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1 Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, Texas 77843–1114, USA
2 Department of Chemistry, Texas A&M University, College Station, Texas 77843–3255, USA
Reprint requests to: Hagan Bayley, Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA; e-mail: bayley{at}tamu.edu; fax: (979) 847-9481.
(RECEIVED October 25, 2001; FINAL REVISION December 18, 2001; ACCEPTED December 18, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.4360102.
3 These authors contributed equally to this work. 
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Abstract |
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-hemolysin (HL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the HL pore. It is also pertinent to the mechanism of assembly of ß-PFT pores and suggests new possibilities for engineering these proteins.
Keywords: ß barrel; leukocidin; membrane protein; pore-forming toxin; protein-protein interaction; staphylococcal -hemolysin; subunit stoichiometry
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Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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-hemolysin (HL), a ß-PFT comprising a single polypeptide of 293 amino acids, has been studied in great detail (Gouaux 1998; Bhakdi et al. 2000). The crystal structure of the heptameric pore formed by HL in detergent has been determined at 1.9-Å resolution (Song et al. 1996). The pore has also been shown to be a heptamer on erythrocyte membranes (Gouaux et al. 1994), in planar lipid bilayers (Krasilnikov et al. 2000) and supported bilayers (Fang et al. 1997), and after spontaneous assembly in solution (Cheley et al. 1997). It remains conceivable that a small fraction of the HL pore is a hexamer (Czajkowsky et al. 1998). The protein has been subjected to extensive structure-function investigations by mutagenesis and targeted chemical modification. This work has shed light both on the assembly process (Walker et al. 1995; Cheley et al. 1997; Olson et al. 1999) and the properties of the assembled pore (Braha et al. 1997; Movileanu et al. 2000; Howorka et al. 2001). The significance of HL as a pathogenic factor has also been examined (Bhakdi et al. 2000), and engineered HL pores are emerging as useful tools in biotechnology (Eroglu et al. 2000; Bayley and Cremer 2001).
On the whole, the leukocidins have been less thoroughly investigated at the molecular level. Like HL, the leukocidins have been implicated as virulence factors, primarily in wounds and infections of soft tissues (König et al. 1997; Alouf and Freer 1999). Both components of these binary toxins are in the ß-PFT family. There are at least five class-F components that share 71%–79% identity at the amino acid level and six class-S components that share 59%–79% identity (Gouaux et al. 1997; Alouf and Freer 1999). The extent of sequence identity between members of one class and members of the other is 20%–30%. No member of either class is >30% identical to HL (Fig. 1A
). Regions of similarity are dispersed throughout the aligned polypeptides (Fig. 1B). The structures of the water-soluble monomeric forms of two class-F components, LukF (HlgB) and LukF-PV, were determined recently (Olson et al. 1999; Pédelacq et al. 1999). Excluding the stem and N-terminal latch domains, both polypeptides display folds that closely resemble the fold of an individual protomer in the fully assembled HL pore (Fig. 1C). The majority of strictly conserved residues in HL, LukF, and LukS are clustered within the hydrophobic core of the structure and are most likely required to preserve the fold. Together the structures of the LukF monomers and the HL heptamer serve as prototypes for the beginning and end points of ß-PFT assembly (Olson et al. 1999; Pédelacq et al. 1999; Heuck et al. 2001).
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). Recently, it was shown that a leukocidin pore has a unitary conductance of 2.5 ns in 1 M KCl (Miles et al. 2001). As this value is over three times greater than the conductance of HL, the diameter of the leukocidin pore can be estimated to be nearly twice that of HL, arguing in favor of more than six subunits. However, it is possible that the large conductance of the leukocidin pore arises from the lack of side chains equivalent to those in the central constriction of HL: Glu 111, Lys 147, and Met 113 (Fig. 1E). To settle this question, we have determined the subunit composition of the leukocidin pore formed by the class-F component HlgB (here LukF; Cooney et al. 1988, 1993) and the class-S component HlgC (here LukS; Cooney et al. 1988, 1993). |
Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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The Bacillus tail does alter the electrophoretic mobility of leukocidin oligomers enough to permit separation of heteromeric pores with differing subunit combinations. Oligomers containing various ratios of LukF to LukF-TL with wild-type LukS were obtained by cotranslation in the presence of rabbit erythrocyte membranes. The concentration of LukS plasmid in the translation was equal to the total concentration of LukF and LukF-TL plasmids. At least a fraction of leukocidin oligomers are stable in SDS (Sugawara et al. 1997; Miles et al. 2001), and analysis by SDS-polyacrylamide gel electrophoresis and autoradiography revealed five bands (Fig. 2A). Each increment in electrophoretic mobility must correspond to the incorporation of one LukF-TL subunit into the oligomeric complex. The fastest migrating band represents the native SDS-stable leukocidin oligomer containing only LukF and LukS subunits; whereas the band of lowest mobility represents the leukocidin oligomer containing LukF-TL and LukS. Therefore, the three additional bands, in order of decreasing mobility, represent heteromers containing one, two, and three LukF-TL subunits. The result indicates that the leukocidin oligomer contains four LukF subunits.
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). In this case, the result indicates that the leukocidin oligomer contains four LukS subunits. Taken together, the electrophoresis experiments show that the leukocidin pore is an octamer consisting of four subunits of Luk F and four subunits of LukS (Fig. 2C).
The gel-shift experiments are certainly not as clean as those performed previously with HL (Gouaux et al. 1994; Braha et al. 1997). However, leukocidin oligomers are more difficult to form and to handle, and the clarity of the bands could not be improved, even though a wide range of experimental variations were tested, including several different N- and C-terminal extensions and numerous conditions for electrophoresis. Gel-shift experiments involving all four constructs, LukF, LukF-TL, LukS, and LukS-TL, lacked sufficient resolution for individual bands to be distinguished (data not shown).
Subunit composition of leukocidin by targeted chemical modification and single-channel recording
An independent method was used to elucidate the subunit composition of the leukocidin pore. Cysteine residues were engineered with side chains projecting into the presumed lumen (Fig. 1E) and reacted with the sulfhydryl-specific reagent MTSES (sodium (2-sulfonatoethyl)methanethiosulfonate) during single-channel recording in planar bilayers. In the case of cysteine substitutions near the trans mouth of the transmembrane barrel (Fig. 1E), distinct current steps were obtained with MTSES, but mutations at several other positions and alternative reagents gave less favorable results. The voltage-dependent gating of the leukocidin pore differs from that of HL. At positive potentials, above +40 mV, a complex gating profile is seen (Miles et al. 2001). Further, the single-cysteine mutants used here gate at potentials more negative than -80 mV. Therefore, experiments were performed at a holding potential of -60 mV, at which the pores remain open for long periods and carry sufficient current to permit detection of the modifications.
Single-channel recordings were performed on gel-purified leukocidin oligomers containing LukF-S124C and wild-type LukS (Fig. 3A). The unitary conductance was 2420 ± 100 pS (n = 12). When MTSES (1.5 mM) was added to the trans side of the lipid bilayer, a stepwise decrease in the current was seen (Fig. 3A). Four clearly resolved levels were observed, with an average step blockade of 3.5 ± 0.7 pA or 2.4% ± 0.4% (n = 12; Table 1). In 11 of 12 experiments, four steps only were observed over the course of 
5 sec; no further events were seen in the next 45 min. In one case, three events were observed. The cysteine modification could be reversed in a stepwise fashion by the addition of DTT (10 mM; Fig. 3A). The results confirm that the leukocidin oligomer contains four LukF subunits.
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). The unitary conductance was 2560 ± 90 pS (n = 9). Again, four clearly resolved steps to lower conductance were observed upon addition of MTSES (1.5 mM) to the trans chamber, in this case in all nine experiments. The average step size was 3.3 ± 0.6 pA or 2.1% ± 0.3% (n = 9; Table 1). Again, the cysteine modification could be reversed in stepwise fashion by the addition of DTT (10 mM; Fig. 3A). The results confirm that the leukocidin oligomer also contains four LukS subunits.
The total current blockade produced by MTSES modification of the cysteine-substituted leukocidin pores was investigated at the macroscopic current level. For LukF-S124C/wild-type LukS and wild-type LukF/LukS-A122C, the values were 9.9% ± 1.2% (n = 4) and 8.5% ± 0.7% (n = 4), respectively (Fig. 4; Table 1). These results are consistent with the total extent of block seen in the single-channel experiments, suggesting that the majority of leukocidin pores in a population are octamers containing four LukF subunits and four LukS subunits, in agreement with the gel-shift experiments.
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HL pore (Gouaux et al. 1994; Braha et al. 1997). Gel-shift experiments based on genetically engineered truncations or extensions have proved useful in other cases (Heginbotham et al. 1997; Zitzer et al. 1999; Miyata et al. 2001), and this approach was adopted here. However, it was not possible to count all the subunits in a single experiment because the leukocidin oligomers are more difficult to form and to separate. Therefore, the LukF and LukS subunits were counted separately, which in any case led to a complete description of the stoichiometry. Similarly, it was difficult to count all the subunits at once in the bilayer experiments. In experiments with oligomers in which both components were mutated (LukF-S124C/LukS-A122C), we performed one experiment with seven steps, four with six steps, and one with five steps (data not shown). Krasilnikov and colleagues experienced similar difficulties in examining the HL heptamer, in which only 9 out of 38 counts were complete (Krasilnikov et al. 2000), perhaps because of steric or coulombic repulsion between the incoming reagent and the residues modified initially. Therefore, the LukF and LukS subunits were again counted separately and 20 out of 21 experiments revealed four subunits, which we believe to be the full count in each case.
Permutation of the subunits about the central axis
The experiments performed here do not reveal the arrangement of the LukF and LukS subunits around the central axis. In the absence of additional information, we favor the simplest scheme in which LukF and LukS alternate (Fig. 5). This arrangement requires only two types of subunit–subunit interaction; whereas all other arrangements and random assortment generate four different interfaces. Unless a structure of the pore can be determined, resolution of this question will require cross-linking experiments or visualization by electron or atomic force microscopy. It can be noted that both possibilities have been found in membrane proteins: The and ß subunits of the extramembraneous F1 portion of ATP synthase alternate around a threefold axis (Abrahams et al. 1994); whereas the subunits of the nicotinic acetylcholine receptor pentamer (2ß
) are in different environments (Karlin 1993).
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HL, we propose that the transmembrane domain of leukocidin is a 16-stranded ß barrel with two strands contributed by each subunit. In the HL pore, S = N, where S is the shear number (Murzin et al. 1994a) and N the number of ß strands (Song et al. 1996). Assuming S = 16 for the leukocidin pore, the calculated diameter (C–C) is 28 Å (Murzin et al. 1994a,b). By a similar estimation, the diameter of the HL barrel is 25 Å (Song et al. 1996). A simplistic calculation of the expected ratio of conductance values for the leukocidin and HL pores can be made based on these values: D22/D21 = 1.3. This is far lower than the measured conductance ratio of 3.3 (Miles et al. 2001). Therefore, the increased diameter of the barrel caused by the additional subunit only accounts in part for the increased conductance of the leukocidin pore.
Previous determinations of leukocidin stoichiometry
Previous attempts to determine the ratio of LukF to LukS subunits in the leukocidin pore proved unconvincing. Densitometric data from immunoblots of the oligomeric species provided an estimate of 2:1 for the molar ratio of LukF to LukS (Ozawa et al. 1995; Kaneko et al. 1997). On the other hand, SDS gel electrophoresis of pores purified on sucrose gradients have indicated that the ratio is 1:1 (Sugawara et al. 1997, 1999; Ferreras et al. 1998). Studies by electron microscopy were inconclusive. Ring-shaped structures have been observed on erythrocyte membranes, and it has been speculated that they are hexamers (Sugawara et al. 1997, 1999). Finally, the pore has been modeled as a hexamer but with little justification (Pédelacq et al. 1999).
Our results suggest that the majority of the SDS-resistant leukocidin pores are octamers, but it remains possible that a small fraction are hexamers or of other stoichiometries. For example, a fraction of HL pores may be hexamers (Czajkowsky et al. 1998; Bhakdi et al. 2000). Membrane proteins made from large rings of subunits are known to have variable numbers of subunits, as in the case of the cholesterol-dependent pore-forming toxins (Bayley 1997; Heuck et al. 2001) or a fixed number of subunits that varies between homologs, as in the case of the FO domain of ATP synthase (Stock et al. 1999; Seelert et al. 2000; Jiang et al. 2001; Stahlberg et al. 2001). Other proteins with small rings of subunits also have variable numbers of subunits; for example, the Hs1VU protease of Escherichia coli can form hexameric and heptameric rings (Rohrwild et al. 1997), and viral capsid proteins can be located in different environments in the same particle, for example around fivefold or pseudo-six-fold axes in T = 3 icosahedra (Harrison 2001).
Evolutionary considerations
It seems likely that the LukF and LukS genes were formed by an ancient duplication (Archibald et al. 2000). The HL gene, which has a similar extent of sequence identity, must have diverged very soon before or after (Fig. 1A). Presumably, a mutation affecting the interface between the subunits then occurred in one subunit, making it unable to interact with the second. A complementary mutation must have occurred in the second subunit, allowing the first subunit back into the oligomer and fixing the system genetically as a ring of alternating subunits. In accord with this possibility, several F and S genes occur in single transcription units, permitting coordinated regulation of expression (Alouf and Freer 1999; Bronner et al. 2000). Because F and S subunits can mix and match (Prévost et al. 1995), the leukocidin heteromer can display a wide variety of combinations and permutations (Ferreras et al. 1998; Menestrina et al. 2001), which might offer a selective advantage by contributing to the various cellular and species specificities that have been observed (König et al. 1997; Gravet et al. 1998).
Future prospects
Knowledge of the subunit composition of the leukocidin pore raises interesting questions about the assembly process. In the case of HL, monomers first form a heptameric prepore (Walker et al. 1992, 1995). Nothing is known about intermediates in heptamer formation. For example, in one possibility, the heptamer may assemble through multiple pathways involving random collisions of individual subunits and incomplete oligomers. In a second possibility, the sequential addition of individual subunits may occur until a ring is completed. In the case of the leukocidin pore, it is possible that specific dimers are first formed. Because the prepore-to-pore conversion is the rate-determining step, for HL at least (Walker et al. 1995), and the intermediates are short-lived, it will be a challenge to settle this issue.
Knowledge of the subunit composition also opens up new prospects for the engineering of ß-PFTs. HL has been a productive target for engineering studies (Bayley 1999; Eroglu et al. 2000; Bayley and Cremer 2001). However, new methods for forming and purifying heteromeric pores had to be developed to circumvent difficulties arising from the presence of seven identical subunits. For example, procedures have been refined for placing single mutations (Braha et al. 1997) or chemical modifications (Movileanu et al. 2000) within the HL pore. Extensions of these procedures with the leukocidin pore should allow more complex structures to be made involving the modification of two neighboring subunits. It might also be possible to gain control over the number of subunits in a pore by engineering the subunit interfaces.
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Materials and methods |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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Analysis of leukocidin hetero-oligomers
Hetero-oligomers of leukocidin subunits containing LukF and/or LukF-TL with LukS and/or LukS-TL were prepared by mixing the DNA constructs in various molar ratios prior to transcription and translation in a cell-free system in the presence of rabbit erythrocyte membranes (Cheley et al. 1999). The washed membrane pellets were solubilized with sample buffer (Laemmli 1970) and subjected to SDS-polyacrylamide gel electrophoresis in a 5% gel. An autoradiogram was made of the dried gel.
Gel-purified leukocidin oligomers
Leukocidin oligomers were prepared by in vitro expression in the presence of rabbit erythrocyte membranes as described above. As a precaution against the oxidation of cysteine residues, 2 mM DDT was added to the buffer used to wash the membrane pellet (Movileanu et al. 2001). The oligomers were purified by SDS-polyacrylamide gel electrophoresis in an 8% gel in the presence of 0.1 mM sodium thioglycolate (Miles et al. 2001; Movileanu et al. 2001). The oligomers were stored at -80°C in 10 mM Tris.HCl at pH 8.0 containing 2 mM DTT.
Electrical recordings
Single-channel recordings were carried out with planar lipid membranes, as described earlier (Montal and Mueller 1972; Braha et al. 1997). Both the cis and trans chambers of the apparatus contained recording buffer: 1 M KCl, 50 mM Tris.HCl, 200 µM DTT, 100 µM EDTA at pH 8.0. The bilayer was formed from 1,2-diphytanoyl-sn-glycerophosphocholine (Avanti Polar Lipids, Inc.). Protein was added to the cis chamber, which was at ground. Single-channel and macroscopic currents were recorded by using a patch clamp amplifier (Axopatch 200B, Axon Instruments). A Pentium PC equipped with a DigiData 1200 A/D converter (Axon Instruments) was used for data acquisition. The current traces were low-pass filtered with an 8-pole Bessel-filter (Model 900, Frequency Devices) at 0.5 kHz for single-channel currents and 1 kHz for macroscopic currents, and acquired directly by the computer at a sampling rate of 5 kHz by using Clampex8.0 software (Axon Instruments). Measurements were performed at a temperature of 23° ± 0.5°C. Fresh stock solutions (100 mM) of sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES, Toronto Research Chemicals) in recording buffer without DTT were prepared for each experiment and kept on ice.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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