The Identification and Structure of the Membrane-spanning Domain of the Clostridium septicum Alpha Toxin

Alpha toxin (AT) is a pore-forming toxin produced by Clostridium septicum that belongs to the unique aerolysin-like family of pore-forming toxins. The location and structure of the transmembrane domains of these toxins have remained elusive. Using deletion mutagenesis, cysteine-scanning mutagenesis and multiple spectrofluorimetric methods a membrane-spanning amphipathic β-hairpin of AT has been identified. Spectrofluorimetric analysis of cysteine-substituted residues modified with an environmentally sensitive fluorescent probe via the cysteine sulfydryl showed that the side chains of residues 203-232 alternated between the aqueous milieu and the membrane core when the AT oligomer was inserted into membranes, consistent with the formation of an amphipathic transmembrane β-hairpin. AT derivatives that contained deletions that removed up to 90% of the β-hairpin did not form a pore but were similar to native toxin in all other aspects of the mechanism. Furthermore, a mutant of AT that contained an engineered disulfide, predicted to restrict the movement of the β-hairpin, functioned similarly to native toxin except that it did not form a pore unless the disulfide bond was reduced. Together these studies revealed the location and structure of the membrane-spanning domain of AT. Alpha toxin (AT) is a pore-forming toxin produced by Clostridium septicum that belongs to the unique aerolysin-like family of pore-forming toxins. The location and structure of the transmembrane domains of these toxins have remained elusive. Using deletion mutagenesis, cysteine-scanning mutagenesis and multiple spectrofluorimetric methods a membrane-spanning amphipathic β-hairpin of AT has been identified. Spectrofluorimetric analysis of cysteine-substituted residues modified with an environmentally sensitive fluorescent probe via the cysteine sulfydryl showed that the side chains of residues 203-232 alternated between the aqueous milieu and the membrane core when the AT oligomer was inserted into membranes, consistent with the formation of an amphipathic transmembrane β-hairpin. AT derivatives that contained deletions that removed up to 90% of the β-hairpin did not form a pore but were similar to native toxin in all other aspects of the mechanism. Furthermore, a mutant of AT that contained an engineered disulfide, predicted to restrict the movement of the β-hairpin, functioned similarly to native toxin except that it did not form a pore unless the disulfide bond was reduced. Together these studies revealed the location and structure of the membrane-spanning domain of AT. Clostridium septicum is a Gram-positive anaerobe that typically causes fulminant, often fatal infections such as nontraumatic gas gangrene or necrotizing enterocolitis in compromised patients (1Stevens D.L. Musher D.M. Watson D.A. Eddy H. Hamill R.J. Gyorkey F. Rosen H. Mader J. Rev. Infect. Dis. 1990; 12: 286-296Google Scholar). More recently it has also been shown to be a superinfection of hemolytic uremic syndrome (2Barnham M. Weightman N. Emerg. Infect. Dis. 1998; 4: 321-324Google Scholar). Alpha toxin (AT) 1The abbreviations used are: AT, alpha toxin; TMD, transmembrane domain; CDCs, cholesterol-dependent cytolysins; NBD, N,N′-dimethyl-N-(iodoacetyl-N′-(7-nitrobenz-2-oxa-1,3-diazoyl)ethylendiamine; IAF, 5-iodoacetamidofluoroscein; FI, fluorescence intensity; D1-D4, domains 1-4; DOXYL, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy; DSA, DOXYL-stearic acid; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; MES, 4-morpholineethanesulfonic acid; HBSS, Hanks' balanced salt solution; DTT, dithiothreitol. is the only known lethal factor secreted by C. septicum (3Ballard J. Bryant A. Stevens D. Tweten R.K. Infect. Immun. 1992; 60: 784-790Google Scholar). AT belongs to a distinctive class of related cytolytic toxins whose prototype is aerolysin, secreted by the Gram-negative bacterium Aeromonas hydrophila (4Howard S.P. Buckley J.T. Biochemistry. 1982; 21: 1662-1667Google Scholar). Interestingly, enterolobin, a cytolytic protein derived from the Brazilian Enterolobium contortisiliquum tree also appears to be related to aerolysin (5de Sousa M.V. Morhy L. An. Acad. Bras. Cienc. 1989; 61: 405-412Google Scholar, 6Fontes W. Sousa M.V. Aragao J.B. Morhy L. Arch. Biochem. Biophys. 1997; 347: 201-207Google Scholar). The mechanisms of AT and aerolysin have been shown to be highly similar. Both toxins bind to cells via glycophosphatidylinositol-anchored protein receptors (7Gordon V.M. Nelson K.L. Buckley J.T. Stevens V.L. Tweten R.K. Elwood P.C. Leppla S.H. J. Biol. Chem. 1999; 274: 27274-27280Google Scholar, 8Wichroski M.J. Melton J.A. Donahue C.G. Tweten R.K. Ward G.E. Infect. Immun. 2002; 70: 4353-4361Google Scholar), although aerolysin appears to bind to some receptors that are not recognized by AT and vice versa. Following cell binding both toxins are activated by the proteolytic cleavage of an amino-terminal propeptide by furin or furin-like protease (9Gordon V.M. Benz R. Fujii K. Leppla S.H. Tweten R.K. Infect. Immun. 1997; 65: 4130-4134Google Scholar, 10Abrami L. Fivaz M. Decroly E. Seidah N.G. Jean F. Thomas G. Leppla S.H. Buckley J.T. van der Goot F.G. J. Biol. Chem. 1998; 273: 32656-32661Google Scholar). Activation then allows the toxin monomers to oligomerize on the membrane and form a pore (11Ballard J. Sokolov Y. Yuan W.-L. Kagan B.L. Tweten R.K. Mol. Microbiol. 1993; 10: 627-634Google Scholar, 12Howard S.P. Buckley J.T. J. Bacteriol. 1985; 163: 336-340Google Scholar). The difference seen in receptor specificity of the two toxins appears to be linked to the presence of an amino-terminal peptide of aerolysin that is not conserved in AT. The crystal structure of aerolysin, the only member of this toxin family whose crystal structure has been solved (13Parker M.W. Buckley J.T. Postma J.P.M. Tucker A.D. Leonard K. Pattus F. Tsernoglou D. Nature. 1994; 367: 292-295Google Scholar), shows that it is a bi-lobal protein consisting of four distinct domains (D1-D4), three of which are rich in β-sheet structure (see Fig. 1). The small lobe of aerolysin is missing from the amino terminus in AT, and this implies that AT is a single-lobed structure consisting of three domains that are homologous to D2-D4 of aerolysin (see Fig. 1). The small lobe of aerolysin (D1) contains a lectin-binding domain that enables it to bind to various receptors that are not recognized by AT (14Rossjohn J. Buckley J.T. Hazes B. Murzin A.G. Read R.J. Parker M.W. EMBO J. 1997; 16: 3426-3434Google Scholar, 15Diep D.B. Nelson K.L. Lawrence T.S. Sellman B. Tweten R.K. Buckley J.T. Mol. Microbiol. 1999; 31: 785-794Google Scholar). However, fusion of D1 of aerolysin to the amino terminus of AT converts it to a molecule with aerolysin-like receptor specificity and activity (15Diep D.B. Nelson K.L. Lawrence T.S. Sellman B. Tweten R.K. Buckley J.T. Mol. Microbiol. 1999; 31: 785-794Google Scholar). Comparatively little is known about the cytolytic mechanism of enterolobin, but its primary structure appears to be more related to aerolysin than AT. Enterolobin displays sequence similarity with both the small and large lobes of aerolysin and appears to form a dimer in solution (6Fontes W. Sousa M.V. Aragao J.B. Morhy L. Arch. Biochem. Biophys. 1997; 347: 201-207Google Scholar, 16Bittencourt S.E. Silva L.P. Azevedo R.B. Cunha R.B. Lima C.M. Ricart C.A. Sousa M.V. FEBS Lett. 2003; 549: 47-51Google Scholar). Although many aspects of the cytolytic mechanism of AT and aerolysin have been elucidated, the one or more domains of these toxins directly involved in the formation of the pore have yet to be identified. Two structural motifs used by pore-forming toxins to span membranes are amphipathic β-strands and α-helices. Both motifs have been identified in toxins as the secondary structures used to cross the membrane during pore formation. For example, the pore-forming domain of the colicins and the T-domain of diphtheria toxin use a pair of amphipathic α-helices to span the membrane (17Parker M.W. Pattus F. Tucker A.D. Tsernoglou D. Nature. 1989; 337: 93-96Google Scholar, 18Oh K.J. Zhan H. Cui C. Hideg K. Collier R.J. Hubbell W.L. Science. 1996; 273: 810-812Google Scholar), whereas the protective antigen component of anthrax toxin (19Benson E.L. Huynh P.D. Finkelstein A. Collier R.J. Biochemistry. 1998; 37: 3941-3948Google Scholar), the α-hemolysin from Staphylococcus aureus (20Song L.Z. C. H. Science. 1996; 274: Scholar, A. A. B. M. H. M. EMBO J. 1996; Scholar), and from Clostridium J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: Scholar, J. Parker M.W. Tweten R.K. 1999; all use amphipathic β-strands that to the formation of a transmembrane (see Fig. 1). However, of the has not been or The of the cholesterol-dependent was to be of a J. Tweten R.K. Parker M.W. 1997; Scholar), or an amphipathic M. A. M. J. Biol. Chem. 1996; and J. Parker M. 1999; that the did not the membrane The pore-forming of the was shown to be of two amphipathic in of the crystal structure that are derived from α-helices of the J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: Scholar, J. Parker M.W. Tweten R.K. 1999; Scholar, J. Tweten R.K. Parker M.W. 1997; Scholar). was that the of aerolysin the membrane and the of aerolysin (13Parker M.W. Buckley J.T. Postma J.P.M. Tucker A.D. Leonard K. Pattus F. Tsernoglou D. Nature. 1994; 367: 292-295Google Scholar). More Y. C. M. J. Parker M.W. van der Goot F.G. Biol. 2002; also that the the of aerolysin on an of was to be a oligomer of However, to have been that directly the of a of aerolysin, or the related AT or enterolobin, with the The structural of AT and the aerolysin crystal structure a amphipathic that is in of AT and the of aerolysin 1). Using multiple fluorescent and have that this amphipathic in of AT, of residues to an amphipathic β-hairpin that the membrane and is pore formation. of residues in the of AT, to the of aerolysin that it is only with the membrane and is to directly in the formation of the transmembrane and AT was into the and into cells as Kagan B.L. Tweten R.K. Mol. Microbiol. 1997; Scholar). The cell was a of of were from and all from unless of and deletion in AT were a mutagenesis 1989; except that was used as the and was were the with and and into with and of AT, was the mutagenesis and the was cysteine were produced as the mutagenesis or mutagenesis and of and of native AT and the various AT derivatives were to Sellman Kagan B.L. Tweten R.K. Mol. Microbiol. 1997; Scholar). The cell were in of of cells was in an was removed by of the AT from the was a and was as Kagan B.L. Tweten R.K. Mol. Microbiol. 1997; Scholar). from the were in a with a membrane and were MES, For cysteine-substituted was in the was to the toxin was by a of K. cells were in with and of and of cells were to the cells were and to a of in Hanks' balanced salt solution were by the cell in an membranes were by was and the cell membranes were by the membrane in of and Following the the membranes were with to an of of the AT of AT was on the crystal structure of C. J. J. and M. W. The sequence which the related enterolobin, and were the aerolysin structure as a the of on a The was in two of of sequence between AT and aerolysin and to these and to the the sequence to aerolysin is a of peptide The were to the M. A. Scholar), the side and was by of with on the by with Nature. 1992; Scholar). The of the was and the R. D. Nature. 1992; Scholar). The similar the sequence the and of the AT are of a of AT with or cysteine-substituted of AT were in was removed the unique of AT mutant with the of the fluorescent or of toxin was a in or was then to the protein to a The was in the and a in to The of was an of or an of of and Scholar). was by a of K. cells were in with and of and cells were into the of a in a of cells of was in a in which the were with of was into the of a and up to of with The toxin was in the of the from was to of the the cells and Following of was to and The were and the of toxin that in cell lethal from cells with mutant toxins were as a of the from cells with AT. fluorescence was an with a in the a and a with the The was and the was were between and both and of For the was by the fluorescence of an For toxin was with a The was with the of a of the protease of toxin was or in the presence of of membranes and up to of with C. were in the to and to to For were into with of and into the a of of by a was the core of the into the membrane as and were to a of in membrane were in the solution by directly the to the The membranes were and For of toxin was with of membranes in a of up to with in the The were into with of and into the a of were between and with an of to the that the is in membranes in were used as on of were and from the to the of and of cells was a binding of were with up to in and Following cells were and with of to toxin and up to a of with The fluorescence of the cells was by analysis on a and with a was in and all the of four binding was by toxin to of toxin and this was from that with toxin to was used analysis to an and of toxin or deletion to binding with to cells was a modified of the binding was with of or mutant toxin to and up to a of in to with were and the fluorescence was was in the the fluorescence of the cells was the of was used analysis of with a Activation and of and on and of toxin in the presence of membranes was as and mutant was activated a of The was and the was by the of a of the protease membranes were to the activated and the was to with and and of were to the and on a The were to and the was with primary was removed by the three in and then secondary to was to the and the was an The was three in to of the recognized by the was by the with the solution to between and between and amphipathic α-helices and β-strands are by the of the of side chains with the membrane and aqueous residues of an amphipathic with the membrane K.J. Zhan H. Cui C. Hideg K. Collier R.J. Hubbell W.L. Science. 1996; 273: 810-812Google Scholar), whereas residues of an amphipathic with the membrane J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: Scholar). to of AT of an amphipathic transmembrane β-hairpin. The of J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: was used to this the membrane and then to the and its secondary it is inserted into the of the environmentally sensitive of the fluorescent The fluorescence of is when it is in an aqueous such as the of the but when it is in a such as the core of the to were to and the were and with The of mutant was in the and presence of membranes that one or more receptors AT (7Gordon V.M. Nelson K.L. Buckley J.T. Stevens V.L. Tweten R.K. Elwood P.C. Leppla S.H. J. Biol. Chem. 1999; 274: 27274-27280Google Scholar). Fig. shows from and in the and an amphipathic β-hairpin the side of is predicted to with the during pore whereas the side of is predicted to in the of the with this in was seen its with membranes whereas the of the The fluorescence NBD, when to the that were all residues this are in Fig. The in the of was from the of the in the and an of in which the side chains of these residues to in and of a The is between residues to and to of AT the an in the of movement of the into a such as the it not the location of the in the The membrane location of residues shown to a was by are of as shown by J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: Scholar), when to the of be into membranes and only to the core J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: Scholar, J. Parker M.W. Tweten R.K. 1999; Scholar). seen in Fig. the of by DOXYL-stearic was consistent with the seen the in Fig. The of was seen between residues and which did not an in on membranes, was by the that the side in a in the of AT, the of the cysteine this not as the side from its location in the to its in the on the of and be to the not into with the both a small of when with these the is this and then it is that the the two transmembrane β-strands the span of residues from to as a these residues be only to the The cytolytic activity of of the was on the residues and in or and activity not that or activity are in and and are predicted to the membrane when inserted and to the of activity by these were not of between and on the of AT it appears that deletion of the amphipathic not of AT, it not to be a core domain with that deletion of these residues only pore formation the residues are in the membrane-spanning this were of the and The deletion removed of the predicted deletion were to cytolytic activity on cells not The three deletion did not to be receptor and were not of the binding showed that to a class of receptors these cells with a of showed that the deletion similar to that AT the deletions did not receptor Furthermore, the deletion were activated in by similar to and on membranes as as or than AT, structure not been compromised by the deletions by also did not that were not of the native toxin in solution not the structure of these was not to an that in the of and of deletion and disulfide the AT deletion and or the disulfide mutant were proteolytic with and the formation of on of a between the and the of studies on the in of aerolysin showed that it be to the of the molecule by of a disulfide bond between the and the of the molecule J. Nelson K.L. F.G. Parker M.W. Buckley J.T. Biochemistry. 1998; 37: Scholar). the disulfide and pore formation it was with these it was that the from the β-sheet in aerolysin to form a and in similar was with AT, in which residues to to cysteine in aerolysin on the AT structural in Fig. were with was to be in its but of the activity of native toxin when not activated by and with membranes, and on cells both and in a similar to native AT movement of the in of AT is only pore formation and not binding or of AT to the of aerolysin was to be of the which a that the membrane (13Parker M.W. Buckley J.T. Postma J.P.M. Tucker A.D. Leonard K. Pattus F. Tsernoglou D. Nature. 1994; 367: 292-295Google Scholar). has been in a of an aerolysin mutant that a in which an was to the of a transmembrane that was to be the propeptide Y. C. M. J. Parker M.W. van der Goot F.G. Biol. 2002; Scholar). of aerolysin is homologous to of AT, residues this of AT to with the membrane during pore formation. Two residues in of aerolysin, and were to with the membrane when are residues three residues on β-strands which were Y. C. M. J. Parker M.W. van der Goot F.G. Biol. 2002; to up an as is in J.A. G. Biochem. Scholar). and on two β-strands in aerolysin and are not conserved in AT. For of aerolysin, the in AT is a whereas the homologous is a a more it to a cysteine and the mutant with with and in the presence of membranes, a in that was not of to the membranes that it not with the membrane during pore formation. β-strands between and of AT on one of these β-strands Fig. the predicted residues of were on the which the of the of AT the to of the residues this with the the membrane then these which are on the as the predicted location of the with the membrane toxin residues were in the as in and seen in Fig. all of the residues on this except a in on membranes with the with membranes the membrane DSA, of the residues little or that these residues not with the membrane to a of AT, to of aerolysin, was also predicted to with the membrane Y. C. M. J. Parker M.W. van der Goot F.G. Biol. 2002; Scholar). is on a than Fig. two residues of to this with the membrane during pore formation. Using the as both residues showed an in with membranes but little or when was in the which on in the of of AT directly the on this are predicted to be by the propeptide its and are one of the residues of the showed a in that was not on membranes the residues of the β-strands not to have with the The formation of transmembrane by various toxins has been a of in the the solution of the crystal structure of the pore of the Staphylococcus aureus α-hemolysin by (20Song L.Z. C. H. Science. 1996; 274: Scholar). The structure of the α-hemolysin membrane pore showed the amphipathic of monomers of the toxin an amphipathic structure in the that only anthrax toxin (19Benson E.L. Huynh P.D. Finkelstein A. Collier R.J. Biochemistry. 1998; 37: 3941-3948Google and J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: Scholar, J. Parker M.W. Tweten R.K. 1999; have been shown to transmembrane to form a the have shown that residues 203-232 of AT also in the formation of a membrane-spanning β-hairpin with aureus the of the monomers of the AT oligomer and into the membrane to the pore-forming with this is the in the of the to cysteine-substituted residues that were predicted to the membrane a in than the residues predicted to the of the Furthermore, analysis with a that the residues identified by were with the of the The of the AT is also similar in to that the transmembrane of aureus α-hemolysin (20Song L.Z. C. H. Science. 1996; 274: Scholar), the anthrax protective antigen (19Benson E.L. Huynh P.D. Finkelstein A. Collier R.J. Biochemistry. 1998; 37: 3941-3948Google Scholar), and the two transmembrane of J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: Scholar, J. Parker M.W. Tweten R.K. 1999; Scholar). The fluorescence were by deletion analysis of the on the crystal structure of the related aerolysin and the of AT predicted that deletion of the transmembrane from AT not the of AT and have its to bind proteolytic and to form membrane these all the of native except that were to form a pore and were anthrax protective antigen when its transmembrane was Collier Biochemistry. 1999; Scholar). this is to the formation of the AT but not the other toxin that to pore formation. into the membrane this from the J. Nelson K.L. F.G. Parker M.W. Buckley J.T. Biochemistry. 1998; 37: shown that the of a disulfide into aerolysin by the of residues and with pore formation. disulfide the in aerolysin to the that this disulfide of aerolysin and that this in aerolysin to of the disulfide, the aerolysin its cytolytic on the of AT, the residues of AT were to the disulfide in AT pore formation when However, the deletion it the to bind membranes, proteolytic and to form the aerolysin cytolytic activity when the disulfide was reduced. it also appears that this transmembrane from the of AT to the β-hairpin to into the difference between the aerolysin and AT disulfide be that oligomerize when the engineered disulfide is whereas the disulfide mutant of aerolysin not to oligomerize J. Nelson K.L. F.G. Parker M.W. Buckley J.T. Biochemistry. 1998; 37: Scholar). difference the that aerolysin as a dimer in solution (13Parker M.W. Buckley J.T. Postma J.P.M. Tucker A.D. Leonard K. Pattus F. Tsernoglou D. Nature. 1994; 367: 292-295Google Scholar), whereas AT as a A. Melton and R. K. and the aerolysin dimer to dimer formation in via its small lobe (13Parker M.W. Buckley J.T. Postma J.P.M. Tucker A.D. Leonard K. Pattus F. Tsernoglou D. Nature. 1994; 367: 292-295Google Scholar, D.B. Lawrence T.S. J. S.P. Buckley J.T. Mol. Microbiol. 1998; Scholar), which is missing in AT. the small lobe is removed from aerolysin-like AT, it as a in solution D.B. Lawrence T.S. J. S.P. Buckley J.T. Mol. Microbiol. 1998; Scholar). it appears that the movement of the of aerolysin also be the of the aerolysin dimer receptor binding R. A. J. Buckley J.T. J. Biol. Chem. Scholar). the amphipathic structure of the AT conserved in aerolysin and Both aerolysin and an amphipathic structure that is in primary structures and is conserved with that of AT Although contains an in the of the predicted this the predicted and the aqueous However, it also the membrane with the of the side and the to the similar to that in the J. Parker M.W. Tweten R.K. Biochemistry. 1998; 37: Scholar). it appears AT, aerolysin and also use an amphipathic β-hairpin structure to form transmembrane has been (13Parker M.W. Buckley J.T. Postma J.P.M. Tucker A.D. Leonard K. Pattus F. Tsernoglou D. Nature. 1994; 367: 292-295Google Scholar, Y. C. M. J. Parker M.W. van der Goot F.G. Biol. 2002; that the of aerolysin a that and the membrane and of AT and aerolysin, the of these two and the proteolytic cleavage of the propeptide has been shown to be toxin by by the propeptide S.P. Buckley J.T. J. Bacteriol. 1985; 163: 336-340Google Scholar, Tweten R.K. Mol. Microbiol. 1997; Scholar, Buckley J.T. Infect. Immun. Scholar). Although a of some residues of AT of with the the not a as the of AT. the that the of AT and its to bind and oligomerize pore formation is also consistent with the of in the of AT Tweten R.K. Mol. Microbiol. 1997; and in the of aerolysin S.P. Buckley J.T. J. Bacteriol. 1985; 163: 336-340Google Scholar, M.W. Buckley J.T. Postma J.P.M. Tucker A.D. Leonard K. Pattus F. Tsernoglou D. Nature. 1994; 367: 292-295Google Scholar, Tweten R.K. Mol. Microbiol. 1997; Scholar, Buckley J.T. Infect. Immun. Scholar). studies the the location and structure of the transmembrane domain of a member of the aerolysin family of cytolytic C. septicum alpha amphipathic also the structures of the related aerolysin and toxins that also an amphipathic β-hairpin to span the that this family of toxins belongs to the class of toxins Tweten R.K. Biochemistry. that an amphipathic β-hairpin to form a membrane-spanning the of

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.