JOURNAL OF STRUCTURAL BIOLOGY ARTICLE NO. SB983959
121, 110–122 (1998)
a-Hemolysin from Staphylococcus aureus: An Archetype of b-Barrel, Channel-Forming Toxins Eric Gouaux Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032 Received January 5, 1998
brane-inserted heptamer (Gouaux et al., 1994). The sensitivity of different cells to aHL varies over many orders of magnitude: human erythrocytes require 1 µM aHL for lysis, while human platelets and rabbit erythrocytes are lysed at an aHL concentration of 1 nM (Hildebrand et al., 1991). The sensitivity of rabbit erythrocytes to aHL suggests that they possess a specific aHL receptor, although it has not been conclusively identified. In contrast, the binding of aHL to human erythrocytes is nonspecific and reflects the inherent membrane-binding activity of aHL (Hildebrand et al., 1991). The cytotoxic properties of aHL are mediated by the channel-forming heptamer and the primary mechanisms of cell damage and death are (i) leakage of ions, water, and low molecular weight molecules out of and into the cell, and (ii) cell lysis (Bhakdi and Tranum-Jensen, 1991). aHL forms relatively large, water-filled channels in cell membranes (Menestrina, 1986). The interior surface of the channel, although polar in overall character, was predicted to contain hydrophobic patches (Bezrukov et al., 1996). From the analysis of the conductance of single oligomers (,60 pS, 115 mV, 0.1 M KCl, pH 7.0), an effective diameter of 11.4 6 0.4 Å has been estimated (Menestrina, 1986). aHL channels formed on cell membranes display partial rectification, modest anion selectivity, and rapid fluctuations to a higher single channel conductance state at acidic pH (Kasianowicz and Bezrukov, 1995; Korchev et al., 1995; Menestrina, 1986). Slow, reversible voltage-dependent channel closure can be induced by low pH or by the presence of di- and trivalent cations (Korchev et al., 1995; Menestrina, 1986). Measurements carried out using planar lipid bilayers and erythrocyte membranes on the G130C single cysteine mutant indicate that residue 130 defines a portion of the trans mouth of the channel (Krasilnikov et al., 1997) and, together with fluorescence energy transfer experiments (Ward et al., 1994), suggest that residue 130 is located near the lipid head groups on the inner leaflet. The heptameric channel, which is sufficiently large to permit
a-Hemolysin, secreted from Staphylococcus aureus as a water-soluble monomer of 33.2 kDa, assembles on cell membranes to form transmembrane, heptameric channels. The structure of the detergentsolubilized heptamer has been determined by X-ray crystallography to 1.9 Å resolution. The heptamer has a mushroom-like shape and measures up to 100 Å in diameter and 100 Å in height. Spanning the length of the molecule and coincident with the molecular sevenfold axis is a water-filled channel that ranges in diameter from D16 to D46 Å. A 14 strand antiparallel b-barrel, in which two strands are contributed by each subunit, defines the transmembrane domain. On the exterior of the b-barrel there is a hydrophobic belt approximately 30 Å in width that provides a surface complementary to the nonpolar portion of the lipid bilayer. The extensive protomer–protomer interfaces are composed of both salt-links and hydrogen bonds, as well as hydrophobic interactions, and these contacts provide a molecular rationalization for the stability of the heptamer in SDS solutions up to 65°C. With the structure of the heptamer in hand, we can better understand the mechanisms by which the assembled protein interacts with the membrane and can postulate mechanisms of assembly. r 1998 Academic Press Key Words: transmembrane channels; heptamer; membrane protein; bacterial toxin; a-toxin
1. INTRODUCTION
a-Hemolysin (aHL) is a self-assembling, channelforming toxin secreted from Staphylococcus aureus as a water-soluble monomer of 33.2 kDa (Bayley, 1995; Bhakdi et al., 1996; Bhakdi and TranumJensen, 1991; Gouaux, 1997; Lesieur et al., 1997). Upon binding to the membrane of a susceptible cell, such as that provided by human platelets (Bhakdi et al., 1988), peripheral blood monocytes (Bhakdi et al., 1989), keratinocytes (Walev et al., 1993), and endothelial cells (Suttorp et al., 1988), the membrane-bound monomer oligomerizes to form a 232.4-kDa mem1047-8477/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
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STRUCTURE AND FUNCTION OF STAPHYLOCOCCAL a-HEMOLYSIN
the passage of single-strand DNA and RNA (Kasianowicz et al., 1996), provides a useful scaffold for the introduction of channel-modifying groups via protein engineering (Bayley, 1995; Braha et al., 1997). Numerous experiments have illuminated critical features of the assembly mechanism of aHL (see below) and have implicated key residues in the steps of membrane binding, assembly, and channel formation and function. a1 Watersoluble monomer
=
a*1
=
a*7
=
a7
Membrane- Heptameric Heptameric bound prepore pore monomer (a,b,c)
In terms of secondary structure, circular dichroism (CD) shows that there is no large net change in the primarily b structure upon conversion of a1 to a7 (Ikigai and Nakae, 1985; Tobkes et al., 1985), although Ikigai and Nakae note that a1 and a7 do differ in their respective CD spectra between 190 and 210 nm. Of course there may be substantial rearrangements of secondary structure between a1 and a7, which give rise to only minor net changes in the CD. In contrast to the limited differences in the CD spectra between a1 and a7, a1 and a7 vary greatly when probed by trypsin and proteinase K: a1 is readily cleaved within the amino latch (A1-V20) and the glycine-rich region (K110-Y148); a*1 and a*7 are only digested within the amino latch; and a7 is remarkably resistant to proteolysis. In fact, a heptameric aggregation state and proteolytic sensitivity at only the amino latch are hallmarks of a*7 a,b,c, the proposed prepore intermediates (Valeva et al., 1997a; Walker et al., 1995). Recent experiments indicate that three preporelike intermediates separate a*1 and a7 (a*7 a,b,c) and that these intermediates differ in terms of the degree of protomer interface formation at residue 35 and the extent to which the glycine-rich region has moved into a hydrophobic environment (Valeva et al., 1997a). Valeva et al. also argue that although prepore formation is a cooperative process, the final step in which the b-strands are inserted into the membrane and the channel is formed is not cooperative and each protomer acts independently (Valeva et al., 1997a). In other words, in the case of a heteroheptamer in which one subunit is defective at a protomer interface but otherwise native-like, the assembled channel has a stem b-barrel composed of only 12 strands and the 2 strands of the mutant promoter do not insert into the bilayer. b-Barrel formation is also dependent on cell type. For example, even though aHL forms heptamers on both aHL-sensitive cells and on aHL-resistant cells, it is only on the sensitive
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cells that formation of the transmembrane b-barrel occurs (Valeva et al., 1997c). Bacterial channel-forming toxins that employ closed b-barrels (b-channel forming toxins b-CFTs); represent a growing family of proteins involved in bacterial pathogenesis (Gouaux, 1997; Lesieur et al., 1997; Parker, 1996). Other members of this group include aerolysin (Parker et al., 1996), anthrax protective antigen (Petosa et al., 1997), Clostridium septicum a-toxin (Ballard et al., 1995), and probably Clostridium perfringens b-toxin (Hunter et al., 1993) and iota-b toxin (Perelle et al., 1993), Bacillus cereus hemolysin-II (Sinev et al., 1993), and vegetative insecticidal protein toxins from B. cereus and B. thuringiensis. Thus, with the first structure of an assembled b-CFT in hand, we can study the structural features and molecular interactions that define a self-assembling, oligomeric transmembrane channel. 2. HEPTAMER AND PROTOMER STRUCTURE
With a shape resembling that of a mushroom, the aHL heptamer measures approximately 100 Å in height and up to 100 Å in diameter (see Fig. 1). A solvent-filled channel runs along the sevenfold axis and ranges from ,15 to ,46 Å in diameter. The overall size and shape of the heptamer as determined from the 1.9 Å crystal structure (Song et al., 1996) is in good agreement with the dimensions of the assembled toxin determined from low resolution electron microscopy studies (Ward and Leonard, 1992). However, results from electron microscopy as well as from other experiments have been interpreted in terms of a hexameric subunit stoichiometry (see for example: Bhakdi et al., 1981; Ikigai and Nakae, 1985; Tobkes et al., 1985; Ward and Leonard, 1992). Although it is clear from crystallographic and chemical modification studies that the predominate, if not the only assembled oligomer is a heptamer (Gouaux et al., 1994), under certain conditions a SDS-stable oligomer of a different subunit stoichiometry may form, such as a hexamer. Alternatively, productive and nonproductive assembly intermediates that are hexamers or other oligomers may form under some conditions. Comprising the heptamer are the cap, rim, and stem domains. The stem domain, a 14 strand antiparallel b-barrel, defines the transmembrane channel. The cap domain protrudes from the extracellular surface and forms a large hydrophilic domain and the 7 rim domains define the underside of the cap and are in close proximity if not in direct contact with the outer leaflet of the cell membrane. A basic and aromatic amino acid-rich crevice between the rim domains and the stem domain forms a binding site for phospholipid headgroups (Song et al., 1996).
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FIG. 1. (A) Ribbon representation of the aHL heptamer viewed perpendicular to the sevenfold axis of noncrystallographic symmetry and showing the putative location of the membrane bilayer; one protomer is a darker shade with its amino (N) and carboxyl termini (C) labeled. The cap, rim, and stem domains are also indicated. The heptamer is approximately 100 Å in height. (B) View of the heptamer down the sevenfold axis. (C) Ribbon representation of a protomer from the aHL structure with the amino latch, b-sandwich domain, triangle region, rim, and stem labeled. The b-sandwich forms the core of the protomer and the triangle domain bridges the stem b-strands and the protomer core. Additional links between protomers are formed by the amino latches. The rim domain, located below the b-sandwich, contains a number of exposed tyrosine and tryptophan residues. The b-strands (1–16) and short turns of a- or 310-helix (A–D) are labeled.
While the protomer b-sandwich domains make extensive interprotomer contacts and define the cap domain, and b-strands 7 and 8 constitute the stem domain, the 7 rim domains participate in only a few protomer–protomer contacts. The structure of a protomeric subunit abstracted from the heptamer is composed of 16 b-strands (52.9%), four turns of a- or 310-helix (4.3%), and substantial non-a, non-b elements of polypeptide structure (42.8%), in good agreement with estimates from circular dichroism data (Ikigai and Nakae, 1985; Tobkes et al., 1985). A 5 1 6 b-sandwich constitutes the core of a protomer from which the amino latch (A1-V20) and b-strands 7 and 8 make pronounced excursions. As shown in Fig. 1C, the rim domain is located beneath the b-sandwich and is continuous, in terms of three elements of secondary structure (b-strands 5, 14, 15), with the b-sandwich domain. Nevertheless, there are a number of reasons to delineate the rim domain as a region distinct in terms of structure, properties, and function. For example, (i) the rim domain does not have as extensive a hydrophobic core as the b-sandwich domain, (ii) the residues located in the rim domain have significantly higher temperature factors than the residues in the b-sandwich domain, (iii) there are a relatively large number of solvent-exposed tryptophan and tyrosine residues, thus rendering the surface of the rim domain distinct from the surface of the b-sandwich domain, (iv) the rim domain is composed of a large fraction of non-a, non-b polypeptide structure, and (v) we speculate that the rim domain may undergo significant conformational rear-
rangements in the a1 to a7 transition. Aerolysin, another heptameric b-CFT, also has a domain that contains a number of exposed aromatic residues. However, the aromatic amino acid-rich region of aerolysin has been predicted to be distal to the membrane surface (Parker et al., 1994). The membrane-spanning stem domain is composed of 14 antiparallel b-strands arranged in a right hand, closed b-barrel that is 52 Å high and 26 Å in diameter, measured from Ca to Ca (Song et al., 1996). Located near the beginning and at the end of the stem-forming b-strands are two tyrosine residues, Y112 and Y148. The aHL b-barrel has a shear number (S; McLachlan, 1979; Murzin et al., 1994a, 1994b) of 14 (S 5 n), which stands in contrast to the porin b-barrels, which have shear numbers of n 1 4, where n is the number of strands (Pauptit et al., 1991; Schirmer et al., 1995). The stereochemistry of the aHL b-barrel is reminiscent of the 20 strand, pentameric b-barrel of GMP cyclohydrolase (Nar et al., 1995) and the 12 strand b-barrels of CksHs2 (Parge et al., 1993) and 6-pyruvoyl tetrahydropterin synthase (Nar et al., 1994). Constrained by the rotational noncrystallographic symmetry, as well as by interactions within and between b-strands, it is likely that the b-barrels of the other b-CFTs will also have shear numbers which equal the number of strands. 3. CHANNEL, MEMBRANE, AND INTERPROTOMER SURFACES
As illustrated in Fig. 2, the solvent-filled channel varies from as narrow as ,15 Å in diameter at the
STRUCTURE AND FUNCTION OF STAPHYLOCOCCAL a-HEMOLYSIN
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FIG. 2. (A) Cylindrical projection of the b-strands of the stem domain. Residues that project their a-carbon substituent to the interior and exterior of the channel are shown on the left and right, respectively. Nonpolar and aromatic residues are depicted in black letters, charged amino acids are in black italicized letters, and polar and glycine residues are in gray. The residues that define the membrane-inserted region of the stem as judged by fluorescence studies of acrylodan-derivatized single cysteine mutants (Y118 and V140) are labeled with asterisks (Valeva et al., 1996). N121, a residue proposed to line the interior of the channel (Valeva et al., 1996), is marked with an arrow. (B) Sagittal section of a solvent accessible surface of the heptamer, colored using the same code as in 2A. Interestingly, the interior of the stem is polar yet uncharged with the exception of the rings of basic and acidic residues at the stem neck and base. (C) Side view. In contrast to the polar and charged nature of the cap domain, the stem domain exhibits a nonpolar belt of residues about 30 Å in width. Exposed aromatic amino acids in the rim domain may interact with the membrane. (D) Intracellular view. This is a view from the membrane looking toward the putative membrane binding surface of the stem and rim domain. On the membrane-facing surfaces of the stem and the rim domains are a preponderance of basic and aromatic amino acid residues. Parts B, C, and D of this figure were made by M. R. Hobaugh and L. Song.
top of the stem to ,46 Å in diameter within the cap domain. Nevertheless, throughout most of the stem domain, the diameter is relatively constant at ,18 Å. Within the stem domain, the residues that line the interior surface of the channel are mostly serine, threonine, and asparagine residues. Notable exceptions are the rings of acidic and basic residues located at the base and top of the stem, the bands of nonpolar residues defined by M113 and L135, and the abundance of glycines. On the exterior of the stem, there is a band of nonpolar residues ,30 Å wide that defines the bilayer-spanning portion of the protein. This ,30 Å
belt matches the hydrophobic portion of a bilayer composed of dipalmitoylphosphatidyl choline lipids (Franks, 1976; Pastor et al., 1991). On the surface of the rim domain there are numerous solvent-exposed aromatic residues, such as Y68, W179, Y182, W187, Y190, W260, W265, and W274 (see Fig. 3). Fluorescence studies on site-specifically modified mutants (Valeva et al., 1996), and additional spectroscopic experiments (Ve´csey-Semje´n et al., 1997), suggest that portions of the rim may insert into the lipid bilayer. Indeed, the lipid binding site located between the top of the stem and the rim (Song et al., 1996) would bring the central region of lipid acyl
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FIG. 3. Ribbon representations of a single protomer from the aHL heptamer structure. (A) Schematic view of a protomer showing the tyrosine, tryptophan, and F120 residues in ball-and-stick mode. Note the abundance of tyrosine and tryptophan groups in the rim domain. (B) Amino acids implicated in membrane binding are located primarily on the outer surface of the rim domain (Walker and Bayley, 1995a). With the exception of R200, the putative membrane-binding or receptor-binding sites are a significant distance (.10 Å) from the proposed location of the membrane in the assembled heptamer. Thus, a substantial reorientation of the protomers may take place following membrane binding or the aHL receptor may project an equally significant distance ‘‘up’’ from the membrane plane. (C) Shown are the residues that when either mutated to cysteine or when mutated to cysteine and derivatized with a ,600-Da reagent diminish or abolish heptamer assembly and cell lysis (Walker and Bayley, 1995a).
chains in close proximity to the exposed aromatic residues of the rim domain and could explain the observation that 9,10-brominated lipids quench the tryptophan fluorescence of the assembled heptamer (Ve´csey-Semje´n et al., 1997). The contacts between subunits are extensive in number, diverse in character, and primarily involve residues in the N-terminal 200 amino acids of each protomer. As illustrated in Fig. 3 and documented in Table I, residues on many distinct portions of the molecule affect cell binding, cell lysis, and heptamer assembly, although here we will focus most of the attention on processes that can be directly related to the heptamer structure. Each protomer participates in about 120 salt bridges and hydrogen bonds, and in approximately 850 van der Waals interactions. In terms of solvent accessible surface area, an isolated protomer (17,120 Å2 ) abstracted from the heptamer structure buries almost one-third of its surface area (5664 Å) in the heptamer. All regions and domains of a protomer, the amino latch, the b-sandwich, the triangle region, the rim, and the stem participate in protomer–protomer contacts. At the amino terminus, a deletion of only the first two residues (AD . . .) abolishes hemolytic activity, although the mutant is still able to bind to cells (Walker et al., 1992a). However, individual charged residues in this region, such as D2, do not appear to play essential roles (Walker et al., 1992a). In agreement with the heptamer crystal structure, a sitedirected fluorescence study showed that the amino termini come into close proximity to each other on
the same time scale as heptamer formation (Valeva et al., 1997b). H35 is a site that plays a key role in assembly and is particularly sensitive to substitution; conservative changes diminish heptamer formation and lytic activity while nonconservative changes abolish both activities (Jursch et al., 1994; Krishna-
TABLE I Residues Implicated in Membrane Binding, Cell Lysis, and Heptamer Formation a Binding R66C d E70C d R200C s D254C s D255C s D276C s
Lysis K8C s D24C d H35C d K37C s H48C s R66C s E70C d D100C s R104C s D108C s K110C d E111C s D127C s D128C s
Heptamer Formation T129C s G130C s K131C s K147C s D152C d D162C s K168C s D183C s R184C s D185C s R200C s D254C s D255C s D276C s
H35C d R184C s R200C s D254C s D255C s
a Residues that, when mutated to cysteine and derivatized with 4-acetamino-48-((iodoacetyl)amino)stilbene-2,28-disulfonate, are significantly diminished in membrane binding, cell lysis, and heptamer forming activity. The open or closed circle following each table entry indicates if the behavior of the underivatized mutant is similar to or significantly reduced from the wild-type protein, respectively. These data are taken from Walker and Bayley, J. Biol. Chem. 270, 23065–23071 (1995).
STRUCTURE AND FUNCTION OF STAPHYLOCOCCAL a-HEMOLYSIN
sastry et al., 1994; Menzies and Kernodle, 1994). The side chain of residue 35 is positioned between bstrands 6 and 9, at the center of a neighboring b-sandwich. Recalling that strands 6 and 9 lead into and out of the triangle, respectively, changes at position 35 may not only affect the stability of a critical interface, but they may also perturb the structure of the triangle region on a neighboring protomer. Additional important protomer–protomer contacts are formed by (i) H48 and D24, and by (ii) K37 and K58 on one protomer interacting with D100 on a neighbor. The triangle region mediates numerous contacts between protomers. These interactions underscore the central role it is likely to play in the assembly and stability of the heptameric channel. For example, (i) K110 interacts with the main chain carbonyl oxygen of Q150 and the side chain groups of D152 and N173 and (ii) N105 forms an intraprotomer hydrogen bond with the main chain nitrogen of L157 and an interprotomer hydrogen bond with the main chain carbonyl of G223. In addition, I107 defines one side of a hydrophobic cluster that faces F153, L219, and V169 on an adjacent subunit. Since mutation of K110 or K152 to cysteine diminishes or abolishes hemolytic activity (Walker and Bayley, 1995a), one would expect that disruption of other interactions in the triangle region would significantly diminish the heptamer formation and lytic activity of aHL. Hopefully, the relevance of particular interactions determined from the aHL heptamer structure will be tested by site-directed mutagenesis and other methods. 4. MECHANISM OF ASSEMBLY
Evidence accumulated to date suggests that the assembly of aHL proceeds from the water-soluble monomer (a1 ) to the membrane-bound monomer (a*1 ), to the prepore states (a*7 a,b,c) and finally to the assembled heptamer (a7; Valeva et al., 1997a, 1995; Walker et al., 1995, 1992a). Although a species with a size similar to that of a heptamer forms in heated aqueous solution and the species retains some hemolytic activity (Cooper et al., 1966), it is not clear how similar the structure of the water-soluble aggregate is to the structure of the membrane-inserted heptamer. Thus, for aHL under physiological conditions, membrane binding precedes oligomerization. Other b-CFTs, such as aerolysin (van der Goot et al., 1993) and anthrax protective antigen (Milne et al., 1994), form water-soluble, channel-competent heptamers in aqueous solution without heat treatment. The water-soluble monomer. In the absence of a high resolution X-ray or NMR structure, what can we say about the structure of a1? On the basis of partial proteolysis, we know that sites in the amino
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latch (K8) and in the glycine-rich linker (K131) are susceptible to trypsin. The chemical modification of cysteine mutants (Krishnasastry et al., 1994; Valeva et al., 1997b, 1996; Walker and Bayley, 1995a) indicates that the amino latch and the glycine-rich linker are accessible to derivatization by sulfhydryl-directed reagents under nondenaturing conditions. Interestingly, the single cysteine mutants between residues 118 and 124 and at residue 140 demonstrate an increased tendency to oligomerize at room temperature in aqueous solution (Valeva et al., 1996). One interpretation of these observations is that the mutation disrupts some element of structure that masks a protomer interface in the heptamer and thus facilitates assembly. Reversible dimerization, via reversible disulfide bond formation, might also promote heptamer formation and could also explain the observations of Valeva and coworkers (Valeva et al., 1996). On the basis of surface labeling studies (Krishnasastry et al., 1994), the carboxyl terminus becomes more reactive in the assembled state, suggesting that the environment around residue T292 changes significantly upon assembly. In fact, T292 is within 8 Å of L25 on an adjacent protomer and therefore the conformation and reactivity of this site might be perturbed by assembly. If we adopt the notion that the water-soluble monomer inhibits heptamer formation in aqueous solution by masking, either by conformational rearrangement or by occlusion, key protomer interfaces present in the heptamer, then we would predict that the stem bstrands may fold back so as to interact with the edges of the b-sandwich domain. However, the rearrangement of the b-strands in a1 should not obscure the outer surface of the rim domain, a region that contains numerous residues important for cell binding (Walker and Bayley, 1995a). Membrane-bound intermediates. Upon membrane-binding, aHL undergoes a number of significant changes prior to the formation of a7: (i) the central, glycine-rich region is no longer susceptible to proteolysis although the residues within this section have not inserted into the membrane (Valeva et al., 1996); (ii) the amino latch remains sensitive to proteolysis (Tobkes et al., 1985; Walker et al., 1995, 1992a); (iii) the reactivity of a single cysteine mutant at H35 (H35C) to a dianionic sulfhydryl reagent (IASD) is reduced significantly and the reactivity of the mutant T292C to the same reagent is substantially increased (Krishnasastry et al., 1994); and (iv) spectroscopic data suggest that tryptophan and tyrosine residues become more solvent-exposed (Ve´cseySemje´n et al., 1997). If the location of residues implicated in cell binding suggest an initial contact site, then membrane or receptor binding occurs on the outer surface of the rim domain (see Fig. 3B).
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TABLE II a-Hemolysin Homologs Protein a
Class b
Strain c
Accession no. d
Mature N-Terminus e
No. of A.A. f
MW g
pI h
References
aHL Hl IIi HlgB HlgB HlgB (LukF) LukF-R LukF-I LukF-PV LukF-PV-like HlgA HlgA (gHLII) HlgA LukM LukS-PV LukS-R HlgC HlgC LukS-I HlgC (LukS) b-Toxin
— — F F F F F F F S S S S S S S S S S —
S.a., Wood 46 B.c., VKM-B771 S.a., ATCC 49775 S.a., Smith 5R S.a., MRSA S.a., P83 S.i., ATCC 51874 S.a., ATCC 49775 S.a., P83 S.a., ATCC 49775 S.a., MRSA S.a., Smith 5R S.a., P83 ATCC 31890 S.a., ATCC 49775 S.a., P83 S.a., ATCC 49775 S.a., Smith 5R S.i., ATCC 51874 S.a., MRSA C.p., type C (CN 5386)
152953 1930114 550424 295156 484391 477911 628931 288292 1262748 550422 484389 295154 577649 551670 477585 550423 295155 628930 400204 410019
*ADSDIN... MKKAKG... *AEGKIT... AEGKIT... *AEGKIT... *AEGKIT... *ANQITP... *AQHITP... AQHITP... *ENKIED... ENKIED... ENKIED... TTNAED... *DNNIEN... *ANDTED... *ANDTED... *ANDTED... *ANTIEE... *ANDTED... *NDIGKT...
293 412 300 300 298 300 300 301 296 280 280 280 280 284 286 286 286 281 286 309
33.25 45.66 34.18 34.12 33.98 34.20 33.86 34.38 33.72 31.81 31.92 31.92 32.01 32.39 32.69 32.54 32.55 32.14 32.53 34.86
7.94 9.02 9.17 9.18 9.25 (9.17) 7.25 8.96 8.99 9.52 9.53 9.53 9.17 8.77 (9.23) 9.32 9.18 9.37 9.10 5.52
(Walker et al., 1992b) (Sinev et al., 1993) (Pre´vost et al., 1995b) (Cooney et al., 1993) (Rahman et al., 1992) (Supersac et al., 1993) (Pre´vost et al., 1995a) (Pre´vost et al., 1995b) (Kaneko et al., 1997) (Pre´vost et al., 1995b) (Rahman et al., 1993) (Cooney et al., 1993) (Choorit et al., 1995) (Pre´vost et al., 1995b) (Supersac et al., 1993) (Pre´vost et al., 1995b) (Cooney et al., 1993) (Pre´vost et al., 1995a) (Rahman et al., 1991) (Hunter et al., 1993)
a
Protein name with synonym in parenthesis. F and S, fast and slow eluting from cation exchange chromatography resin, respectively. c S.a., Staphylococcus aureus; MRSA, methicillin resistant Staphylococcus aureus; S.i., Staphylococcus intermedius; C.p., Clostridium perfringens. d Accession No. is the National Center for Biotechnology Information sequence identification number. e Sequence of the amino terminal 6 residues of the mature protein, after cleavage of the signal sequence. Sequences marked with an asterisk have been determined by N-terminal amino acid sequencing. f No. of A.A., the number of amino acid residues in the mature protein. g MW, the molecular weight of the mature protein (kDa) calculated from the amino acid sequence. h pI, the isoelectric point of the mature protein calculated from the amino acid sequence (Bjellqvist et al., 1993, 1994). i Mature N-Terminus, No. of A.A., MW and pI are for the preprotein. b
Subsequent to membrane binding, conformational rearrangements of the glycine-rich region and probably other interacting portions of the protein expose tyrosine and tryptophan residues in the rim and stem domains, occlude the glycine-rich region from proteolysis, although not from reaction with watersoluble sulfhydryl compounds, and partially bury residue 35 while further exposing position 292 to chemical modification. Of course the changes in reactivity at positions 35 and 292 may also be due to changes in the chemistry of the environment. Most importantly, at the a*1 stage there is no evidence to suggest that any portion of the protein inserts into the lipid bilayer.
Data on the structural features of the membranebound, heptameric prepore intermediates come primarily from proteolysis (Tobkes et al., 1985; Walker et al., 1995, 1992a), chemical modification (Krishnasastry et al., 1994), and spectroscopic experiments (Valeva et al., 1997a, 1995, 1997b, 1996; Ve´cseySemje´n et al., 1997), which frequently have employed site-directed mutants that cannot form the SDS-stable, membrane-inserted channel. These experiments show that the a*7 a,b,c species have an amino latch that is readily severed by trypsin (Walker et al., 1995). On the basis of a recent series of fluorescence experiments using wild-type aHL, single cysteine mutants (H35C, G130C), or other modified
FIG. 4. Alignment of Staphylococcal aHL (Alpha-HL), leukocidin (LukF-PV, LukF-P83, LukF-I, LukS-I, LukM, LukS-PV), and g-hemolysin (HlgB-5R, HlgB-MRSA, HlgB-49775, HlgA-5R, HlgA-MRSA, HlgA-49775) sequences along with hemolysin-II from Bacillus cereus (HL-II) and b-toxin from Clostridium perfringens (Beta-toxin). Within the group of Staphylococcal toxins, there are 28 strictly conserved residues. The approximate correspondence of the elements of secondary structure determined from the aHL heptamer structure are shown above the aligned sequences. Identical residues present in one-half or more of the sequences are shaded in black while conservative substitutions are shaded in gray. The apparently nonhomologous carboxyl termini of hemolysin II from Bacillus cereus was omitted from the figure. Residue numbers relative to the aHL polypeptide are indicated above the aHL sequence. Clustal W (Thompson et al., 1994) was used to perform the alignment and Boxshade 3.21 (http://ulrec3.unil.ch/software/BOX_form.html) was employed to create the figure.
STRUCTURE AND FUNCTION OF STAPHYLOCOCCAL a-HEMOLYSIN
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forms of aHL (Valeva et al., 1997a), it was proposed that in a*7 a, the environment around residue 35 becomes slightly more hydrophobic; in a*7 b, the stem has entered a more hydrophobic proteinaceous environment; and in a*7 c, the context of residue 35 is significantly more hydrophobic and the resulting heptamer has acquired SDS stability. The conversion of a*7 c to a7 is marked by the insertion of the glycine-rich region into the membrane, the folding of the amino latch into the upper-cap portion of the channel, and transmembrane channel formation. A striking conclusion reached by Valeva and coworkers (Valeva et al., 1997a) is that while the transition of a*7 b to a*7 c is cooperative, the transition of a*7 c to a7 is not cooperative. Thus, if a heteroheptamer has a single subunit with an altered protomer interface in which the perturbation is as far from the stem as it is in the case of the H35R mutation, then according to Valeva et al., the two potentially stem-forming strands on the mutant subunit neither enter the membrane nor form a portion of the b-barrel (Valeva et al., 1997a). In other words, the b-barrel formed from a heteroheptamer with one H35R subunit and six wild-type subunits has 12 strands and not 14. In the absence of high resolution structures of the assembly intermediates a*1 and a*7 a,b,c, it is difficult to define structural features of these intermediates. Nevertheless, with the structure of a7 in hand (Song et al., 1996), we can make some observations on the assembly mechanism in general and on the transition from a*7 c to a7 in particular. To begin, it should be emphasized that when using site-directed mutants or other perturbed molecules to elucidate a mechanism, the perturbation may change the mechanism, as has been seen in the study of enzymatic mechanisms (Knowles, 1991). In the instance of aHL, Valeva and colleagues report that the transition from a*c 7 to a7 is not cooperative, as mentioned in the preceeding paragraph. However, it may be that formation of a wild-type-like protomer interface at residue 35 is necessary for insertion of the glycinerich region into the membrane and that, in the wild-type protein, the transition from a*7 c to a7 is highly cooperative. On the basis of the structure of a7, it is clear that the protomer interface region around residue 35 is intimately linked to the triangle region and the top of the stem domain. Perturbations at and near residue 35 are, at least in part, necessarily interprotomer in nature because of the location of residue 35 at a protomer–protomer interface. Consequently, if the interface region around H35 is malformed due to a mutation or a mutation combined with chemical modification, the structure of the triangle region in an adjacent subunit may be disrupted, thus preventing the incorporation of the b-strands into the stem
TABLE III Residues Conserved between a-Hemolysin, g-Hemolysin, and Leukocidin a Domain b
Residue c
Location d
b-Sandwich
F42 L53 G59 I61 K164 W167 F224 P226 F228 Y249 Y282 W286 Y101 P103 (cis) N105 Q150 W80 S186 F196 F210 L219 D254 D45 K50 Y118 G122 G134 Y148
Core, inner sheet Core, inner sheet Core, inner sheet Core, inner sheet Surface, outer sheet Surface, outer sheet Core, inner sheet Core, inner sheet Core, inner sheet Core, outer sheet Core, outer sheet Core, outer sheet Interprotomer contact Triangle vertex Interprotomer contact Interdomain contact, triangle/rim Core, coil Coil, partially exposed Core, coil Core, coil Interprotomer, buried Rim/b-sandwich juncture Cap top, surface Cap top, surface Transmembrane, exterior Transmembrane, exterior Transmembrane, exterior Interprotomer
Triangle
Rim
Cap top Stem
a
Conserved residues according to Fig. 4. Domains as approximately defined in Fig. 1c. c Single letter amino acid abbreviation. d Location and relevant interactions of conserved residues. b
b-barrel. Assuming for the moment that the transition from a*7 c to a7 is cooperative, it could be that the H35R mutant used in the study of Valeva and colleagues (Valeva et al., 1997a) so severely disrupts the interface such that the key interprotomer, i.e., potentially cooperative, interactions can not form. This might be the reason why the glycine-rich, stem-forming strands of the H35R mutant protomer do not insert into the membrane. Clearly, further studies must be carried out in order to elucidate the mechanism of wild-type aHL assembly. 5. THE FAMILY OF b-CFTS FROM S. AUREUS
aHL, gHL, and leukocidin from S. aureus form a group of lytic b-CFTs that are related in primary sequence (Gouaux et al., 1997; Hunter et al., 1993) and three-dimensional structure (Table II and Fig. 4; for recent reviews see: Gouaux et al., 1997; Tomita and Kamio, 1997). Additional members of this family of bacterial toxins are probably b-toxin from C. perfringens (Hunter et al., 1993) and hemolysin II from B. cereus (Sinev et al., 1993). General, although
STRUCTURE AND FUNCTION OF STAPHYLOCOCCAL a-HEMOLYSIN
not totally conserved features of the primary structure of these proteins are a molecular mass of approximately 32–34 kDa, a basic pI, a sequence with no prominent hydrophobic segments, a glycinerich region intermediate between the amino and carboxyl termini, and a preponderance of aromatic residues in the carboxyl terminal half of the protein. However, even the most closely related proteins do not share more than ,30% amino acid identity with aHL and one member, hemolysin II from B. cereus, is substantially larger than the other members of the group. The proteins most similar to one another, in terms of their amino acid sequences, are the leukocidins and gHLs. Within the group of toxins that include only those from S. aureus, there are 28 strictly conserved residues. The locations of the conserved residues in the context of the aHL heptamer structure strengthen the notion that proto-
119
meric units of these b-CFTs have similar folds and structures, at least in regions that correspond to the b-sandwich domain in aHL (see Table III and Fig. 5). Yet because there are many differences in primary structure between these toxins, and since the conserved residues are primarily on the interior of an aHL protomer and not at the protomer–protomer interfaces, the structures of the assembled oligomers may diverge from that of the aHL heptamer. 6. aHL AND PROTEIN ENGINEERING
Specific properties and features of aHL, such as (i) the propensity to self-assemble, (ii) the proteasesensitive, glycine-rich region that lines the lumen of the channel, (iii) the absence of cysteine residues in the wild-type protein, and (iv) the relatively large, solvent-filled transmembrane channel have been
FIG. 5. Ribbon drawing of three protomers, viewed from the lumen of the channel, showing the location of the 28 strictly conserved residues in the family of Staphylococcal toxins. For clarity, the conserved residues are not all depicted on each protomer. Instead, the locations of the residues were chosen to emphasize that the conserved amino acids are primarily clustered in three groups: one centered near F196, another around F224 and F228, and the third localized to L53. This figure was made by L. Song.
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ERIC GOUAUX
exploited by Hagan Bayley and his coworkers in their creative production of novel forms of aHL (for a review see (Bayley, 1995)). Forms of aHL have been created that are activated by specific proteases (Panchal et al., 1996; Walker and Bayley, 1994), by photolysis (Chang et al., 1995), and by site-directed chemical modification (Walker and Bayley, 1995b). One particularly interesting variant which contains five consecutive histidine residues in the glycine-rich region can be blocked from assembly by Zn12 ions and, in the assembled state, the channel is switched off and on by the addition and removal of Zn12 ions, respectively (Walker et al., 1995, 1994). These reversible channels function in both planar lipid bilayers and in plasma membranes (Russo et al., 1997). Further elegant experiments have demonstrated a procedure for making heteroheptamers. As an example of this technology, Bayley et al. have documented the behavior of a heteroheptamer in which a single protomer contains four histidines that may form a Zn12 binding site (Braha et al., 1997). Indeed, the success in engineering channels with desired properties indicates that the future prospects for creating novel aHL channels are excellent. 7. CONCLUSION
The crystal structure of the aHL heptamer has shown, at the level of molecular detail, how a watersoluble protein can create a transmembrane channel. Features such as the bilayer-spanning, 14 strand b-barrel, the putative phospholipid-binding crevice between the rim and the stem domains, and the extensive subunit interfaces, taken as examples, further our understanding of the relationship between structure and mechanism in aHL. The aHL structure adds to our knowledge and understanding of related channel-forming toxins such as leukocidin and g-hemolysin, as well as to more distant relatives, such as anthrax protective antigen and aerolysin. Protein engineering investigations are additional avenues of research that will benefit from a high resolution view of the aHL heptamer. The work by L. Song and M. R. Hobaugh on the structure determination of the a-hemolysin heptamer, together with the collaborative efforts of the Bayley laboratory, have made this review possible. Financial support for work on the a-hemolysin project in particular and on membrane proteins in general has been provided by the Office of Naval Research, the National Institutes of Health, the Chicago Community Trust, the National Science Foundation, and the Alfred P. Sloan Foundation. Past and present members of the Gouaux laboratory are gratefully acknowledged for providing a stimulating research environment. REFERENCES Ballard, J., Crabtree, J., Roe, B. A., and Tweeten, R. K. (1995) The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin, Infect. Immun. 63, 340–344.
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