| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, England, United Kingdom
Reprint requests to: Hagan Bayley, Department of Chemistry, University of Oxford, Oxford OX1 3TA, England, United Kingdom; e-mail: hagan.bayley{at}chem.ox.ac.uk; fax: +44-1865-275708.
(RECEIVED June 11, 2005; FINAL REVISION June 11, 2005; ACCEPTED June 25, 2005)
 |
Abstract |
|---|
 TOP Abstract IntroductionResults and DiscussionMaterials and methodsElectronic supplemental materialReferences |
|---|
-hemolysin (HL) and leukocidin (Luk) polypeptides are members of a family of related 
-barrel pore-forming toxins. Upon binding to susceptible cells, HL forms water-filled homoheptameric transmembrane pores. By contrast, Luk pores are formed by two classes of subunit, F and S, rendering a heptameric structure displeasing on symmetry grounds at least. Both the subunit stoichiometry and arrangement within the Luk pore have been contentious issues. Here we use chemical and genetic approaches to show that (1) the predominant, or perhaps the only, form of the Luk pore is an octamer; (2) the subunit stoichiometry is 1:1; and (3) the subunits are arranged in an alternating fashion about a central axis of symmetry, at least when a fused LukS-LukF construct is used. The experimental approaches we have used also open up new avenues for engineering the arrangement of the subunits of -barrel pore-forming toxins.
Keywords: -barrel; chemical cross-linking; concatameric subunits; leukocidin; pore-forming toxin; staphylococcal -hemolysin; subunit stoichiometry; subunit arrangement
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051648505.
|
Introduction |
|---|
|
TOPAbstractIntroductionResults and DiscussionMaterials and methodsElectronic supplemental materialReferences |
|---|
-Hemolysin (HL) and leukocidin (Luk) are -barrel pore-forming toxins (PFT), which are secreted by Staphylococcus aureus as water-soluble monomers (Alouf and Freer 1999; Bhakdi et al. 2000; Menestrina et al. 2001; Montoya and Gouaux 2003; Kaneko and Kamio 2004). Upon binding to susceptible cells, they assemble into transmembrane pores that cause cell permeation and, in some cases, lysis. The proteins are pathogenic factors in various diseases (König et al. 1997; Prévost et al. 2001, 2003).
HL is a 293-residue polypeptide, which assembles on biological membranes, on lipid bilayers, and in detergent to form homoheptameric pores (Gouaux et al. 1994; Song et al. 1996; Fang et al. 1997; Krasilnikov et al. 2000). Under certain circumstances, a fraction of the oligomer may be hexameric (Czajkowsky et al. 1998). The sensitivity of cells to attack by HL varies over many orders of magnitude, suggesting the existence of a receptor that facilitates assembly (Hildebrand et al. 1991). The receptor on red blood cells remains unidentified, but caveolin may play a role with other cell types (Pany et al. 2004; Vijayavargia et al. 2004a,b). Upon binding to membranes, HL monomers first form an inactive heptameric prepore (Walker et al. 1992, 1995; Olson et al. 1999; Kawate and Gouaux 2003). The prepore then inserts into the lipid bilayer to form the active heptamer. The crystal structure of the HL pore has been solved at 1.9 Å resolution and currently serves as a prototype for the end point in the assembly of PFT (Song et al. 1996). The HL pore is emerging as a useful tool in biotechnology (Bayley and Cremer 2001; Bayley and Jayasinghe 2004). For example, it has been extensively engineered for stochastic sensing, by which a wide variety of analytes is detected at the single molecule level through the modulation of the current flowing through a single pore. Because of their importance in medicine and technology, it is important to understand the assembly and structure of HL and related PFTs in detail.
Unlike HL, leukocidins are bicomponent toxins and the co-assembly of one class F component with one class S component is necessary to form a functional hetero-oligomeric pore (Montoya and Gouaux 2003; Kaneko and Kamio 2004). There are at least six class F proteins (LukF-PV, LukF-R, LukD, LukF'-PV, HlgB, and LukF-I) and seven class S proteins (LukS-PV, LukS-R, LukE, LukM, HlgA, HlgC, and LukS-I) associated with various strains of Staphylococcus aureus (Alouf and Freer 1999; Prévost et al. 2003; Guillet et al. 2004). The F and S proteins share a common ancestor (Kaneko and Kamio 2004). Proteins within each class (F or S) share <70% identity at the amino acid level, while the identity drops to < 27% between members of the two different classes (Prévost et al. 2001, 2003). Members of neither class are > 30% identical to HL (Gouaux et al. 1997; Prévost et al. 2003).
Although the structure of a Luk oligomer is yet to be solved, the structures of two water-soluble class F monomers and one class S monomer have been determined. The LukF (HlgB) structure has been solved at 1.8 Å and 2.3 Å resolution and the fold resembles that of the HL protomer in the heptameric pore, with the exception of the amino latch and pre-stem domains, which are involved in intersubunit interactions and the formation of the transmembrane barrel, respectively (Olson et al. 1999). The LukF-PV structure has been solved at 2.0 Å resolution and is almost identical to LukF (Pédelacq et al. 1999). The LukS-PV structure has been determined recently at 2.0 Å resolution (Guillet et al. 2004). Although most of the fold of LukS-PV is similar to that of LukF, the rim domain shows a significantly different conformation. The lack of a crystal structure for the oligomeric state has limited the use of Luk in protein engineering studies. The similarity between Luk and HL would allow such studies to move ahead in the absence of a structure if two issues were settled: (1) the number of subunits present in the oligomer from each of class F and class S and (2) the arrangement of those subunits around the central axis of the pore.
Numerous experiments have been carried out to determine the stoichiometry and the subunit arrangement of Luk pores (comprising the F component, HlgB or LukF, and the S component, HlgC or LukS) and the closely related 
-hemolysin pore (HL, comprising the F component, HlgB or LukF, and the S component, HlgA or HLII). Densitometric data from immunoblots of HL assembled on human erythrocyte membranes and run on SDS–polyacrylamide gels, suggested a 2:1 molar ratio of LukF to LukS components (Ozawa et al. 1995; Kaneko et al. 1997). Ring-shaped structures of HL were later observed by electron microscopy on human erythrocytes (Sugawara et al. 1997). Densitometric analysis performed on the heat-dissociated rings, isolated from a sucrose density gradient and run on SDS gels, revealed a 1:1 molar ratio of the LukF to LukS components. Based on these findings, a hexameric structure was proposed for the HL pore with very little justification (Sugawara et al. 1997). Similar ring-shaped structures of Luk oligomers were then observed on human polymorphonuclear leukocytes and rabbit erythrocytes (Sugawara et al. 1999). In this case, densitometric analysis and immunoblotting data again suggested a 1:1 molar ratio of LukF to LukS. Kinetic data on membrane permeabilization were subsequently used to justify hexameric structures for both Luk and HL pores assembled on lipid vesicles (Ferreras et al. 1998). Quantification of the heat-dissociated subunits after SDS–polyacrylamide electrophoresis of the oligomers revealed a 1:1 molar ratio of the F and S components (Ferreras et al. 1998).
Contradictory heptameric and hexameric model structures later accompanied the crystal structures of monomeric LukF and LukF-PV, respectively (Olson et al. 1999; Pédelacq et al. 1999). Later, however, gel-shift electrophoresis and single-channel recording experiments, in which the subunits were counted by in situ chemical modification, provided clear evidence for an octameric pore containing four subunits each of LukF and LukS (Miles et al. 2002b). Nevertheless, images of purified HL pores on human erythrocyte membranes obtained by electron microscopy were interpreted as heptamers (Sugawara-Tomita et al. 2002). The results of cross-linking experiments with glutaraldehyde were taken in support of a 3:4 or 4:3 molar ratio of the F to S components. High-resolution electron microscopy of pores with glutathione S-transferase fused to S or F subunits revealed a heptameric arrangement with six of the subunits in an alternating configuration. A model was proposed for the assembly of heptamers, in which an open heterohexamer with an alternating arrangement of F and S subunits is first formed. This is transformed into a closed circular form upon binding of the seventh (S or F) subunit (Sugawara-Tomita et al. 2002). However, the images were not entirely convincing. Because a heptameric arrangement must tolerate some identical neighbors, it seems likely that the full spectrum of subunit combinations should be permitted in this case, including homo-oligomers.
The assembly of LukF and HLII to form the HL pore on erythrocyte membranes has also been investigated at the single-molecule level with fluorescently labeled subunits (Nguyen et al. 2003). Fluorescence resonance energy transfer (FRET) between acceptor and donor dyes attached to the two monomers revealed the formation of LukF-HLII heterodimers and subsequently tetramers, but not homodimers of either LukF or HLII. The investigators again favored a hexameric or heptameric pore.
In the present paper, we present convincing evidence that the Luk pore, formed by the class F component HlgB (LukF) and the class S component HlgC (LukS) (Cooney et al. 1988, 1993) and assembled on rabbit red cell membranes, is an octamer. We further show that there are four LukF and four LukS subunits. Finally, at least when a tandem LukS-LukF construct is used, the subunits are arranged in an alternating fashion around the central axis of the pore.
|
Results and Discussion |
|---|
|
TOPAbstractIntroductionResults and DiscussionMaterials and methodsElectronic supplemental materialReferences |
|---|
; Table 1). Importantly, the forms of higher symmetry have a reduced probability of formation because they correspond to a lower number of linear forms (see Electronic Supplemental Material for details).
|
|
). Unlike HL, neither LukF nor LukS forms homo-oligomericpores (Alouf and Freer 1999;Sugawara et al. 1999). Therefore, it is reasonable to assume that neither F nor S subunits can interact with their own type. If LukF interacts only with LukS as in arrangement J, there are only two distinct interfaces between the two types of subunit (Fig. 1B, b-c and d-a). If two LukF or two LukS subunits can interact with each other, as in any other arrangement (Fig. 1A, A–I), there are four interfaces (Fig. 1B, b-c, d-a, d-c, and b-a). If the d-c and b-a interfaces exist, it seems likely that both LukF and LukS should form homo-oligomeric pores. Against this, it might be argued that the F and S subunits each take up a slightly different fraction of space around the central axis and that four of each in any arrangement are required to close the circle at 360°. It should also be noted that there are other oligomeric membrane proteins in which a particular subunit is arranged in more than one way, e.g., the subunit of the pentameric nicotinic acetylcholine receptor, which occurs in two copies and is flanked (clockwise from the top) by either the and subunits or the 
and subunits (Karlin 1993).
Subunit arrangement of the leukocidin pore by chemical cross-linking of adjacent subunits
To determine whether the leukocidin pore has an alternating arrangement of subunits, we performed a cross-linking experiment. Previous attempts with glutaraldehyde gave an ~50% yield of cross-linked subunits (Sugawara-Tomita et al. 2002). In an effort to improve the yield, we sought to make cross-links between Cys residues introduced at specific sites on adjacent subunits. We used the structure of the hemolysin heptamer to identify Asp-2 and Arg-56 as proximal residues in adjacent subunits. Arg-56 is on a central strand of a -sheet of the cap domain and probably has limited mobility. Therefore, we chose Asp-2 on the flexible amino latch as the site to complete the cross-link (Fig. 2A). Then we identified the corresponding residues in LukF and LukS, Lys-55 and Asn-2, by analyzing the sequence homology between HL, LukF and LukS, and examining the crystal structures (Fig. 2B; Song et al. 1996; Olson et al. 1999; Pédelacq et al. 1999; Guillet et al. 2004). Based on unpublished experiments, we also knew that LukS-Y113H assembles more efficiently and shows higher hemolytic activity when oligomerized with either wild type LukF or LukF-Y117H (data not shown). According to the crystal structures, Tyr-113 in LukS and Tyr-117 in LukF correspond to Tyr-118 in HL. Therefore, where indicated, we used the double mutant LukS-N2C/Y113H to obtain the improved oligomerization yield required for success in the cross-linking experiments.
|
, step A). Various attempts to cross-link the cysteine residues were made with the intact oligomer (Materials and Methods; Fig. 2C, step B). Noncovalent intersubunit interactions were then disrupted by heating the oligomer in SDS. If the LukF and LukS subunits alternated, we expected to see LukS-LukF dimers at this stage by SDS–polyacrylamide gel electrophoresis performed in the absence of thiols (Fig. 2C, step C). We first used the disulfide-forming Cu-orthophenanthroline (Cu-OPA) as the cross-linking agent (Lee et al. 1994; Hamdan et al. 2002). In this case, <50% of the oligomer was converted to dimer and the rest to monomer, according to phosphorimager analysis (Fig. 3A, lane B). We then conducted experiments with 4-sulfophenylarsine oxide (SPAO) as the cross-linking agent. Trivalent arsenic compounds had not previously been used to form intersubunit cross-links in proteins. They have, however, been used in many studies to explore closely spaced protein thiols within individual subunits. For example, phenylarsineoxide binds to thiols in the active site of human thioredoxin with Kd = 370 nM (Donoghue et al. 2000) and methylarsenous acid binds to two thiols in -helical polypeptide chains with Kd = 30–200 nM (Cline et al. 2003). In agreement with the experiments with Cu-OPA, we also observed a dimer after treatment with SPAO and heat dissociation (Fig. 3B, lane B). Phosphorimager quantification revealed that the yield of dimers had been improved to ~65%. As expected, the -S2- or -S-As-S- bonds were cleaved by treatment with dithiothreitol (DTT) (Fig. 2C, step D) to yield the monomeric constituents, LukS and LukF, in a ratio of 1:1 (Fig. 3A,B, lane C).
|
, arrangement J), a maximum of 100% oligomer to dimer conversion by cross-linking would be possible. The lower value we observed could be due to one or more of the following reasons:
, step C), we treated the samples with IASD. Upon SDS–polyacrylamide gel electrophoresis, a shift in both the LukF and LukS monomer bands was observed suggesting that they contained free, reactive cysteine residues (Fig. 3C). ), based on the arguments made earlier. Therefore, we explored the possibility of a random arrangement of subunits in more detail.
Is the leukocidin subunit arrangement random?
First, we calculated the expected oligomer to dimer conversion if the subunit arrangement of the Luk pore was random (Fig. 2E; Table 1; Electronic Supplemental Material). In the HL crystal structure, the N termini of the assembled monomers interact with neighboring subunits in the clockwise direction only (looking into the structure from the cap side) (Song et al. 1996), and we assume that the situation is the same in the leukocidin oligomer. In our mutants, the cysteine residue of LukS is positioned at a specific site in the N terminus and we expect a dimer only if LukS precedes LukF in a clockwise manner (Fig. 2E). We calculated the frequency of occurrence of each octameric form containing four LukF and four LukS subunits generated by random assembly (Table 1). We then estimated the number of potential dimers that would be formed from each arrangement and from that we evaluated the percentage of the subunits from all 10 forms that would be converted to dimers if cross-linking were complete. The value is 57%. Since the value we obtained, 65%, is higher, it is reasonable to argue that the subunit arrangement of the leukocidin octamer is not random and most likely alternating (with <100% efficient cross-linking). Another possibility is a "semirandom" or biased arrangement. Because of the uncertainty, we performed additional experiments that pertain to this issue.
Confirmation that the oligomers contain an equal number of LukF and LukS subunits
As explained earlier, if the subunit arrangement is random, there should be LukS–LukS and LukF–LukF interactions in the oligomers, in addition to LukF–LukS interactions (Fig. 1B). If that were the case, it might be possible for more or less than four subunits of each type to be incorporated into the oligomer. To test this possibility, we oligomerized LukF and LukS in widely different starting ratios (Fig. 4A). We then heated the gel-purified oligomers in SDS to disrupt the inter-subunit interactions. The dissociated LukF and LukS subunits were then separated in an additional gel and their ratios determined by phosphorimager analysis to be close to 1:1 in all cases (Fig. 4B).
|
). In the hemolysin pore, a 37-Å linker is the minimum length required to connect the N terminus of one subunit to the C terminus of the adjacent subunit, assuming that no rearrangement of the polypeptide chain occurs (Fig. 5B). Therefore, we decided to use a 15-residue serine/glycine linker (~48 Å) to connect LukF and LukS.
|
). Interestingly, the activity of the dimer was found to be at least 50 times higher than that of the wild-type subunits (Fig. 5C), although the extent of oligomerization of the dimer was not notably more efficient than that of the wild-type subunits (Fig. 5D). During the assembly of the wild type Luk pore, the two types of membrane-bound monomers must diffuse on the membrane surface, searching for partners with which to interact in the proper orientation to form the prepore. Linking the two subunits together presumably facilitates this process. The unaltered electrophoretic mobility of the oligomer formed from LukS-LukF suggests the structure X (Fig. 6A). Structures of type Z have been proposed when the , , and subunits of the epithelial sodium channel (ENaC) have been concatenated as a trimer (--) (Firsov et al. 1998). In our case, a linker of at least 60 Å would be required to place the LukS-LukF dimer in the structure Z (Fig. 6A, based on the dimensions of the HL pore).
|
), the extreme case is shown where eight subunits are excluded. In other cases where concatenated subunits have been examined, "excluded" subunits have been observed. For example, in the study of ENaC stoichiometry, the three subunits of one -- trimer formed functional channels by incorporating the subunit of a second -- trimer, but excluding the and subunits of the latter (Firsov et al. 1998). Similar exclusion of subunits has been observed in a pentameric neuronal acetylcholine receptor (Groot-Kormelink et al. 2004).
Presumably a structure such as Y (Fig. 6A) would result in an altered electrophoretic mobility of the oligomer. Nevertheless, we decided to perform a gel shift experiment to count the number of LukS-LukF dimers in the oligomer formed from them by using normal dimers and dimers with an extended polypeptide chain. This approach works well with HL monomers, but produced less clear results with LukF and LukS monomers (Miles et al. 2002b). We attached the same 94-residue polypeptide sequence from Bacillus cereus hemolysin II (BacTL) that had been used in these cases at the C terminus of the LukS-LukF dimer. Like the unmodified LukS-LukF dimer, the extended construct, LukS-LukF-BacTL, displayed enhanced hemolytic activity (Fig. 5C, row C, and 5D, lane C). We mixed the unextended and extended dimers in different ratios and allowed them to assemble on rabbit RBCMs, which was followed by examination by SDS–polyacrylamide gel electrophoresis. With the dimer arrangement X (Fig. 6A), we expected to separate five different oligomers corresponding to the five possible combinations of subunits (Fig. 6B, P,Q,R,S,T). We did indeed observe five distinct bands (Fig. 6C), which comprised LukS-LukF and LukS-LukF-BacTL in different ratios as shown by heating oligomers before electrophoresis (Fig. 6D). These results clearly demonstrate that the oligomer contains only four LukS-LukF dimers, which provides additional strong evidence for the octameric stoicheometry of the leukocidin pore and an alternating arrangement of subunits.
In summary, our study proves (1) there are equal numbers of F and S subunits in the Luk pore (by assembly in different starting ratios; Fig. 4); (2) there are four of each type of subunit in the pore (subunit counting experiment [Miles et al. 2002b]; the tandem construct forms an "octamer" that is of the same mobility in an SDS–gel as the wild type Luk pore [Fig. 6]); (3) the F and S subunits can be in an alternating arrangement about a central axis (tandem gene experiment; Figs. 5, 6); and (4) when the Luk pore is formed from individual F and S subunits on red cells, the structure is at least biased toward the alternating arrangement (cross-linking experiment; Figs. 2, 3).
Future prospects for engineering
Knowledge of the crystal structure paved the way to precise engineering of the HL pore (Bayley and Jayasinghe 2004) and prospective applications, for example in biosensor technology (Bayley and Cremer 2001). Leukocidin is also an attractive target for protein engineering, with properties that differ from HL:
HL pore and, even after taking this into account, have an unexpectedly high conductance (Miles et al. 2001). It may be possible, for example, to accommodate larger molecular adapters within the Luk pore than is possible with the HL pore, CDs rather than CDs perhaps. HL; it is possible to engineer one subunit without affecting the other. In the case of HL, it is straightforward to make homoheptamers and heteroheptamers containing one mutant subunit (Fig. 7A). However, other combinations of subunits have more than one permutation about the central axis (Braha et al. 1997). In the case of the Luk pore, the new knowledge about the subunit arrangement will allow four of the eight subunits to be altered and distributed symmetrically around the central axis (Fig. 7B). Eight alterations of two types can also be introduced in an alternating arrangement (Fig. 7C). The HL, LukS, and LukF genes can be aligned (Gouaux et al. 1997), permitting mutations in the LukF and LukS polypeptides to be placed at the same or different levels within the barrel of the assembled pore. PFTs. In the simplest case, mutations in two adjacent subunits can be placed in the assembled Luk pore (Fig 7D). Clearly, this approach might be extended to longer chains of subunits and mixtures of them, or to the HL pore. Active concatamers might be also be useful in studying the assembly of PFTs (Walker et al. 1995; Olson et al. 1999; Nguyen et al. 2003) because they might resemble assembly intermediates.
|
|
Materials and methods |
|---|
|
TOPAbstractIntroductionResults and DiscussionMaterials and methodsElectronic supplemental materialReferences |
|---|
Coupled in vitro transcription and translation
All proteins were generated by coupled in vitro transcription and translation (IVTT) by using an E. coli T7-S30 extract system for circular DNA (Promega). The complete 1 mM amino acid mixture minus cysteine and the complete 1 mM amino acid mixture minus methionine were mixed in equal volumes to obtain the working amino acid solution required to generate high concentrations of the proteins. The amino acids (2.5 µL) were mixed with premix solution (10 µL), [35S]L-methionine (1 µL, MP Biomedicals, 1175 Ci/mmol, 10 mCi/mL), plasmid DNA (4 µL, 400 ng/µL) and T7 S30 extract (7.5 µL) supplemented with rifampicin (20 µg/mL) (Cheley et al. 1997). Synthesis was carried out for 60 min at 30°C to generate 25 µL of IVTT protein. To form hetero-oligomeric proteins containing LukF wild type and LukS wild type, or LukF-K55C and LukS-N2C or LukS-N2C/ Y113H, plasmid DNAs encoding each component were mixed in equal amounts, and a portion of the mixture (4 µL) was used for IVTT. In the cases of the proteins from LukS-LukF and LukS-LukF-BacTL, 4 µL of plasmid DNA was used.
Purification of oligomers for cross-linking experiment
35S-labeled heterooligomers containing LukF-K55C and LukS-N2C or LukS-N2C/Y113H were generated by IVTT (200 µL total volume) in the presence of rabbit erythrocyte membranes (Cheley et al. 1999). The washed membrane pellet was solubilized with sample buffer (Laemmli 1970) and subjected to SDS–polyacrylamide electrophoresis in a 5% gel. An autoradiogram was made from the unfixed gel after it had been dried at room temperature. Using the autoradiogram as the template, the Luk oligomer band was cut from the gel and the fragment placed in 200 µL elution buffer (50 mM Tris. HCl [pH 6.8], containing 1 mM DTT for SPAO cross-linking and 50 mM sodium phosphate [pH 7.3], containing 1 mM DTT for Cu-OPA cross-linking). The rehydrated gel was crushed using a sterile pestle and agitated overnight at 4°C. The suspension was then transferred to a 0.2 µm cellulose acetate Microfilter-fuge tube (Rainin) and centrifuged at 21,000g for 30 min (Miles et al. 2001). The eluted protein solution was transferred to a Microcon YM-10 centrifugal filter device (Millipore) and centrifuged at 14,000g for 20 min. The concentrated sample in the filter device was diluted twice with the desired buffer (300 µL) and reconcentrated (30 min, 14,000g). Finally, the sample was recovered by centrifugation of the inverted filter device (75 µL final volume).
Cross-linking experiments
A portion of the purified oligomer (25 µL) was kept as a control sample (A). The remainder (50 µL) was incubated with either Cu-OPA (20 µL of 15 mM CuSO4; 45 mM 1,10-phenanthroline; 50 mM Na phosphate [pH 7.3]; 10% [w/v] glycerol) (Lee et al. 1994) or 35 mM SPAO (20 µL in 35 mM MOPS [pH 8.0]) (Oneto 1938; Shin et al. 2002) for 10 min at room temperature. After the addition of gel-loading buffer (70 µL) (Laemmli 1970), the samples were heated at 95°C for 5 min and divided into two portions of 70 µL each (B and C). Sample B was not further treated, while sample C was treated with DTT (10 µl, 100 mM) for 10 min at room temperature to cleave the cross-links. Samples A, B, and C were subjected to SDS–polyacrylamide gel electrophoresis in 9% or 10% gels, as indicated (Fig. 3A,B).
Cross-linking with aqueous iodine (Pakula and Simon 1992; Betanzos et al. 2002), bis-((N-iodoacetyl)piperazinyl)-sulfonerhodamine (Corrie et al. 1998), and dibromobimane (Bhattacharjee and Rosen 1996) was carried out by similar procedures. In these cases, the proteins were obtained as indicated above in 50 mM Tris. HCl (pH 6.8), containing 1 mM DTT (75 µL). A portion of this (25 µL) was kept as a control (A). The remaining protein (50 µL) was treated with (1) aqueous iodine: 20 µL of 10 mM aqueous iodine for 15 min at room temperature; (2) bis-((N-iodoacetyl)piperazi-nyl)sulfonerhodamine (Molecular Probes): 20 µL of a 5 mM solution in dimethylformamide for 20 min at room temperature; (3) dibromobimane (Molecular Probes): 20 µL of a 10 mM solution in dimethylformamide for 20 min at room temperature. After cross-linking, the samples were treated and analyzed as indicated earlier.
IASD modification
Dimers of LukF-K55C and LukS-N2C/Y113H were generated by cross-linking of oligomers formed on red cell membranes with SPAO as described above (sample B). After heating at 95°C in gel loading buffer to dissociate the oligomers, the sample was divided into two portions. One half was kept as the control and the other half was incubated with 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonic acid, disodium salt (IASD, Molecular Probes) for 10 min at a final concentration of 10 mM (Fig. 3C). The samples were subjected to SDS–polyacrylamide gel electrophoresis in 10% gels.
Luk hetero-oligomers formed from different ratios of LukF and LukS monomers
Hetero-oligomers containing wild type LukF and wild type LukS were prepared by mixing in various molar ratios the plasmids containing the F and S genes prior to IVTT in the presence of rabbit RBCMs (Cheley et al. 1999). The washed membrane pellets were subjected to SDS–polyacrylamide gel electrophoresis in 5% gels (Fig. 4A). Oligomers were obtained from the gels as described (Miles et al. 2001), concentrated using a vacuum centrifuge, and heated for 10 min at 95°C in the presence of sample buffer (Laemmli 1970). The heated samples were subjected to SDS–polyacrylamide gel electrophoresis in 12.5% gels (Fig. 4B).
Hemolytic assay and oligomerization of Luk dimers
Wild type LukS, wild type LukF, LukS-LukF dimers, and LukS-LukF-BacTL were obtained by IVTT. The protein (25 µL or 12.5 µL each of wild type LukS and wild type LukF in the case of Fig. 5C, row A) was diluted with MBSA buffer (75 µL; 10 mM MOPS titrated with NaOH, 150 mM NaCl [pH 7.4], containing 1 mg/ml bovine serum albumin) in the first well of each row of a microtiter plate (Fig. 5C). MBSA (25 µL) was used as the blank (Fig. 5C, row D). The proteins were then subjected to two-fold serial dilution with the same buffer across each row, leaving 50 µL in each well. Hemolysis was initiated by the addition of an equal volume of washed rabbit RBCs (1% in MBSA) to each well and monitored by observing the decrease in light scattering at 595 nm with a Bio-Rad micro-plate reader (model 3550-UV) (Cheley et al. 1999).
Oligomerization was examined on rabbit RBCM. Wild type LukS and wild type LukF proteins obtained by IVTT were mixed in an equal ratio. This mixture (12.5 µL), LukS-LukF dimer (12.5 µL of IVTT), and LukS-LukF-BacTL (12.5 µL of IVTT) were each incubated with washed rabbit RBCM (5 µL, 4.2 mg/mL of protein) in MBSA (50 µL) for 1 h at 30°C. After centrifugation, the resulting membrane pellets were washed with MBSA and subjected to SDS–polyacrylamide gel electrophoresis in 8% gels (Fig. 5D) (Cheley et al. 1999).
Subunit stoichiometry examined with Luk dimers
Hetero-oligomers containing the LukS-LukF and LukS-LukF-BacTL fusion proteins were prepared by mixing in various molar ratios plasmids encoding two polypeptides, prior to IVTT in the presence of rabbit RBCMs (30 µL total volume) (Cheley et al. 1999). After centrifugation, the resulting membrane pellets were resuspended in MBSA, recovered by centrifugation and dissolved in electrophoresis sample buffer (60 µL). A portion of each (40 µL) was subjected to SDS–polyacrylamide gel electrophoresis in a 5% gel (Fig. 6C). The remainders (20 µl) were heated for 10 min at 95°C and subjected to SDS–polyacrylamide gel electrophoresis in a precast 7% Tris-acetate gel (Bio-Rad).
|
Electronic supplemental material |
|---|
|
TOPAbstractIntroductionResults and DiscussionMaterials and methodsElectronic supplemental materialReferences |
|---|
|
Footnotes |
|---|
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPAbstractIntroductionResults and DiscussionMaterials and methodsElectronic supplemental materialReferences |
|---|
Baida, G., Budarina, Z.I., Kuzmin, N.P., and Solonin, A.S. 1999. Complete nucleotide sequence and molecular characterization of hemolysin II gene from Bacillus cereus. FEMS Microbiol. Lett. 180: 7–14.[CrossRef][Medline]
Bayley, H. and Cremer, P.S. 2001. Stochastic sensors inspired by biology. Nature 413: 226–230.[CrossRef][Medline]
Bayley, H. and Jayasinghe, L. 2004. Functional engineered channels and pores. Mol. Membrane Biol. 21: 209–220.[CrossRef][Medline]
Betanzos, M., Chiang, C.-S., Guy, H.R., and Sukharev, S. 2002. A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension. Nature Struct. Biol. 9: 704–710.[CrossRef][Medline]
Bhakdi, S., Walev, I., Palmer, M., and Valeva, A. 2000. Staphylococcal toxin. In Bacterial protein toxins (eds. K. Aktories and I. Just), pp. 509–527. Springer, Berlin.
Bhattacharjee, H. and Rosen, B.P. 1996. Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase. J. Biol. Chem. 271: 24465–24470.
Braha, O., Walker, B., Cheley, S., Kasianowicz, J.J., Song, L., Gouaux, J.E., and Bayley, H. 1997. Designed protein pores as components for biosensors. Chem. Biol. 4: 497–505.[CrossRef][Medline]
Cheley, S., Malghani, M.S., Song, L., Hobaugh, M., Gouaux, J.E., Yang, J., and Bayley, H. 1997. Spontaneous oligomerization of a staphylococcal -hemolysin conformationally constrained by removal of residues that form the transmembrane barrel. Protein Eng. 10: 1433–1443.
Cheley, S., Braha, O., Lu, X., Conlan, S., and Bayley, H. 1999. A functional protein pore with a "retro" transmembrane domain. Protein Sci. 8: 1257–1267.[Abstract]
Cline, D.J., Thorpe, C., and Schneider, J.P. 2003. Effects of As(III) binding on -helical structure. J. Am. Chem. Soc. 125: 2923–2929.[CrossRef][Medline]
Cooney, J., Mulvey, M., Arbuthnott, J., and Foster, T. 1988. Molecular cloning and genetic analysis of the determinant for -lysin, a two component toxin of Staphylococcal aureus. J. Gen. Microbiol. 134: 2179–2188.[Medline]
Cooney, J., Kienle, Z., Foster, T.J., and O’Toole, P.W. 1993. The - hemolysin locus of Staphylococcus aureus comprises three linked genes, two of which are identical to the genes for the F and S components of leukocidin. Infect. Immun. 61: 768–771.
Corrie, J.E.T., Craik, J.S., and Munasinghe, V.R.N. 1998. A homobifunctional rhodamine for labeling proteins with defined orientations of a fluorophore. Bioconjugate Chem. 9: 160–167.[CrossRef][Medline]
Czajkowsky, D.M., Sheng, S., and Shao, Z. 1998. Staphylococcal -hemolysin can form hexamers in phospholipid bilayers. J. Mol. Biol. 276: 325–330.[CrossRef][Medline]
Donoghue, N., Yam, P.T.W., Jiang, X.-M., and Hogg, P.J. 2000. Presence of closely spaced protein thiols on the surface of mammalian cells. Protein Sci. 9: 2436–2445.[Abstract]
Dorigo, B., Schalch, T., Kulangara, A., Duda, S., Schroeder, R.R., and Richmond, T.J. 2004. Nucleosome arrays reveal the two-start organization of the chromatin fiber. Science 306: 1571–1573.
Fang, Y., Cheley, S., Bayley, H., and Yang, J. 1997. The heptameric prepore of a staphylococcal -hemolysin mutant in lipid bilayers imaged by atomic force microscopy. Biochemistry 36: 9518–9522.[CrossRef][Medline]
Ferreras, M., Höper, F., Dalla Serra, M., Colin, D.A., Prévost, G., and Menestrina, G. 1998. The interaction of Staphylococcus aureus bi-component -hemolysins and leucocidins with cells and lipid membranes. Biochim. Biophys. Acta 1414: 108–126.[Medline]
Firsov, D., Gautschi, I., Merillat, A.M., Rossier, B.C., and Schild, L. 1998. The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J. 17: 344–352.[CrossRef][Medline]
Gouaux, J.E., Braha, O., Hobaugh, M.R., Song, L., Cheley, S., Shustak, C., and Bayley, H. 1994. Subunit stoichiometry of staphylococcal -hemolysin in crystals and on membranes: A heptameric transmembrane pore. Proc. Natl. Acad. Sci. 91: 12828–12831.
Gouaux, E., Hobaugh, M., and Song, L. 1997. -Hemolysin, -hemolysin and leukocidin from Staphylococcus aureus: Distant in sequence but similar in structure. Protein Sci. 6: 2631–2635.[Abstract]
Groot-Kormelink, P.J., Broadbent, S.D., Boorman, J.P., and Sivilotti, L.G. 2004. Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors. J. Gen. Physiol. 123: 697–708.
Guillet, V., Roblin, P., Werner, S., Coriaola, M., Menestrina, G., Monteil, H., Prévost, G., and Mourey, L. 2004. Crystal structure of leucocidin S component. New insight into the staphylococcal -barrel pore-forming toxins. J. Biol. Chem. 279: 41028–41037.
Hamdan, F.F., Ward, S.D., Siddiqui, N.A., Bloodworth, L.M., and Wess, J. 2002. Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor. Biochemistry 41: 7647–7658.[CrossRef][Medline]
Hildebrand, A., Pohl, M., and Bhakdi, S. 1991. Staphylococcus aureus -toxin. Dual mechanism of binding to target cells. J. Biol. Chem. 266: 17195–17200.
Howorka, S. and Bayley, H. 1998. Improved protocol for high-throughput cysteine scanning mutagenesis. Biotechniques 25: 766–772.
Jiang, W., Hermolin, J., and Fillingame, R.H. 2001. The preferred stoichiometry of c subunits in the rotary motor section of Escherichia coli ATP synthase is 10. Proc. Natl. Acad. Sci. 98: 4966–4971.
Jones, D.H. 1995. PCR mutagenesis and recombination in vivo. In PCR primer: A laboratory manual (eds. Dieffenbach C.W. and Dveksler G.S.), pp. 591–601. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Jones, P.C., Jiang, W., and Fillingame, R.H. 1998. Arrangement of the multicopy H+-translocating subunit c in the membrane sector of the Escherichia coli F1FO ATP synthase. J. Biol. Chem. 273: 17178–17185.
Kaneko, J. and Kamio, Y. 2004. Bacterial two-component and heteroheptameric pore-forming cytolytic toxins: Structures, pore-forming mechanism, and organization of the genes. Biosci. Biotechnol. Biochem. 68: 981–1003.[CrossRef][Medline]
Kaneko, J., Ozawa, T., Tomita, T., and Kamio, Y. 1997. Sequential binding of staphylococcal -hemolysin to human erythrocytes and complex formation of hemolysin on the cell surface. Biosci. Biotech. Biochem. 61: 846–851.[Medline]
Karlin, A. 1993. Structure of nicotinic acetylcholine receptors. Curr. Opinion Neurobiol. 3: 299–309.[CrossRef][Medline]
Kawate, T. and Gouaux, E. 2003. Arresting and releasing staphylococcal -hemolysin at intermediate stages of pore formation by engineered protein disulfide bonds. Protein Sci. 12: 997–1006.
König, B., Prévost, G., and König, W. 1997. Composition of staphylococcal bi-component toxins determines pathophysiological reactions. J. Med. Microbiol. 46: 479–485.[Abstract]
Krasilnikov, O.V., Merzlyak, P.G., Yuldasheva, L.N., Rodrigues, C.G., Bhakdi, S., and Valeva, A. 2000. Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus -toxin in planar lipid bilayers. Mol. Microbiol. 37: 1372–1378.[CrossRef][Medline]
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.[CrossRef][Medline]
Lee, G.F., Burrows, G.G., Lebert, M.R., Dutton, D.P., and Hazelbauer, G.L. 1994. Deducing the organization of a transmembrane domain by disulfide crosslinking: The bacterial chemoreceptor Trg. J. Biol. Chem. 269: 29920–29927.
Maurer, J.A., Elmore, D.E., Lester, H.A., and Dougherty, D.A. 2000. Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL). J. Biol. Chem. 275: 22238–22244.
Menestrina, G., Dalla Serra, M., and Prévost, G. 2001. Mode of action of barrel pore-forming toxins of the staphylococcal -hemolysin family. Toxicon 39: 1661–1672.[Medline]
Miles, G., Cheley, S., Braha, O., and Bayley, H. 2001. The staphylococcal leukocidin bicomponent toxin forms large ionic channels. Biochemistry 40: 8514–8522.[CrossRef][Medline]
Miles, G., Bayley, H., and Cheley, S. 2002a. Properties of Bacillus cereus hemolysin II: A heptameric transmembrane pore. Protein Sci. 11: 1813–1824.
Miles, G., Movileanu, L., and Bayley, H. 2002b. Subunit composition of a bicomponent toxin: Staphylococcal leukocidin forms an octameric transmembrane pore. Protein Sci. 11: 894–902.
Minier, F. and Sigel, E. 2004. Techniques: Use of concatenated subunits for the study of ligand-gated ion channels. Trends Pharmacol. Sci. 25: 499–503.[CrossRef][Medline]
Montoya, M. and Gouaux, E. 2003. -Barrel membrane protein folding and structure viewed through the lens of -hemolysin. Biochim. Biophys. Acta 1609: 19–27.[Medline]
Nguyen, V.T., Kamio, Y., and Higuchi, H. 2003. Single-molecule imaging of cooperative assembly of -hemolysin on erythrocyte membranes. EMBO J. 19: 4968–4979.[CrossRef]
Olson, R., Nariya, H., Yokota, K., Kamio, Y., and Gouaux, E. 1999. Crystal structure of staphylococcal LukF delineates conformational changes accompanying formation of a transmembrane channel. Nat. Struct. Biol. 6: 134–140.[CrossRef][Medline]
Oneto, J.F. 1938. Sulfophenylarsonic acids and certain of their derivatives. I. p-sulfophenylarsonic acid. J. Am. Chem. Soc. 60: 2058–2059.[CrossRef]
Ozawa, T., Kaneko, J., and Kamio, Y. 1995. Essential binding of LukF of staphylococcal -hemolysin followed by the binding of HII for the hemolysis of human erythrocytes. Biosci. Biotech. Biochem. 59: 1181–1183.[Medline]
