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Crystallization of the delta toxin of Staphylococcus aureus
.I. .bJol. h’iol. (1986) 192. 675-676
Crystallization of the Delta Toxin of Staphylococcus aweus Delta toxin
is a small cytolytic polypeptide produced and secreted by the organism aureus and belongs to a family of surface-active toxins that exhibit pronounced effect’s on a wide variety of cellular membranes. Although t’his class of prot’eins has been much studied by a wide variety of physical techniques, no consensus has been reached on their mode of action. Therefore, in order to investigate their role in causing membrane damage, a structural analysis of the delta t*oxin has been initiated. Crystals of and this prot
,Stnphylococcus
~Staphylococcus ~WPUS produces a wide variety of t~strac~t~llu1arprot,eins. many of which are important in the pathogenesis of staphylococcal infections. L4mongst them are a group of exotoxins that irtc4udt~ the membrane-damaging agents alpha. beta, gamma and delt,a toxins (also called cytoIJGns. haemolysins or Iysins). The delta toxin is produced by most pathogenic strains of coagulasepositive staphylocsocci and a high proportion of (.oagl”lasf,-nrgativc isolates (Jeljaszewicz. 1972: (~enimc~ll ~4crl.. 1976). This protein is strongly surface active. relativeI! stable to boiling and readily soluble in both aqueous and organic solvents, such as chloroform/methanol. Amino acid sequrnce determination has shown that thrk toxin c,onsists of a Sresidue polypeptide chain cvit h no arginine. coysteine, histidine. proline or tyrosinr. ant1 has a formylated S-t,erminal met)hiotrine residue (Fitton rt al., 1980). Assembly of t,he prot,ein into higher molecular weight aggregat’es has been observed try physicochemical studies using it number of spectroscopic techniques. These indicate, i hat the toxin exist’s in a number of states of asso&t,ion. ranging from a tetramer t’o higher molecular wright multimers (up to Nr 200.000) in aqueous solution. depending on pH or organic solvent concentration (Fitton. 1981). The purified delta toxin is caytolytic for a wide variety of membra,nes including bacterial protoplasts. erythroc+es, tissue culture cell lines, Iysosotnrs and liposomes (Kreger et al., 1971) and in thrse respects its cytolytic properties resemble mrlittin. the princ~ipal toxic component, of bee venom (Thelestam & Miilley. 1975a,h). In addition to their similarities in biological activity, both delta toxin a~rd mrlittin share a number of other common features. Firstly. I)oth are 26 amino acid residue polypeptitles and. secondly, X-ray structure analysis of melitt in at 2.0 L%resolution has shown that it caonsists of a t’etramer under the conditions of’ crystallization (Anderson et al.: 1980) with each pol>*pr~ptitir folded into a single bent alpha-helix. the si ruct ure being organized into a four-helical
bundle wit’h 222 symmetry (Terwilliger 8 Eisenberg. 1982a,b). Each hehx displays a striking amphipathic molecular surface. and within the melit tin tetramer the hydrophobic residues are buried inside the core of bhe molecule. Analvsis of the delta toxin sequence and the use of predictive algorithms suggest that this polypeptide exists also in w helical conformation and. although no direct’ sequence homology with melittin can be discerned, a similar pattern of hydrophobic and hydrophilic sidtb-chains can he seen (Fitton, 1981). This view has been confirmed by recent proton nuclear magnetic resonance experiments on the delta toxin in rnixrd micelles with perdeuterated dodecylphosphocholine (Lee it al., unpublished results). These results show that residues 5 to 23 of the toxin form an extended helix with an amphipathica distribution of Ijolar and non-polar amino acid sidrchaitls. Despite the availabilitS of this structural information, a clear picture of the mode of acation of this (alass of toxins has not emerged. \Vhilst the pronounc~cd amphipathic surface of these helicrs provide a potential struct’ure with faces that could interact with both the membrane and its aqueous environment. the mechanism t,- which the nwm branes are destabilized by such an interaction is unclrar. Furthermore, investigations into the cahanyes in the electrical properties of planar lipid bilayrrs induced by melitt,in appear to indicat,e that the interaction between melittin and the membrane is such as t’o form channels. t’hrouph which ions can move (Tosteson & Tosteson. 1981). Thus the assembly of this class of toxins into channel-like structures spanning the membrane in a similar arrangement to that, proposed for alamethicin (Fox & Richards. 1982) remains a dist,inc*t possibility for their mode of sction. M’P initiat,ed our studies in an attempt’ to shed more light on this area by an examination of t’he st ructurr oft he delta toxin. Delta tosin was purified from cult uw snpef.natants of S. ouwus 186X using R growth mediultl
as described by Gladstone $ van Heyningen (I 957). but omitt’ing sodium lactat,e. Aft’er incubat’ing cultures at 37°C” for 24 hours. the culture supernatant was removed and heated to WY’ for one hour. Following removal of t,he denat’ured protein by centrifugation, t,he pooled supernatants were precipitated with 65O’, saturated ammonium sulphat,e and allowed to stand at 4°C overnight. The precipitate was redissolved and separated on an n-octyl Sepharose low-pressure liquid chromatography system (Nolte & Kapral, 1981). The toxin was further purified on an n-octyl Aquapore RP300 semi-preparative column using an LKK HPLC system. For crystallization, samples of freeze-dried delta toxin were dissolved in distilled water to give a, protein concent’rat,ion of approximately 10 mgjml and 2-methylpentan-2,4-diol added t,o this solut’ion to give a range of concentrat,ions from 10 to 200,, (v/v). Using 50.~1 dialysis buttons. the delta toxin solut,ions were dialysed against tnixtures of methlypentan-diol and distilled wat(er in t,he range 30 to 550/, (v/v) with respect t’o met’hylpentandiol concentrat’ion, at a constant temperature of 19°C. Crystals appeared wit’hin a few days using the highest concentration; however. in order to produce crystals large enough for X-ray study, the buttons in 35 to 409b methylpentan-diol had to he left undisturbed for several months. Well-shaped rot]like crystals of maximum dimensions 0.15 mm x 0.5 mm x 0.07 mm could be cut from the clusters of crystals that had grown and these were then stabilized in 759;, methylpentan-dial. S-ray precession phot’ographs of the crystals show that they belong to the monoclinic system, and hkl reflections are svstematically absent when k +k = 2,~+ 1. identifying the space group as (‘2. with cell dimensions *. = 62.2 a. h = 23.2 A. c = 34.0 w and fi = 97.3”. The cell volume is 4.86 x 104,A3 which. if the cell contains eight molecules of 2977 &I,. gives a I;, value of 2.04 A3 dalt,on-‘. which compares with the most common value for 76 proteins of molecular weight’ less t*ha,n 20,000 of 2.1 B3 dalton-’ and a range of values from 1.75 to 2.90 A3 dalton-’ (Matthews, 196X). Since the unit, cell contains four equivalent positions, this would indicate that the asymmetric unit contains two polypeptide chains, as the values of LZ for 4 and 12 molecules in the unit cell, 4.09 and 1.36. respectively, fall outside this empirically determined range. We have tried measuring the density of crosslinked toxin crystals in order t’o independent’ly determine the number of molecules in t,he unit cell. However, problems associated with the small size of these crystals have preventrd an aecurat’e determination of the density but our results indicate a value of between 8 and 12 molecules in the unit cell. In view of the very low value of I’,,, for 1% molecules in the cell and the extremely tightly packed structure that this would imply. we believe t’hat the most likely value for the number of molecules in the asymmetric3 unit is two.
The carystals, although small. diffract ~~xceetlingl:, well. t,o beyond 2.0 X resolution. Furtherrnorc~. the>appear to be very resist’ant to radiation damage. showing little decay of t)he high-angle refltvtiow after several da,ys exposure to X-rays. In iLd(iiti0tl. since the crystals are grown from methylpetltaIrdiol, a naturally cryogenic solvent. they (‘at1 I)t, cooled easily to low temperatures t,o reduce tht radiation damage further without the need for their transfer from their native mother liquor S.0 :\ diffractometer data hare been collected OII the crystals. and heavy-atom tlerivativw itt’(’ I)tsing assessed. The result,s of this st,ruc*t)ural investigation will provide valuable further information ilr the st,udy of the mechanism of acation of this c*lnss of toxins. Re are grateful t,o the hlR,C for t,heir support ot’ this work through project, grant.s to D.W.R. and .l.E.F. and to
Professor A. C. T. h’orth and F. Korbrr of’ Leeds ITniversity for provision of ditfractomrtrr tiat,a c~ollt~c%ion facilities.
D. H. Thomas D. W. Rice J. E. Fitton? Department of’ Kiochemistrg University of Sheffield Sheffield, SIO 2T?j, England References Anderson, I).. Terwilliger. T. C‘.. Wickner. \V. & Eisenberg, D. (1980). ,J. Hiol. (/hem. 255. 257% %58d. Fitton. ?J.E. (1981). FBRS Letters, 130, 257.-260. Fitton. *J. E.. Dell, A. & Shaw. W. V. (1980). FEW Letters, 115, 209-216. Fox. R. 0. & Richards. F. M. (1982). .Vatnru {London). 300. 325 330. (:emmell, C”. G., Thelestam. Jl. & IVadstrom. T. (1976). In Staphylococci and Staphylococci Diaeasu,s (tJrljaszewicz, J.. rd.). pp. 133-136. Zbl. Kakt.. suppl. 5. Gustav Fischer Verlag, Stuttgart. Gladstone, G. P. &, van Heyningen. 14’. E. (1957). Hr. .J. Exp.
Pathol.
38, 1% 137.
Jeljaszewicz. .I. (1972). In The AYaphylocorci. ((‘ohm. J. 0.. rd.). p. %O, Wiley. Bew York. Kreger. A. K.. Kim, K. S.. Zaboretzky. F. & Kernheirnrr, A. I\‘. (1971). Infect. Immunol. 3. 449.-465. Matthews. H. W’. (196X). J. Mol. Riol. 33, 491 -497. Nolte, F. S. & Kapral, F. A. (1981). Infect. Immunol. 31. 1086 1093. Terwilliger. T. C. & Eisenberg. I). (1!)89a). .J. Hiol. (‘hem. 257. 6010.~601.5. Terwilliger. T. c’. 8: Eisenberg. 1). (1982b). .1. Biol. (‘//c,w. 257. 6016~6OdZ. Thelestam, M. & Mfilley. R. (1975a). Infect. Jmrnunol. 11. 640-648. Thelestam. hl. & Mijlley. R. (19756). Inject. /mmuno/. 12. 225.-232. Tosteson. 11. T. & Tostrsou. I). (‘. (1981). l~iophyys. J. 36.
109~116. t Present address: I(‘1 Pharmawutic& Division. Mrwsidt~ Alderley Park. Macclesfield. Cheshire SKI0 1’1’(:. England.
Crystallization of the Delta Toxin of Staphylococcus aweus Delta toxin
is a small cytolytic polypeptide produced and secreted by the organism aureus and belongs to a family of surface-active toxins that exhibit pronounced effect’s on a wide variety of cellular membranes. Although t’his class of prot’eins has been much studied by a wide variety of physical techniques, no consensus has been reached on their mode of action. Therefore, in order to investigate their role in causing membrane damage, a structural analysis of the delta t*oxin has been initiated. Crystals of and this prot
,Stnphylococcus
~Staphylococcus ~WPUS produces a wide variety of t~strac~t~llu1arprot,eins. many of which are important in the pathogenesis of staphylococcal infections. L4mongst them are a group of exotoxins that irtc4udt~ the membrane-damaging agents alpha. beta, gamma and delt,a toxins (also called cytoIJGns. haemolysins or Iysins). The delta toxin is produced by most pathogenic strains of coagulasepositive staphylocsocci and a high proportion of (.oagl”lasf,-nrgativc isolates (Jeljaszewicz. 1972: (~enimc~ll ~4crl.. 1976). This protein is strongly surface active. relativeI! stable to boiling and readily soluble in both aqueous and organic solvents, such as chloroform/methanol. Amino acid sequrnce determination has shown that thrk toxin c,onsists of a Sresidue polypeptide chain cvit h no arginine. coysteine, histidine. proline or tyrosinr. ant1 has a formylated S-t,erminal met)hiotrine residue (Fitton rt al., 1980). Assembly of t,he prot,ein into higher molecular weight aggregat’es has been observed try physicochemical studies using it number of spectroscopic techniques. These indicate, i hat the toxin exist’s in a number of states of asso&t,ion. ranging from a tetramer t’o higher molecular wright multimers (up to Nr 200.000) in aqueous solution. depending on pH or organic solvent concentration (Fitton. 1981). The purified delta toxin is caytolytic for a wide variety of membra,nes including bacterial protoplasts. erythroc+es, tissue culture cell lines, Iysosotnrs and liposomes (Kreger et al., 1971) and in thrse respects its cytolytic properties resemble mrlittin. the princ~ipal toxic component, of bee venom (Thelestam & Miilley. 1975a,h). In addition to their similarities in biological activity, both delta toxin a~rd mrlittin share a number of other common features. Firstly. I)oth are 26 amino acid residue polypeptitles and. secondly, X-ray structure analysis of melitt in at 2.0 L%resolution has shown that it caonsists of a t’etramer under the conditions of’ crystallization (Anderson et al.: 1980) with each pol>*pr~ptitir folded into a single bent alpha-helix. the si ruct ure being organized into a four-helical
bundle wit’h 222 symmetry (Terwilliger 8 Eisenberg. 1982a,b). Each hehx displays a striking amphipathic molecular surface. and within the melit tin tetramer the hydrophobic residues are buried inside the core of bhe molecule. Analvsis of the delta toxin sequence and the use of predictive algorithms suggest that this polypeptide exists also in w helical conformation and. although no direct’ sequence homology with melittin can be discerned, a similar pattern of hydrophobic and hydrophilic sidtb-chains can he seen (Fitton, 1981). This view has been confirmed by recent proton nuclear magnetic resonance experiments on the delta toxin in rnixrd micelles with perdeuterated dodecylphosphocholine (Lee it al., unpublished results). These results show that residues 5 to 23 of the toxin form an extended helix with an amphipathica distribution of Ijolar and non-polar amino acid sidrchaitls. Despite the availabilitS of this structural information, a clear picture of the mode of acation of this (alass of toxins has not emerged. \Vhilst the pronounc~cd amphipathic surface of these helicrs provide a potential struct’ure with faces that could interact with both the membrane and its aqueous environment. the mechanism t,- which the nwm branes are destabilized by such an interaction is unclrar. Furthermore, investigations into the cahanyes in the electrical properties of planar lipid bilayrrs induced by melitt,in appear to indicat,e that the interaction between melittin and the membrane is such as t’o form channels. t’hrouph which ions can move (Tosteson & Tosteson. 1981). Thus the assembly of this class of toxins into channel-like structures spanning the membrane in a similar arrangement to that, proposed for alamethicin (Fox & Richards. 1982) remains a dist,inc*t possibility for their mode of sction. M’P initiat,ed our studies in an attempt’ to shed more light on this area by an examination of t’he st ructurr oft he delta toxin. Delta tosin was purified from cult uw snpef.natants of S. ouwus 186X using R growth mediultl
as described by Gladstone $ van Heyningen (I 957). but omitt’ing sodium lactat,e. Aft’er incubat’ing cultures at 37°C” for 24 hours. the culture supernatant was removed and heated to WY’ for one hour. Following removal of t,he denat’ured protein by centrifugation, t,he pooled supernatants were precipitated with 65O’, saturated ammonium sulphat,e and allowed to stand at 4°C overnight. The precipitate was redissolved and separated on an n-octyl Sepharose low-pressure liquid chromatography system (Nolte & Kapral, 1981). The toxin was further purified on an n-octyl Aquapore RP300 semi-preparative column using an LKK HPLC system. For crystallization, samples of freeze-dried delta toxin were dissolved in distilled water to give a, protein concent’rat,ion of approximately 10 mgjml and 2-methylpentan-2,4-diol added t,o this solut’ion to give a range of concentrat,ions from 10 to 200,, (v/v). Using 50.~1 dialysis buttons. the delta toxin solut,ions were dialysed against tnixtures of methlypentan-diol and distilled wat(er in t,he range 30 to 550/, (v/v) with respect t’o met’hylpentandiol concentrat’ion, at a constant temperature of 19°C. Crystals appeared wit’hin a few days using the highest concentration; however. in order to produce crystals large enough for X-ray study, the buttons in 35 to 409b methylpentan-diol had to he left undisturbed for several months. Well-shaped rot]like crystals of maximum dimensions 0.15 mm x 0.5 mm x 0.07 mm could be cut from the clusters of crystals that had grown and these were then stabilized in 759;, methylpentan-dial. S-ray precession phot’ographs of the crystals show that they belong to the monoclinic system, and hkl reflections are svstematically absent when k +k = 2,~+ 1. identifying the space group as (‘2. with cell dimensions *. = 62.2 a. h = 23.2 A. c = 34.0 w and fi = 97.3”. The cell volume is 4.86 x 104,A3 which. if the cell contains eight molecules of 2977 &I,. gives a I;, value of 2.04 A3 dalt,on-‘. which compares with the most common value for 76 proteins of molecular weight’ less t*ha,n 20,000 of 2.1 B3 dalton-’ and a range of values from 1.75 to 2.90 A3 dalton-’ (Matthews, 196X). Since the unit, cell contains four equivalent positions, this would indicate that the asymmetric unit contains two polypeptide chains, as the values of LZ for 4 and 12 molecules in the unit cell, 4.09 and 1.36. respectively, fall outside this empirically determined range. We have tried measuring the density of crosslinked toxin crystals in order t’o independent’ly determine the number of molecules in t,he unit cell. However, problems associated with the small size of these crystals have preventrd an aecurat’e determination of the density but our results indicate a value of between 8 and 12 molecules in the unit cell. In view of the very low value of I’,,, for 1% molecules in the cell and the extremely tightly packed structure that this would imply. we believe t’hat the most likely value for the number of molecules in the asymmetric3 unit is two.
The carystals, although small. diffract ~~xceetlingl:, well. t,o beyond 2.0 X resolution. Furtherrnorc~. the>appear to be very resist’ant to radiation damage. showing little decay of t)he high-angle refltvtiow after several da,ys exposure to X-rays. In iLd(iiti0tl. since the crystals are grown from methylpetltaIrdiol, a naturally cryogenic solvent. they (‘at1 I)t, cooled easily to low temperatures t,o reduce tht radiation damage further without the need for their transfer from their native mother liquor S.0 :\ diffractometer data hare been collected OII the crystals. and heavy-atom tlerivativw itt’(’ I)tsing assessed. The result,s of this st,ruc*t)ural investigation will provide valuable further information ilr the st,udy of the mechanism of acation of this c*lnss of toxins. Re are grateful t,o the hlR,C for t,heir support ot’ this work through project, grant.s to D.W.R. and .l.E.F. and to
Professor A. C. T. h’orth and F. Korbrr of’ Leeds ITniversity for provision of ditfractomrtrr tiat,a c~ollt~c%ion facilities.
D. H. Thomas D. W. Rice J. E. Fitton? Department of’ Kiochemistrg University of Sheffield Sheffield, SIO 2T?j, England References Anderson, I).. Terwilliger. T. C‘.. Wickner. \V. & Eisenberg, D. (1980). ,J. Hiol. (/hem. 255. 257% %58d. Fitton. ?J.E. (1981). FBRS Letters, 130, 257.-260. Fitton. *J. E.. Dell, A. & Shaw. W. V. (1980). FEW Letters, 115, 209-216. Fox. R. 0. & Richards. F. M. (1982). .Vatnru {London). 300. 325 330. (:emmell, C”. G., Thelestam. Jl. & IVadstrom. T. (1976). In Staphylococci and Staphylococci Diaeasu,s (tJrljaszewicz, J.. rd.). pp. 133-136. Zbl. Kakt.. suppl. 5. Gustav Fischer Verlag, Stuttgart. Gladstone, G. P. &, van Heyningen. 14’. E. (1957). Hr. .J. Exp.
Pathol.
38, 1% 137.
Jeljaszewicz. .I. (1972). In The AYaphylocorci. ((‘ohm. J. 0.. rd.). p. %O, Wiley. Bew York. Kreger. A. K.. Kim, K. S.. Zaboretzky. F. & Kernheirnrr, A. I\‘. (1971). Infect. Immunol. 3. 449.-465. Matthews. H. W’. (196X). J. Mol. Riol. 33, 491 -497. Nolte, F. S. & Kapral, F. A. (1981). Infect. Immunol. 31. 1086 1093. Terwilliger. T. C. & Eisenberg. I). (1!)89a). .J. Hiol. (‘hem. 257. 6010.~601.5. Terwilliger. T. c’. 8: Eisenberg. 1). (1982b). .1. Biol. (‘//c,w. 257. 6016~6OdZ. Thelestam, M. & Mfilley. R. (1975a). Infect. Jmrnunol. 11. 640-648. Thelestam. hl. & Mijlley. R. (19756). Inject. /mmuno/. 12. 225.-232. Tosteson. 11. T. & Tostrsou. I). (‘. (1981). l~iophyys. J. 36.
109~116. t Present address: I(‘1 Pharmawutic& Division. Mrwsidt~ Alderley Park. Macclesfield. Cheshire SKI0 1’1’(:. England.