structure of the key toxin in gas gangrenefulltext.calis.edu.cn/nature/nsb/5/8/nsb0898_738.pdf ·...

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738 nature structural biology • volume 5 number 8 • august 1998

1Department of Crystallography, Birkbeck College, Malet Street, London, WC1E 7HX, England. 2Defence Evaluation and Research Agency, CBD Porton Down,Salisbury, Wiltshire, SP4 0QJ, England.

Correspondence should be addressed to A.K.B. email: [email protected]

Over 50 years ago pioneering studies in microbial pathogenesisshowed that the potent α-toxin of Clostridium perfringens wasan enzyme, namely phospholipase C (PLC)1. This was the firstbacterial toxin to be identified as an enzyme. Further studiesproved, however, that not all PLCs are toxic (Clostridium novyiiPLC, for example, is toxic, while that Bacillus cereus PLC is not),and that therefore enzymatic activity alone is not sufficient fortoxicity. While it has long been thought that the key differencebetween toxic and non-toxic bacterial phospholipases C is the

haemolytic function of the former, there has been little advancein understanding the relative toxicities of the various enzymes.The structure of α-toxin, in combination with the knownstructure of the non-toxic B. cereus PC-PLC enzyme2, providesan opportunity to investigate factors influencing toxicity in amore detailed manner.

Studies of α-toxin have shown that it is the key virulencedeterminant of C. perfringens in gas gangrene. Specific mutantsof C. perfringens that do not produce α-toxin are unable tocause disease, and vaccination with a genetically engineeredtoxoid has been shown to induce protection against gas gan-grene3,4. The toxin is also implicated in the pathogenesis of sud-den death syndrome in young animals5. Recently otherbacterial phospholipases C, including those from Listeriamonocytogenes5 and Mycobacterium tuberculosis5, have beenimplicated in the pathogenesis of a number of diseases.

The C. perfringens α-toxin is a zinc metallophospholipase5.Phospholipases within this group show approximately 30%identity over 250 residues. Haemolytic bacterial phospholipas-es C (C. bifermentans PLC, C. novyi γ-toxin, C. perfringens α-toxin) possess an additional C-terminal 120 amino acids,which can confer toxicity6. The crystal structure of the non-

Structure of the key toxin in gas gangreneClaire E. Naylor1, Julian T. Eaton1, Angela Howells2, Neil Justin1, David S. Moss1, Richard W. Titball2 andAjit K. Basak1

Clostridium perfringens α-toxin is the key virulence determinant in gas gangrene and has also been implicated inthe pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme thathas phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of theenzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacilluscereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structuralanalogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain inprokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain inintracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with α-toxin. Finally, a possible mechanism for α-toxin attack on membrane-packed phospholipid is described, whichrationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

Fig. 1 a, Cartoon representationof the α-toxin chain. The sequenceis shaded from blue at residue 1 tored at residue 370. Three blackspheres indicate the zinc ion posi-tions in the active site. Secondarystructural elements are named.b, Stereo α-carbon trace, sameview as for (a), with balls markingevery 10 residues, and the chainnumbered at regular intervals.All figures, with the exceptionof Fig. 5, have been drawn witha modified version ofMolscript31,32, and subsequentlyrendered using Raster3D33,34.

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nature structural biology • volume 5 number 8 • august 1998 739

toxic B. cereus PC-PLC, which does not possess the C-terminalamino acids is known6 and residues involved in the coordina-tion of catalytically essential zinc ions in PC-PLC are con-served in α-toxin. Both enzymes are able to hydrolyzemonodispersed phospholipids and the toxicity of C. perfrin-gens α-toxin is thought to be related to the ability of thisenzyme also to interact with phospholipids in eukaryotic cellmembranes5. Membrane phospholipid hydrolysis results inthe perturbation of cell metabolism leading to activation ofthe arachidonic acid cascade and protein kinase C4. Increasingevidence indicates that other bacterial phospholipases C,which play important roles in diseases such as those caused byL. monocytogenes and M. tuberculosis, also affect host cellmetabolism in this way5.

In this paper we describe the three-dimensional structure of α-toxin, deter-mined by X-ray crystallography. Inaddition to the anticipated structural sim-ilarity of the N-terminal domain toB. cereus PC-PLC, the C-terminal domain,which has no sequence homology withany proteins of known structure, is shownto have a strong structural similarity toeukaryotic C2 domains. This domain is aCa2+-dependent phospholipid bindingdomain found in eukaryotic second mes-senger proteins, but has not been recog-nized previously in prokaryotes7. In thiscase, it is interesting that this domain hasbeen identified in a prokaryotic proteinthat, as a toxin, may indirectly causechanges in the activation state of eukary-otic host proteins possessing C2 domains.

Structure determinationCrystals of C. perfringens α-toxin, can begrown in two forms8: one in space groupC2221 with cell dimensions a = 61.3 Å, b =177.3 Å and c = 79.1 Å and one moleculein the asymmetric unit, which diffracts to1.9 Å; the second in space group R32 withcell a = b = 151.4 Å and c = 195.5 Å, thisform has two molecules in the asymmetricunit and also diffracts to 2.0 Å. Both crys-tal forms have a solvent content of approx-imately 50%. The structure was solvedusing a combination of molecular replace-ment and single isomorphous replace-ment (SIR) phasing. The structure wasrefined in space group C2221, for which

higher resolution data are available, to an R-factor and Rfree of20.8% and 25.5 % respectively, for all data between 25.0 Å and1.9 Å.

OverviewThe structure of α-toxin is shown in Fig.1. All residues of the α-toxin structure could be placed in the electron density with theexception of a five-residue hydrophobic loop at the surface of theprotein (residues 84–88). This loop has been modeled and ener-gy minimized using X-PLOR9. The protein falls into twodomains: an α-helical N-terminal domain (residues 1–246) con-taining the active site, and a β-sandwich C-terminal domain(residues 256–370), which has been implicated in membranebinding10. A flexible linker (residues 247–255), containing a

Fig. 2 Sequence alignment of α-toxin withhomologous or structurally similar proteins. CB-PLC is C. bifermentans phospholipase C35; PC-PLC, B. cereus phosphatidylcholine-preferringphospholipase C36; HA5L, human arachidonate5 lipoxygenase37; Synap, Synaptotagmin I18 and1LO, soybean lipoxygenase-138. Secondarystructural elements for α-toxin are shownabove, and for PC-PLC and Synap and 1LO,below, alignment: cylinders indicate α-helices;arrows, β-strands. Dark gray boxes indicate con-served zinc binding residues, clear boxes with ablack outline; conserved aspartates possiblyinvolved in Ca2+ binding.

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series of highly mobile residues (average B-factor for the linker is46.6 Å2 ), connects the two domains.

N-terminal domainThe N-terminal domain is the catalytic domain; the C-terminaldomain alone is unable to hydrolyze phospholipid6. As predictedfrom a sequence alignment (Fig. 2), the overall fold of α-toxin issimilar to that of PC-PLC (Fig. 3): 190 Cα atoms can be alignedwith a root-mean-square (r.m.s.) deviation of 1.49 Å, and as faras possible we have retained the helix names used for PC-PLC.There are some secondary structural differences: a six residueinsertion relative to PC-PLC (α-toxin residues 179–184) hasformed an additional helix (labeled F1), while a 16-residue dele-tion in α-toxin (between residues 212 and 213) removes PC-PLChelices G and H1.

Active siteZinc ions had been identified as essential for the catalytic activityof α-toxin5 and at the end of refinement the presence of metalions in the active site was indicated by difference Fourier density.The metal ions and their ligand coordination in α-toxin areshown in Fig. 4. One ion (labeled Zn1 in Fig. 4) is most likely tobe Zn2+, but unusually low B-factors for the other two, coupledwith bond distances that were very long for Zn2+ ion binding (anaverage over all ligands of 2.54 Å for these two, compared to2.32 Å for Zn1), indicated that they are most likely Cd2+ ions(Cd2 and Cd3 in Fig. 4). Since CdSO4

was present at 0.05 M, the fact that cad-mium ions, which can replace zincwithout loss of activity (R. Titball, pers.comm.), were found in the active sitewas not surprising. These metal ions arebound to residues that are conservedbetween α-toxin and PC-PLC and havebeen identified previously as zinc-bind-ing both by comparison with the PC-PLC structure2 and by mutagenesisstudies10. The metal ions have geome-

tries similar to those in the PC-PLC active sites and their lig-and–metal distances are within the expected range11: 2.2–2.3 Å,for zinc and 2.4–2.7 Å for cadmium. Accessibility calculationshave shown that the active site region is available to solvent. Fig.5, which shows the electrostatic potential surface of the mole-cule, highlights the positive potential created by the metals ionsat the base of the active site cleft. Within the cleft, Cd2 is 6 Å fromthe protein surface, closer than the other two metal ions. This ionis also more mobile than the other metals (it has a B-factor of 25Å2 compared to 18 Å2 and 19 Å2), and is bound to His 148, aresidue previously suggested to bind a zinc ion involved in catal-ysis rather than maintaining structural integrity5. We thereforebelieve that Cd2 occupies the site normally occupied by a catalyt-ic zinc ion.

Substrate bindingSome researchers12 have suggested that α-toxin may have bothinactive and active conformations, and that the conformationalchange in the active site is triggered by the binding of calciumion. In this structure, the open active site cleft and the accessibil-ity of the active site zinc and cadmium ions (in particular Cd2,which we have associated with catalytic activity) suggest that theenzyme is in the correct conformation for membrane bindingand enzyme turnover. Byberg et al.13 have constructed a model ofa phospholipid (with shortened tailgroups compared to the invivo substrate of the enzymes) bound to the active site of PC-

Fig. 3 Stereo superposition ofBacillus cereus phostidylcholine-preferring phospholipase C on α-toxin.

Fig. 4 Stereo view of the active site, showingmetal ions as grey spheres. Zinc and cadmiumligands are shown as ball-and-stick, with theremaining residues are drawn as blackbonds. Waters are red spheres. Metal–ligandinteractions are shown as dashed lines.

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PLC. This model, with the phospholipid tailgroups extended,was oriented in the α-toxin structure using a transformationmatrix that optimally superposed the active sites of PC-PLC andα-toxin. As seen in Fig. 5, the modeled phospholipid can beaccommodated in the α-toxin active site cleft without majorstereochemical clashes with the protein. All of the residues with-in 5 Å of the phospholipid headgroup are either identical or con-servatively mutated between PC-PLC and α-toxin. The modeledtailgroups fit into hydrophobic grooves on the enzyme surface.One tailgroup requires at least one cis-double-bond abouthalfway along its length to interact maximally with the surface.This model is in accordance with the finding that phospholipidswith unsaturated fatty acyl chains are the preferred substrate forα-toxin14. The other tailgroup should ideally include a doublebond at approximately carbon atom 6 of the chain for goodhydrophobic contact with α-toxin: for example, the ceramidemoiety of sphingomyelin (Fig. 5). In our model, this tailgroupinteracts with the C-terminal residues 295–305 and is thereforeconsistent with both the observation that the presence of the C-terminal domain enhances sphingomyelinase activity6 and thefact that PC-PLC, which has no C-terminal domain, cannothydrolyze sphingomyelin.

C-terminal domain and calcium ion bindingThe C-terminal domain (residues 255–370) is an eight-strandedantiparallel β-sandwich with an immunoglobin-like fold.Calcium ions are known to activate α-toxin, and recent circulardichroism (CD) results have shown that calcium binds specifi-cally to the C-terminal domain and induces a conformationchange (B. Bolgiano, pers. comm.). Several attempts were madeto obtain the crystal structure of calcium-bound α-toxin.However, calcium is insoluble in the crystallization buffer forform I crystals, and soaking form II crystals with even micromo-lar quantities of a calcium salt causes them to shatter. No calciumion sites have, therefore, been identified unequivocally. However,a Cd2+ ion (Cd13), which is ligated by six oxygen atoms in an

octahedral arrangement, has been identified in the C-terminaldomain of the protein and is ligated by Asp 336 (Oδ1); Asp 269(O); Gly 271 (O); Ala 337 (O) and two water molecules (Fig. 6).This metal binding site has ion–ligand distances of 2.3–2.5 Å.Two of the ligands to this site (Asp 269 and Asp 336) have beenidentified recently by Guillouard et al.15 as important for calci-um-mediated activation of α-toxin. We have therefore identifiedthis site as a putative Ca2+ binding site. Although the side chain ofAsp 269 is not currently interacting with the ion, it is situatedsuch that a slight alteration of side chain conformation wouldallow its side chain to become a ligand. We speculate that it is notinteracting with the ion in the structure due to the rigid ligandgeometry required by a Cd2+, but that it would interact with aCa2+ ion. Asp 269 is not conserved in Clostridium bifermentansphospholipase C (Fig. 2), where it is mutated to tyrosine.However, in the PC-PLC sequence, Ala 337 is mutated to aspar-tate, and this residue is positioned such that its side chain couldinteract with a Ca2+ ion at the Cd13 site in a manner similar tothat proposed for Asp 269 in the α-toxin structure.

In addition to Cd13, a number of other Cd2+ ions have beenidentified bound to the surface of the protein in non-specificsites, due to the relatively high concentration of cadmium ions inthe crystallization buffer (0.05 M). Two of these ions (Cd9 andCd12) are close to the Cd13 site, and interact with three otherhighly conserved aspartate residues (273, 274, 293). But sincetheir sites are completed by ligands from symmetry related mol-ecules, and these interactions can have no biological significance(the molecule is active as a monomer), definite identification ofany other Ca2+ binding sites must await further crystallographic,or biochemical data.

Relationship to other proteinsThree-dimensional structural alignment using the program Dali(http://www2.ebi.ac.uk/dali/) with proteins in the BrookhavenProtein Data Bank (PDB) shows that the C-terminal domain isstructurally similar to several other β-sandwich domains. It ismost similar to the C-terminal domain of pancreatic lipase16

(HPL, PDB entry 1GPL, 86 Cα atoms superpose with an r.m.s.deviation of 1.92 Å), and the N-terminal domain of Soybeanlipoxygenase-117 (LO1, PDB entry 2SBL, 76 Cα atoms superposewith an r.m.s. deviation of 2.1 Å). The C-terminal domain of α-toxin also resembles the structures of two C2 domains in thePDB: those of synaptotagmin I18 (PDB entry 1RSY, 60 Cα atomssuperpose with an r.m.s. deviation of 2.4 Å) and the C2 domainof phosphoinositide-specific phospholipase Cδ119; (PI-PLC,PDB entry 1DJI, 60 Cα atoms superpose with an r.m.s. deviationof 2.2 Å). All of these proteins have additional catalytic domains,which show no structural analogy either among themselves orwith the α-toxin C-terminal domain.

The C-terminal 120 amino acids of HPL, 1LO and α-toxinhave identical topologies (Fig. 7a,b), while the remaining twodomains are different (Fig. 7c,d). A PI-PLC-like topology can begenerated from α-toxin by interchanging α-toxin strands 6 and 8during folding; the connectivity is unchanged (Fig. 7c).Synaptotagmin I’s connectivity is a circular permutation of thestrands in PI-PLC, as illustrated in their topology diagrams(Fig. 7d). Despite the differences, all eight strands in α-toxinsuperpose well with their equivalents in synaptotagmin I and PI-PLC, and residues from every strand were used in the calculationof r.m.s. deviations between the structures.

C2 domains are Ca2+ and/or phospholipid-binding domainscontaining approximately 120 amino acids; they occur in a widerange of eukaryotic signaling proteins, and are involved in a large

Fig. 5 GRASP39 electrostatic potential surface of α-toxin (shown with theactive site cleft and membrane-binding surface uppermost). Active sitecadmiums were included, but surface cadmium ions removed, for the cal-culation. The positive oxyanion hole for the phosphate head group is vis-ible at the base of the cleft, as indicated by a modeled sphingomyelin.Some residues thought to be important for membrane binding havebeen indicated. The electrostatic potential is scaled from red for -20.0 toblue for +20.0.

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range of processes including lipid-second messenger generation,activation of GTPases and phosphorylation control7. It has beensuggested that C2 domains do not exist in prokaryotes becausecalcium regulation of second messenger systems is uncommonin these organisms7, however a relationship between α-toxin andC2 domains was postulated by Guillouard et al.15, and compari-son of the sequences aligned by equivalent strands7 reveals thatthe pattern of non-polar/polar conservation seen in C2 domainsis 80% retained in the sequence of α-toxin C-terminal domain(Fig. 2).

We have already described a likely calcium ion site (Cd13), lig-ated by conserved aspartates and main chain carbonyls from twoloops. When α-toxin and PI-PLC C2 domains are optimallysuperposed, Cd13 is 2.4 Å from the calcium ion site labeled assite II by Essen et al. in PI-PLC20. After optimal superposition ofα-toxin on synaptotagmin I, Cd13 is 8.5 Å from the site labeledCa1 by Shao et al. in synaptotagmin I21. Site II in PI-PLC and Ca1in synaptotagmin, though differing in detail, have beendescribed by Essen et al.20 as equivalent, because in both cases

Ca2+ is ligated by conserved aspartates on equivalent loops.Specifically, Ca1 in synaptotagmin is ligated by Asp 172and Asp 178 in loop β2β3, and by Asp 230 and Asp 232 inloop β5β6. Site II in PI-PLC is ligated by Asp 653 in loopβ1β2 (topologically equivalent to β2β3 in synaptotagmin,Fig. 7c,d), and Asp 706 and Asp 708 in β5β6 (equivalent toβ6β7 in synaptotagmin). In either case, main chain car-bonyls in both loops also contribute to the metal ion bind-ing. Inspection of the structure-based sequence alignmentfor α-toxin and synaptotagmin I shows that, althoughthere is no exact alignment of proposed calcium bindingresidues between α-toxin and synaptotagmin I (Fig. 2),these residues occur on loops β1β2 and β5β6, which aretopologically equivalent to the calcium ion binding loopsfor PI-PLC and synaptotagmin I (Fig. 7a,c,d). Ca2+ istherefore ligated to α-toxin in a manner highly reminis-cent of Ca2+ binding to PI-PLC and synaptotagmin I, in aspatially equivalent position.

Implications of the structural analogy with C2domainsThe evidence presented suggests a functional and structuralanalogy between eukaryotic C2 domains and the C-termi-nal domain of α-toxin, although there is no evidence of anycommon evolutionary ancestor. This is the first prokaryoticexample of a C2-like domain. Phosphokinase C (PKC) con-tains the first identified C2 domain and, recent evidence5

has shown that the diacyl glycerol product of α-toxin attackis able to interfere with the pathways that PKC regulates.This suggests that α-toxin uses its C2-like domain fold tobind membrane in a manner similar to those of the proteinswhose function it disrupts: α-toxin may cause haemolysisby mimicking proteins in the cells which it attacks.

The N-terminal 120 amino acids of human arachidonate5-lipoxygenase (HA5L) is homologous to the C-terminaldomain of α-toxin (there is a 34% identity over the 120amino acids). The structures of two lipoxygenases areknown: that of rabbit 15-lipoxygenase22 (15LO), which hasa 39% identity over its entire sequence with HA5L, and soy-bean 1-lipoxygenase (1LO)17, which has a 30% identity withthe C-terminal domain of HA5L. There is little sequencehomology between the N-terminal domains of 1LO andHA5L, and there is no similarity between the sequences of1LO and 15LO and α-toxin. 15LO and 1LO have identical

topologies and the topologies of 1LO and α-toxin are the same(Fig. 7a,b). The lipoxygenases are all members of the leukotrienesynthetic pathway, which is disrupted by α-toxin.

The similarity between the N-terminal β-sheet domain in1LO and 15LO and C2 domains has not previously been noted,however these two proteins are cytosolic and show no Ca2+

dependence. Instead of interacting with membrane, they inter-act primarily with lipoxygenase-activating lipoprotein. Aninspection of the sequences of 1LO and α-toxin aligned bystructure (Fig. 2) shows that none of the residues identified asinvolved in calcium binding is conserved, and that large inser-tions occur in these loops (Fig. 7b). 15LO also has large inser-tions in the Ca2+ binding loops, and all but one (Asp 336) of theconserved residues are mutated. In this they bear a strong simi-larity to human pancreatic lipase, described earlier as sharingthe same fold as α-toxin C-terminal domain, 1LO and 15LO.HPL also binds a colipase23 protein, and not calcium; HPL doesnot have the aspartates we have identified with membranebinding.

Fig. 6 Model for membrane binding. Phospholipids are shown as red spheres forheadgroups and gray cylinders for tailgroups, except for one short-tailed lipidpictured in the active site as a space filling model. α-toxin is shown with residuesshaded from gray for those distant (>15 Å) to blue for those closest (< 5 Å) to themembrane, the active site metal ions are shown as black spheres. Some residuesexpected to be important for membrane binding are illustrated, and the proba-ble calcium binding site is shown in more detail in the insert.

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In contrast, HA5L is calcium dependent and membranebinding24. In its sequence (Fig. 2) four of the five Asps that wehave associated as possibly involved in Ca2+ binding are con-served. Of the Cd13 ligands, only Asp 269 is mutated to pheny-lalanine, and in HA5L, as for PC-PLC, residue 337 is anaspartate, and could replace Asp 269 in the binding site. Thereare no large insertions in the calcium binding loops.Understanding of HA5L is therefore also likely to benefit fromcomparison with C2 domain mechanisms of action.

In the light of these structures, α-toxin C-terminal domain(and HA5L) can be seen as an intermediate between two dis-tinct groups of β-sandwich structures. While it has the sametopology as the lipoxygenase group, the function of α-toxin is

quite different from the β-sheet domains in these proteins. Thefunction of α-toxin is identical to that of the eukaryotic C2domains, although its topology is different.

Membrane–protein interactionsTwo mechanisms have been suggested for the binding of C2domains to membranes7. The first proposal is that calciumand/or contact with the membrane catalyzes a large conforma-tional change which exposes a hydrophobic surface for mem-brane insertion. The second suggestion is that calcium ionsmediate interactions with the phospholipid headgroups, creat-ing a charge-charge interaction. This structure supports thesecond possibility.

a b

c d

Fig. 7 Comparison of α-toxin C-terminal domain with other similar folds. a, α-toxin C-terminal domain is shown in blue with human pancreatic lipase(HPL) superposed in yellow. Positions of possible Ca2+ binding sites shown as spheres. b, The N-terminal domain of soybean lipoxygenase-1, notelarge insertions, and the C2 domains of c, Synaptotagmin I and d, PI-PLC. The position of calcium bound to these domains is indicated by greyspheres. A topology diagram for each fold is shown to its upper right, positions of calcium binding residues on these are indicated by red circles.

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Inspection of the α-toxinstructure revealed two extreme-ly exposed hydrophobicresidues; Trp 214 and Phe 334have surface accessible areas of151 Å2 and 164 Å2 respectively.The plane defined by these tworesidues and Cd13, indicatesthe approximate position of amembrane binding surface(Fig. 6), with the open activesite close to, and facingtowards, phospholipid. Thissuggested penetration of Phe334 into the membrane is mir-rored by a proposed penetra-tion of the membrane by ahydrophobic loop in the C2domain of the recently solvedstructure of cPLA225. The posi-tion of the membrane bindingsurface was refined by includinga number of additional atoms(the Cβ from Ala 146, Trp 214,Asp 269, Tyr 275, Asp 336 and Phe 334 and Cd13) in the planedefinition. Tyr 275 had previously been suggested to be a mem-brane binding residue by Guillouard et al.15. Using this refinedplane, membrane interacting residues are defined as those thathave an atom with a deviation of 5 Å or less from the plane. InFig. 6 these are highlighted in blue (residue numbers 59–65,78–83, 135, 139–154, 204–205, 208–217, 268–275, 293–301,329–337, and 364). A sequence comparison shows that, whereasthe overall identity between C. bifermentans PLC and α-toxin is50%, for these loops the two sequences are 75% identical.Furthermore, while the overall identity between PC-PLC and α-toxin is 30%, for the putative membrane binding residues in theN-terminal domain (PC-PLC has no C-terminal domain andcannot bind membrane), the sequence identity is 16%.

The previous paragraph describes a protein capable of interact-ing with a membrane surface, both by burying highly exposedlarge hydrophobic residues and by forming a complex with calci-um and phospholipid headgroups. This interaction could insti-gate conformational changes elsewhere in the protein: a highlyhydrophobic, disordered surface loop (residues 84–88, WYLAY)is bordered on one side by active site residues and on the other bycalcium-binding and domain interface residues (Fig. 1). The loopis not present in PC-PLC, which does not possess a membraneinteracting C-terminal domain (Figs 2, 3). Its highly hydrophobicnature suggests involvement in phospholipid tailgroup binding.Given its position between the active site and the C-terminaldomain, another possible function of this loop could be to com-municate calcium and/or membrane-binding to the active site,thereby initiating any conformational changes required to acti-vate enzyme.

Haemolytic activityFinally, the toxic properties of individual zinc metallophospholi-pases C have been correlated with their abilities to cause lysis oferythrocytes5. This model allows us to suggest a molecular expla-nation for the presence or absence of this function. Two examplesare given below.

PC-PLC has no haemolytic activity26. This protein lacks the C-terminal domain present in α-toxin, which is important for

membrane binding, and which we have identified as a C2-likedomain. In addition, sequence identity with the α-toxin N-termi-nal domain is low (16%) in the putative membrane bindingloops. Two of these binding regions have been completelyobscured: PC-PLC helices G and H1 replace the exposed Trp 214,and the loop including residues 78–83 has been deleted (Fig. 3).We propose that this enzyme is not haemolytic because it hasnone of the membrane-interacting regions identified in α-toxin.

C. bifermentans PLC is weakly haemolytic. It shows 47%sequence identity with α-toxin, a figure that rises to 75% close tothe active site and membrane binding regions, suggesting itshould be strongly haemolytic. However, in the α-toxin–mem-brane model there is a loop (residues 329–337) raised above themembrane binding surface of α-toxin (Fig. 6). This loop containsa larger than average number of mutations between the C. per-fringens and C. bifermentans sequences in particular Tyr 331 (C.perfringens) is changed to Ile (C. bifermentans) and Phe 334 (C.perfringens) to Ile (C. bifermentans). The reduction in size of theseresidues, and consequently in the hydrophobic contact areabetween C. bifermentans PLC and phospholipid, should reducethe free energy of membrane–protein complex formation relativeto α-toxin, thereby reducing its activity and therefore toxicity. Wewould suggest then, that these residues may be crucial to the toxi-city of α-toxin, and that the lack of conservation in other relatedproteins could explain the relative inefficiency of other PLCenzymes present in the environment.

ConclusionThe structure of α-toxin has revealed both an expected homologyto B. cereus PLC, and a structural analogy to the C2 domains ofintracellular second messengers in eukaryotic cells, and the β-sheet domain found in lipoxygenases. Thus, α-toxin may beviewed as an intermediate between these two topologies.However, because of its calcium binding function and mem-brane-binding functions, we have identified the α-toxin C-termi-nal domain as the first example of a prokaryotic C2-like domain.Finally, modeling the membrane binding of α-toxin has allowedus to suggest possible reasons for its potent toxicity, in compari-son to other, highly homologous, but harmless, enzymes.

Fig. 8 Stereo view of 2Fo - Fc omit map for the metal ions in the catalytic domain, together with the final co-ordinates. Map contoured at 1σ.

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MethodsCrystallization. C. Perfringens α-toxin strain CER89L43 wasexpressed in Escherichia coli and purified to homogeneity asdescribed8. Two different crystal forms were grown by hanging dropvapor diffusion at 20 ºC. Form I grew in the presence of 0.05 MCdSO4, with 0.1 M Na-HEPES, pH 7.5 as buffer and 1 M Na-acetate asa precipitant, while form II crystals grew in 0.1 M sodium acetatebuffer at pH 4.6, using 1.7 M NaCl as a precipitant. Form I crystalsare in spacegroup C2221 with unit cell dimensions a = 61.3 Å, b =177.3 Å, c = 79.1 Å, α = β = γ = 90º, and one molecule in the asym-metric unit. The solvent content of the crystal is 50%. Form II crys-tals belong to spacegroup R32 with cell dimensions a = b =151.4 Å, c = 195.5 Å, α = β = 90º, γ = 120º. There are two moleculesin the crystallographic asymmetric unit and the solvent content ofthese crystals is also 50%.

Data collection. The high resolution C2221 native data set was col-lected on stations 9.5 and 7.2 of the synchrotron radiation source atDaresbury Rutherford Appleton Laboratory, on a 300 mmMarResearch ImagePlate. All other data sets, for both the crystalforms, were collected using an in house 180 mm MarResearchImagePlate mounted on a Siemens rotating anode generator operat-ing at 40 kV and 90 mA. All data were processed with the DENZO27

program suite. Scaling and merging were carried out withSCALEPACK, from the same suite. Subsequent data manipulationused the CCP4 program package28. Statistics for each data set aregiven in Table 1.

Molecular replacement. Initial phasing for the C2221 structure wasprovided by the placement of a previous, incompletely traced modelof C. perfringens α-toxin, strain NCTC8237 (Basak et al., unpublisheddata) into this crystal form using X-PLOR9 (Initial R-factor and Rfree val-ues following rigid-body refinement were 50% and 53% respectively).Following refinement of this model, also using X-PLOR, R-factor andRfree values fell to 46% and 43% respectively. Two copies of this modelcould then be placed in the R32 crystal form, using AMoRe29 (correla-tion coefficient 0.49 and R-factor 46%, after initial rigid body), relatedby a translation of half the c-axis, with no rotational component.Details for both molecular replacements are given in Table 1.

SIR phasing. The protein was derivatized with ehtylmercury chloride(EMC) by soaking the R32 crystals for 24 hours in the crystallisationbuffer containing 2 mM EMC. Heavy atom positions were identified

from Patterson difference maps, and confirmed by a differenceFourier map, using the R32 molecular replacement phases. Quality sta-tistics for the derivative are shown in Table 1.

Density modification. Cross-crystal averaging (between the C2221

form model phases and the non-crystallographically averaged R32form SIR phases) and solvent flattening were performed prior tomodel building. The solvent flipping option within DM30 was chosen.During an average round of refinement the Rfree fell from 25% to 9%during this process.

Model building and refinement. The improved phases meant that amuch improved model could be constructed (in the C-terminal domainonly 29 Cα atoms had been approximately correctly positioned in theprevious ‘best’ model), and refinement of the C2221 crystal form pro-ceeded smoothly through rounds of manual rebuilding and refine-ment using X-PLOR9. A bulk solvent correction was made (B = 110 Å2, k= 0.43) allowing all data (25.0–1.9 Å) to be included in later rounds ofrefinement. Waters were included in the later stages at stereochemi-cally sensible positions with a difference density peak of at least 3.5σ.A number of very strong difference peaks were seen (>10 times ther.m.s. electron density in a Fourier synthesis) subsequent to completetracing of the chain, both in the active site and at the surface of theprotein. After consideration of the crystallization buffer these peakswere identified as cadmium ions. Model validation statistics are shownin Table 1; four disallowed residues occur and all four are in a highlymobile region immediatily preceding the modeled residues 84–89. Theaverage B-factors for the main and side chain atoms have remainednearly identical, despite the loosening of restraints at the end ofrefinement.

Coordinates. The refined coordinates have been deposited in theBrookhaven Protein Data Bank with accession number 1CA1.

AcknowledgmentsThe authors would like to thank D. I. Stuart (Laboratory of Molecular Biophysics,Oxford University) for help with the original structure and J. Thornton (University andBirkbeck Colleges, London University) for useful comments on the manuscript, R.Esnouf (Rega Institute for Medical Research, Catholic University of Leuven, Belgium)for the most recent version of Bobscript and also G. Wright for help with datacollection. This research is supported by a grant from the BBSRC, and computersused in the structure solution were provided by the Wellcome Trust.

Received 9 February, 1998; accepted 29 May, 1998.

Table 1 Data collection and processing, structure solution and refinement statistics1

max. res. Nobs Nunique Comp. Rmerge2 MFID3 FOM4 <F>/<E>

(Å) (%) (%) (4 Å/3 Å) (4 Å/3 Å)Native (C2221) 1.9 202,958 30,175 91 6.5 - - -Native (R32) 1.98 257,576 56,901 87 5.8 - - -EMC (R32) 2.8 86,712 19,688 92 9.2 13.4 0.33/0.26 1.21/0.88Final modelRes. (Å) Nreflections Natoms R-factor Rfree Deviations from ideality

working test protein other (%) (%) bond lengths (Å) bond angles25.0–1.9 28,514 1,636 3,009 185 20.8 25.5 0.012 2.117ºR.m.s. coord. Ramachandran plot Average B-factor (Å2) Waterserror (Å) % allowed

5 Ndisallowed main side N Bave (Å2)0.29 91 4 28.1 29.8 174 33.9

1Coordinate error as given in SIGMAA40

2Rmerge = ΣhklΣi Ii(hkl)-I(hkl) /ΣhklΣiI i(hkl)3MFID= FPH −FP / FP, ⟨ F⟩/⟨E⟩ =[Σn FH 2/Σn E 2]1/2, = Σn E 2 { FPH( obs)− FPH( calc)} 2

4FOM = F(hkl)best / F(hkl), F(hkl) best= ΣαP(α)Fhkl(α)/ΣαP(α), where, I = intensity, F = structure factor, F = structure factor amplitude, P = protein, H =heavy atom, PH = protein and heavy atom, P = probability and α = phase angle.5In most favored regions.

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