Besides tryptophans, the rim domain contains several tyrosine residues and is thus rich in aromatic residues. We have studied tryptophan fluorescence quenching using the aqueous soluble quenchers, and spin-labeled and brominated membranebound quenchers were used to evaluate tryptophan-containing regions of alpha-toxin in its soluble and membrane-bound forms. Quenching studies using aqueous soluble quenchers indicated that the tryptophan residues of the soluble a-toxin were deeply buried within the protein tertiary structure. N-Bromosuccinimide (NBS) modification, time-resolved fluorescence measurements, and REES confirmed that some of the tryptophan residues were very deeply buried within the monomeric toxin structure. The fluorescence of membrane-bound a-toxin, on the other hand, was quenched by iodide, indicating exposed tryptophan residues. This was also indicated by a shorter average fluorescence lifetime of the tryptophan residues of the membrane-bound toxin in comparison with the native toxin. Although brominated membrane probes failed to quench the tryptophan fluorescence of the membrane-bound toxin, there was significant quenching by spin-labeled probes. Depth calculation using the parallax method (Chattopadhyay and London, 1987) suggested a location of tryptophans at an average depth of 10 A from the center of the bilayer, indicating that the most of the tryptophan residues are located at the membranewater interface. Based on the x-ray structure, Song et al. (1996) suggested that some of the residues in the rim domain might have some contact with the membrane hydrophobic environment. This is supported by studies of fluorescence changes on binding of acrylodan-labeled single cysteine mutant at position 266 (Valeva et al., 1996) and studies of the spectroscopic analysis of conformational changes in alpha-toxin associated with membrane binding and insertion (Vecsey-Semjen et al., 1997).
It is being increasingly recognized that tryptophan residues of membrane-bound proteins are preferentially located at the membrane-water interface (Weiss and Schulz, 1992; Deisenhofer et al., 1995; Ostermeier et al., 1996; Grigorieff et al., 1996). Our studies thus confirm that the membranebound oligomeric pore formed by S. aureus a-toxin is another example corroborating the preference of tryptophan residues to reside in the membrane-water interface.
EXPERIMENTAL PROCEDURES
Reagents of commercial grade and highest purity were used. Spectral grade water obtained using Milli-Q Plus from Millipore Corporation, Bedford, MA, was used in all experiments. Potassium iodide was bought from Loba Chemicals and acrylamide was from SRL, Bombay, India. NBS was purified by recrystallization from water. 9,10-dibromostearic acid was prepared by addition of bromine to oleic acid in CC1^sub 4^ at DoC according to the procedure of Nevenzel and Howton (1957). The product after chromatography on silica gel appeared as a single spot. The product was further characterized by IR, NMR, and mass spectroscopy. Bis-9,10-dibromostearoyl phosphatidylcholine (9,10-BrPC) was prepared from 9,10-dibromostearic acid according to the procedure of Regen et al. (1983) and characterized by NMR. 5- and 12-Doxyl stearic acids were from Molecular Probes (Eugene, OR). Asolectin (Sigma Chemical Company, St. Louis, MO) was further purified by the procedure of Kagawa and Racker (1971) and stored at -20 deg C as a 0.25 M stock solution in chloroform as determined by phosphate assay (Ames and Dubin, 1960). All lipid concentrations expressed are based on phosphate assay. Samples of lyophilized a-toxin were a kind gift from Dr. S. Bhakdi (Institute of Medical Microbiology, University of Mainz, Augustusplatz, Mainz, Germany). Concentration of a-toxin was estimated from absorbance at 280 nm using an A2.. of 1.1 mg ' ml-' (Harshman et al., 1988). All experiments were done at 23 deg C, using 10 mM Tris containing 100 mM NaCI at pH 7.0 (referred to as standard buffer) unless otherwise specified.
Steady-state fluorescence studies
Steady-state fluorescence measurements were done with a Shimadzu RF540 or Hitachi F-4010 spectrofluorometer using a quartz cuvette of 1-cm path length. For quenching experiments, the excitation wavelength was set at 295 nm with a slit width of 5 nm, and the emission range was set between 300 and 500 nm, with the slit width kept at 10 nm. Steady-state fluorescence quenching was carried out by measuring the fluorescence intensities at the emission maxima as a function of the quencher concentration or as a function of time. Increasing concentrations of the quencher were added from a concentrated stock solution of the quencher in water. Fluorescence intensities were corrected for dilution. For acrylamide quenching studies fluorescence measurements were further corrected for the attenuation of the excitation light intensities due the added acrylamide (Parker, 1968), which has a molar extinction coefficient of -0.23 at 295 nm, by multiplying the measured fluorescence by the factor as given,
Red edge excitation shift studies of a-toxin
REES is a powerful tool to gain information about the environment around tryptophan residues of a protein in solution and to monitor structural changes during transformation from a soluble from to a membrane-bound form (Demchenko, 1988; Mukherjee and Chattopadhyay, 1994, 1995). We used REES to get additional information about soluble and membrane-bound a-toxin. Both native and membrane-bound a-toxin showed a 2-nm REES (Fig. 7), indicating that on average, tryptophan residues were not in a motionally restricted environment. It also suggests that there was no drastic change in the average environment around the tryptophan residues on membrane binding especially in terms of solvent reorientation dynamics. The results are shown in Fig. 7.
NBS modification of the native toxin showed that even a 100-fold molar excess of NBS could not abolish the fluorescence of the toxin. The residual fluorescence showed an emission maximum at 328 nm and was blue-shifted with respect to the unmodified toxin, which had an emission maximum of 332 nm. This argued for the fact that some of the tryptophan residues must be in a highly hydrophobic environment and must be facing motional restriction from its surroundings. This was confirmed by REES of NBSmodified toxin, which gave a REES of 5-6 nm. This result is also shown in Fig. 7.
DISCUSSION
Structural aspects of the mechanism of pore formation in membranes by pore-forming toxins can be understood only by the identification of regions of the toxin interacting with the membrane. Song et al. (1996) have recently described the structure of the detergent-solubilized heptameric form of S. aureus alpha-toxin. However, the structure of the watersoluble monomer is not yet known. The a-toxin monomer has been described in terms of a two-domain model by Tobkes et al. (1985). The molecule is thought to be composed of separately folded N-terminal and C-terminal domains connected by a glycine-rich region described as the hinge region. On the basis of the heptamer structure, the protomer core has been described as a beta sandwich formed by separately folded N- and C-terminal domains, connected by the loop region, which forms the stem domain of the heptameric complex (Song et al., 1996; Gouaux, 1998). The toxin is thought to bind to the membrane surface or an unidentified receptor on the surface as a monomer and to oligomerize into a non-lytic heptameric prepore complex. The prepore oligomeric complex is converted to the lyric oligomer by insertion of the glycine-rich loop (residues 110-148) from each protomer, which organizes into a 14stranded beta-barrel in the membrane hydrophobic core.
The protein must go through several structural changes before reaching its functional pore-forming state. The steps we can visualize are 1) binding of monomer to the membrane, probably accompanied by a mild denaturation of the monomer at the interface, resulting in a conformational chance and formation of a molten globule state of the monomer; 2) formation of heptamer by lateral diffusion in the plane of the membrane (interfacial region) and stabilization of the heptamer by formation of intermonomeric contacts; and 3) a second conformational change, resulting in the spontaneous insertion of the loop into the membrane and formation of the membrane active heptameric pore.
Our studies reveal several features of the monomer structure and structural changes occurring in the transition from soluble to a membrane-bound form. In the monomer, the tryptophan residues were buried within a hydrophobic environment, as indicated by an emission maximum of 332 nm, as well as quenching studies using iodide, acrylamide, and TCE. NBS modification and REES of the NBS-modifed toxin supported the conclusion. Upon binding to membranes the structure goes through a conformational change as indicated by the exposure of tryptophan residues (which indicates change in the tertiary structure of the toxin) as suggested by iodide quenching and the shift in the lifetime. Iodide quenching of the membrane-bound alpha-toxin indicated that about 80% of the tryptophan residues were exposed and quenched by iodide. The fluorescence lifetime of the membrane-bound alpha-toxin also supported this conclusion, as the mean lifetime of the tryptophan residues of the membranebound toxin was shorter (1.35 ns) as compared to that of the native toxin (1.79 ns).
Fluorescence quenching by brominated and spin-labeled fatty acids indicated that in the membrane-bound state the tryptophans were not exposed to the lipid hydrocarbon core. One of us (Chattopadhyay and London, 1987) has previously described an elegant method to determine the depth of a fluorophore in a membrane by comparing the quenching by two membrane-bound quenchers, with the quencher group at different depths on the fatty acyl chain. Using this method we calculated the average depth of the membranequenchable tryptophans to be located at a distance of about 10 A from the center of the bilayer. This means that these must lie close to the membrane-water interface. Interestingly, both native and membrane-bound toxins did not show much REES, indicating that on average the tryptophan residues do not encounter motional restriction. As mentioned earlier, most of the tryptophan residues are localized in the rim domain of the pore complex described by Song et al. (1996). It is thought that some regions of the rim domain may dip into the membrane (Song et al., 1996; VecseySemjen et al., 1997). Our measurement of the depth of Trp residues confirms this belief.
The membrane-bound form of a-toxin thus appears to be another example of a protein in which aromatic residues seem to be sequestered at the membrane boundary. Other examples are bacterial porins (Weiss and Schulz, 1992), the bacterial photosynthetic reaction center (Deisenhofer et al., 1995), cytochrome c oxidase (Ostermeier et al., 1996), and bacteriorhodopsin (Grigorieff et al., 1996). Tryptophan is a unique amino acid in that it has the largest nonpolar surface area and is a polar amino acid due to the presence of indole N-H, which gives it the ability to form an H-bond near the interfacial region of the membrane. This duality in chemical structure helps it to float in the interfacial region. Kachel et al. (1995) have analyzed the depths intrinsically favored by tryptophan and tyrosine by studying the location of membrane associating Trp and Tyr analogues using the parallax analysis of fluorescence quenching. These were found to be located at the same depths as Trp and Tyr in membrane proteins. The amphipathic nature of Trp and Tyr residues has been implicated in its interfacial partitioning and acting as anchors or floats for membrane protein inserted into the membrane. This gives stability to the vectorial nature of membrane proteins (Chattonadhvav et al. 1974
For hydrophilic channel-forming proteins such as alpha-toxin, this property of aromatic residues should be one of the factors stabilizing the intermediate membrane-bound monomer. Besides aromatic residues, the crevice between the stem and the rim domain of a-toxin is also rich in basic amino acids. This could provide the basis of the initial electrostatic interaction of the monomer with the membrane surface, besides participating in interactions with the phospholipid headgroups. The anchoring of the protein also could be giving the necessary orientation for the formation of this prepore complex, reducing the collisional requirement from three dimensions to two dimensions and subsequent stabilization of the prepore complex by electrostatic, hydrophobic, and hydrophilic interactions. It is striking to note that some of the bacterial pore-forming toxins whose structures are known, like aerolysin and perfringolysin, are rich in tryptophan residues and have domains rich in tryptophan. These are aerolysin from Aeromonas hydrophila, which has tryptophan-rich domain 2 (Parker et al., 1994), and the perfringolysin O from Clostridium perfringens, which has tryptophans concentrated in domain 4 (Rossjohn et al., 1997). More interestingly, these have been implicated in binding to its membrane receptors. It is therefore tempting to speculate that these tryptophans may be playing a similar role in the process of the transformation of these family of toxins from a water-soluble to a membrane-bound form.
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[Author Affiliation]
Srikumar M. Raja,* Satinder S. Rawat,# Amitabha Chattopadhyay,# and Anil K. Lala* *Biomembrane Laboratory, Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076 and "Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India
[Author Affiliation]
Received for publication 7 July 1998 and in final form 18 December 1998. Address reprint requests to Anil K. Lala, Biomembrane Laboratory, Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India. Tel.: 91-22-5784383; Fax: 91-22-5783480; E-mail: anillala@chem.tb.ernet.in; or to Amitabha Chattopadhyay, Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India. Tel.: 91-40-7172241; Fax: 91-40-7171195; E-mail: amit@ccmb. ap.nic.in.
S. M. Raja's current address is Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago, IL 60612-7213.
[Author Affiliation]
We thank Dr. Sucharit Bhakdi, Institute of Medical Microbiology, University of Mainz, 55101 Mainz, Germany, for providing samples of a-toxin. This work was supported by a grant-in-aid from the Department of Science and Technology and by the Council for Scientific and Industrial Research, Government of India, New Delhi. S.M.R. and S.S.R. are recipients of Research Fellowships from the Council for Scientific and Industrial Research.
