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C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

1999; Elsevier BV; Volume: 274; Issue: 26 Linguagem: Inglês

10.1074/jbc.274.26.18536

1083-351X

Yukiko Shimada, Megumi Nakamura, Yasuhide Naito, Kohji Nomura, Yoshiko Ohno‐Iwashita,

Venomous Animal Envenomation and Studies

Perfringolysin O (θ-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the β-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in θ-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native θ-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 m guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in β-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of θ-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities. Perfringolysin O (θ-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the β-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in θ-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native θ-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 m guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in β-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of θ-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities. Thiol-activated cytolysins (1Alouf J.E. Geoffroy C. Alouf J.E. Freer J.H. Sourcebook of Bacterial Protein Toxins. Academic Press, London1991: 147-186Google Scholar) comprise a family of bacterial protein toxins that are produced by Gram-positive bacteria. They share a high degree of homology in their amino acid sequences (40–70%) (2Tweten R.K. Infect. Immun. 1988; 56: 3235-3240Crossref PubMed Google Scholar, 3Shimizu T. Okabe A. Minami J. Hayashi H. Infect. Immun. 1991; 59: 137-142Crossref PubMed Google Scholar, 4Kehoe M.A. Miller L. Walker J.A. Boulnois G.J. Infect. Immun. 1987; 55: 3228-3232Crossref PubMed Google Scholar, 5Walker J.A. Allen R.L. Falmagne P. Johnson M.K. Boulnois G.J. Infect. Immun. 1987; 55: 1184-1189Crossref PubMed Google Scholar, 6Mengaud J. Chenevert J. Geoffroy C. Gaillard J. Cossart P. Infect. Immun. 1987; 55: 3225-3227Crossref PubMed Google Scholar, 7Geoffroy C. Mengaud J. Alouf J.E. Cossart P. J. Bacteriol. 1990; 172: 7301-7305Crossref PubMed Google Scholar) and have common biological characteristics, cholesterol binding and the formation of oligomeric pores on plasma membranes. Perfringolysin O (472 amino acids), known as θ-toxin, is such a toxin produced by Clostridium perfringens type A. Its cytolytic mechanism is thought to comprise at least four steps: binding to cholesterol in membranes, insertion into the membrane, oligomerization, and pore formation. θ-Toxin binds specifically to cholesterol on plasma membranes with high affinity (K d ∼ 10−9m) (8Ohno-Iwashita Y. Iwamoto M. Mitsui K. Ando S. Nagai Y. Eur. J. Biochem. 1988; 176: 95-101Crossref PubMed Scopus (59) Google Scholar). By forming oligomeric pores on plasma membranes (9Mitsui K. Sekiya T. Nozawa Y. Hase J. Biochim. Biophys. Acta. 1979; 554: 68-75Crossref PubMed Scopus (26) Google Scholar), θ-toxin causes cell disruption. After several attempts to crystallize θ-toxin (10Sugahara M. Sekino-Suzuki N. Ohno-Iwashita Y. Miki K. J. Crystal Growth. 1996; 168: 288-291Crossref Scopus (2) Google Scholar, 11Feil S.C. Rossjohn J. Rohde K. Tweten R.K. Parker M.W. FEBS Lett. 1996; 397: 290-292Crossref PubMed Scopus (15) Google Scholar), its three-dimensional structure was recently revealed by x-ray diffraction (12Rossjohn J. Feil S.C. McKinstry W.J. Tweten R.K. Parker M.W. Cell. 1997; 89: 685-692Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). This analysis showed θ-toxin to be an elongated rod-shaped molecule rich in β-sheets and to consist of four discontinuous domains. Domain 4 (Fig. 1 b) (residues 363–472), the C-terminal domain, is an autonomous structure comprising a continuous amino acid chain. Six of the seven total tryptophan residues reside in domain 4, and three are located in the sequence of ECTGLAWEWWR (residues 430–440), the longest conserved sequence among thiol-activated cytolysins (2Tweten R.K. Infect. Immun. 1988; 56: 3235-3240Crossref PubMed Google Scholar, 3Shimizu T. Okabe A. Minami J. Hayashi H. Infect. Immun. 1991; 59: 137-142Crossref PubMed Google Scholar). From many efforts to achieve mutagenesis of this toxin family (13Sekino-Suzuki N. Nakamura M. Mitsui K. Ohno-Iwashita Y. Eur. J. Biochem. 1996; 241: 941-947Crossref PubMed Scopus (100) Google Scholar, 14Bhakdi S. Bayley H. Valeva A. Walev I. Walker B. Weller U. Kehoe M. Palmer M. Arch. Microbiol. 1996; 165: 73-79Crossref PubMed Scopus (260) Google Scholar, 15Michel E. Reich K.A. Favier R. Berche P. Cossart P. Mol. Microbiol. 1990; 4: 2167-2178Crossref PubMed Scopus (157) Google Scholar, 16Mitchell T.J. Andrew P.W. Boulnois G.J. Lee C.J. Lock R.A. Paton J.C. Witholt B. Alouf J.E. Boulnois G.J. Bacterial Protein Toxins. Gustav Fischer Verlag, Stuttgart, Germany1992: 429-438Google Scholar, 17Owen R.H.G. Andrew P.W. Boulnois G.J. Michell T.J. FEMS Microbiol. Lett. 1994; 121: 217-221Crossref PubMed Scopus (38) Google Scholar), it was shown that all mutations that inhibit cell binding activity reside in domain 4, suggesting that some region in domain 4 binds to membrane cholesterol upon binding to cells. This is consistent with our previous findings that a C-terminal tryptic fragment that contains predominantly domain 4 binds to cholesterol and to cholesterol-containing membrane (18Iwamoto M. Ohno-Iwashita Y. Ando S. Eur. J. Biochem. 1990; 194: 25-31Crossref PubMed Scopus (45) Google Scholar). Our findings that the toxin binding to cholesterol in liposomal membrane triggers a conformational change around tryptophan residues in domain 4 also support this view (19Nakamura M. Sekino N. Iwamoto M. Ohno-Iwashita Y. Biochemistry. 1995; 34: 6513-6520Crossref PubMed Scopus (85) Google Scholar, 20Nakamura M. Sekino-Suzuki N. Mitsui K. Ohno-Iwashita Y. J. Biochem. 1998; 123: 1145-1155Crossref PubMed Scopus (44) Google Scholar). Recently, possible roles of the C-terminal region in cell binding were suggested by a report that a monoclonal antibody thought to bind near the C terminus specifically blocks cell binding, although the exact epitope was not identified (21de los Toyos J.R. Mendez F.J. Aparicio J.F. Vazquez F. Suarez M.M.G. Fleites A. Hardisson C. Morgan P.J. Andrew P.W. Mitchell T.J. Infect. Immun. 1996; 64: 480-484Crossref PubMed Google Scholar). Despite this finding, it is not known whether the C-terminal region plays a role in cholesterol binding or membrane insertion activity, inasmuch as either one could affect toxin binding to cells. Recent x-ray crystallographic analysis showed that there are two β-strands in antiparallel orientation in the C-terminal end and that one of them is composed of 7 amino acids in the C-terminal end (12Rossjohn J. Feil S.C. McKinstry W.J. Tweten R.K. Parker M.W. Cell. 1997; 89: 685-692Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Here, we constructed and analyzed toxin mutants truncated in the C terminus to define the role of the C-terminal region on cholesterol binding activity. Using an ELISA 1The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; CD, circular dichroism; GdnHCl, guanidine hydrochloride; PAGE, polyacrylamide gel electrophoresis; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol; ESI, electrospray ionization; ESI-MS, electrospray ionization mass spectrometry.assay for quantitative analysis of cholesterol binding activity, we show that the C-terminal end is essential for folding of θ-toxin into the native conformation, thus ensuring activities of cholesterol binding and hemolysis. Anti-θ-toxin antibody was obtained by immunizing rabbits as described previously (18Iwamoto M. Ohno-Iwashita Y. Ando S. Eur. J. Biochem. 1990; 194: 25-31Crossref PubMed Scopus (45) Google Scholar). Cholesterol, subtilisin Carlsberg, and isopropylthio-β-d-galactoside were purchased from Sigma. DEAE-Sephacel was from Amersham Pharmacia Biotech (Uppsala, Sweden). Horseradish peroxidase-conjugated anti-(rabbit IgG) serum was purchased from Seikagaku (Tokyo, Japan). Plasmid pNSP10 containing the perfringolysin O gene (pfoA) (13Sekino-Suzuki N. Nakamura M. Mitsui K. Ohno-Iwashita Y. Eur. J. Biochem. 1996; 241: 941-947Crossref PubMed Scopus (100) Google Scholar) was used to construct six pfoA derivatives encoding truncated θ-toxins. Stop codons were introduced at appropriate sites in the pfoA gene by a site directed mutagenesis kit (CLONTECH) based on the unique site elimination method (22Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1079) Google Scholar). The 5′-deoxyoligonucleotide, dGTGACTGGTGAGGCCTCAACCAAGTC, was used to make a unique restriction site for the selection of all mutations. Stop codon insertion was performed using the following mutagenic primers for: Δ471, 5′-deoxynucleotide dCAGTTTTTACTTTAGTaTAcTatTAAGTAATACTAG; Δ470, dCTTTAGTTTAATTtTAtcaAATACTgGATCCAGGGT; Δ468, dGTTTAATTGTAAGTttatCagGATCCAGGGT. Lowercase letters represent bases changed for mutagenesis. The DNA sequences in the resulting plasmids were confirmed by means of the dideoxynucleotide chain-termination method (23Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52671) Google Scholar). Predicted amino acid sequences in the C-terminal ends of the mutant toxins are shown in Fig. 1. Protein production and purification were performed as described previously (13Sekino-Suzuki N. Nakamura M. Mitsui K. Ohno-Iwashita Y. Eur. J. Biochem. 1996; 241: 941-947Crossref PubMed Scopus (100) Google Scholar) with slight modifications. Escherichia coli strains BL21(DE3) and BL21(DE3) harboring pLysS (24Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar) (Novagen, Madison, WI) were used for the overexpression of wild type θ-toxin and mutant toxins. Wild type θ-toxin and mutant toxins were purified from the periplasmic fraction by a series of DEAE-Sephacel chromatographies. In the case of mutant toxins having no hemolytic activity, the fractions eluted from the first DEAE-Sephacel column were analyzed by immunostaining with anti-θ-toxin antibody after SDS-PAGE. Then, the toxin fractions were loaded onto a second DEAE-Sephacel column equilibrated with 20 mm BisTris, pH 6.5, and eluted with the same buffer containing 40 mm NaCl. For further purification, the toxin fractions were applied to a hydroxylapatite column equilibrated with 20 mm sodium phosphate buffer, pH 7.5, and the toxins were eluted with 100 mm sodium phosphate. Then, the toxins were loaded onto a butyl-agarose column equilibrated with 20 mmTris-HCl, pH 7.5, containing 1.7 m(NH4)2SO4 and eluted with 0.5m (NH4)2SO4. The purity of the toxins was checked by SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar). Hemolytic activity was determined as described previously (26Saito M. Ando S. Tanaka Y. Nagai Y. Mitsui K. Hase J. Mech. Aging Dev. 1982; 20: 53-63Crossref PubMed Scopus (11) Google Scholar). The amount of toxin required for 50% hemolysis of 1 ml of 0.5% sheep erythrocytes in 30 min at 37 °C (HD50) was determined using the von Krogh equation (27Mayer M.M. Kabat E.A. Kabat and Mayer's Experimental Immunochemistry. 2nd Ed. Thomas, Springfield, IL1961: 133-240Google Scholar). The hemolytic activity of each toxin was obtained as a 1/HD50 value and expressed relative to the wild type toxin. After activation with 10 mmdithiothreitol for 30 min at 10 °C, each toxin (0.3 μg) was incubated with 0.5% hematocrit sheep erythrocytes in phosphate-buffered saline, pH 7.0, containing 1 mg/ml bovine serum albumin for 20 min at 20 °C. The mixture was centrifuged at 250,000 × g for 20 min at 4 °C, and both the pellet and supernatant fractions were analyzed by Western blotting. The cholesterol binding activity of each toxin was examined by using TLC plates as described previously (13Sekino-Suzuki N. Nakamura M. Mitsui K. Ohno-Iwashita Y. Eur. J. Biochem. 1996; 241: 941-947Crossref PubMed Scopus (100) Google Scholar, 18Iwamoto M. Ohno-Iwashita Y. Ando S. Eur. J. Biochem. 1990; 194: 25-31Crossref PubMed Scopus (45) Google Scholar). The cholesterol binding activity of each toxin was determined by ELISA using the microtiter plate (Immulon 1, Dynatech Laboratories, Alexandria, VA). The wells were coated with various concentrations of cholesterol (12 - 10,000 pmol) and treated with 10 mg/ml fatty-acid-free bovine serum albumin in Tris-buffered saline for 1 h for blocking. Toxins (each 1 ng) were then added to the wells and the mixtures were incubated for 1 h. After washing with Tris-buffered saline, the mixtures in the wells were incubated with anti-(θ-toxin) antibody for 1 h, followed by incubation with peroxidase-conjugated anti-rabbit IgG for 1 h. Toxins bound to the cholesterol on the microtiter plates were detected by measuring the intensity at 410 nm of the color development with 2,2′-azino-di[3-ethyl-benzthiazoline sulfonate(6)] (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) as a peroxidase substrate. Purified toxins (600 ng each) were treated at 22 °C with subtilisin Carlsberg at an enzyme to substrate ratio of 1:60 (28Ohno-Iwashita Y. Iwamoto M. Mitsui K. Kawasaki H. Ando S. Biochemistry. 1986; 25: 6048-6053Crossref PubMed Scopus (44) Google Scholar). At appropriate times, the digestion was stopped by the addition of 1 mmphenylmethanesulfonyl fluoride. The resultant fractions were analyzed by SDS-PAGE and Western blotting by using anti-(θ-toxin) antibody. Circular dichroism (CD) spectra were recorded using a JASCO J-720 spectropolarimeter at room temperature with 1-or 5-mm pathlength cells. Purified proteins were diluted in 10 mm phosphate buffer with or without 150 mm NaCl. Scans from 250 to 190 nm were recorded with 1-mm cells in the absence of NaCl to minimize buffer noise. Molecular ellipticity ([θ]) was calculated based on the mean residue weight and extinction coefficient ( E2800.1%) estimated as 110.8 and 1.6, respectively. The CONTIN program for secondary structure estimation was kindly provided by Dr. S. W. Provencher. Fluorescent studies were performed with a Shimadzu spectrofluorophotometer RF-5000. Emission spectra were measured in the range of 300–400 nm with an excitation wavelength of 280 or 295 nm. Purified toxins were diluted with Hepes-buffered saline, pH 7.0, to a protein concentration of 10 μg/ml. Wild type and mutant toxins were unfolded by treatment with 6 m guanidine hydrochloride (GdnHCl) in 20 mm Tris-HCl, pH 8.0, 150 mm NaCl at room temperature for 10–30 min. Denaturation was confirmed by a red shift in the fluorescence emission wavelength to 350 nm at an excitation wavelength of 280 nm. Refolding was carried out by dialyzing the samples against 20 mm Tris-HCl, pH 8.0, 150 mmNaCl at 4 °C for 20 h. N-Terminal sequences of wild type and mutant toxins were analyzed with a Biosystems protein sequencer 476A. The molecular masses of the toxins were measured by electrospray ionization mass spectrometry (ESI-MS) using a Fourier transform ion cyclotron resonance mass spectrometer BioApex47E (Bruker Instruments) equipped with an external ESI source (Analytica of Branford). Before being injected into the source by a syringe pump operated at 30 μl/h, the samples were desalted on a reverse-phase high pressure liquid chromatography column (Senshu Pak C8–1251-N) eluted with a 10–60% gradient of acetonitrile, 0.1% trifluoroacetic acid. In some cases, the samples were prepared from the SDS-PAGE gel by the method of Nakayama et al. (29Nakayama H. Uchida K. Shinkai F. Shinoda T. Okuyama T. Seta K. Isobe T. J. Chromatogr. A. 1996; 730: 279-287Crossref PubMed Scopus (19) Google Scholar). After SDS-PAGE, the gel was washed with distilled water and stained with 0.3 mCuCl2 for 3 min. The toxin spot was excised from the SDS-PAGE gel and destained successively in 25 mm Tris-HCl, pH 8.3, and 12.5 mm Tris-HCl, pH 8.3. Then, the toxin was extracted from the gel in 50 mm Tris-HCl, pH 8.8, containing 50 mm EDTA and 0.1% SDS. To remove SDS and other impurities, the extracted toxin was applied to a Phenyl-5PW RP column (Tosoh, Tokyo, Japan) and recovered with 80% acetonitrile in 0.1% trifluoroacetic acid. When this method was used, the molecular mass of θ-toxin was determined by subtracting the mass of the adduct of copper, 63.4, from the observed mass. θ-Toxin mutants truncated at the C terminus were constructed and expressed in E. coli as described under "Experimental Procedures" (Figs.1 and 2). θ-Toxin has an intrinsic signal sequence at its N terminus and is secreted into the periplasm when expressed in E. coli (Fig.2 and Ref. 13Sekino-Suzuki N. Nakamura M. Mitsui K. Ohno-Iwashita Y. Eur. J. Biochem. 1996; 241: 941-947Crossref PubMed Scopus (100) Google Scholar). A similar expression profile was observed for Δ471 and amounts comparable to wild type θ-toxin were recovered from the periplasmic fraction (Fig. 2). A slightly smaller amount was recovered in the case of Δ470. Upon expression of the DNA construct for Δ468 and mutants with larger truncations, the amounts of proteins with molecular sizes close to that of intact θ-toxin decreased with concomitant increases in the amounts of degradation products with sizes around 38 kDa (Fig. 2 and data not shown). The results suggest that truncations at the C terminus affect the biosynthesis of θ-toxin and/or its secretion into the periplasm. To further characterize the mutant toxins, the expressed proteins were purified from the periplasmic fraction by DEAE-Sephacel column chromatographies and their molecular masses were determined by ESI-MS (Table I). The observed molecular masses of wild type, Δ471, and Δ470 are within the range of the predicted molecular masses. In contrast, the observed moleculer mass of the protein recovered from the cells harboring the constructed plasmid for Δ468 is smaller than the predicted mass for Δ468 (Table I). This indicates that the production of the protein with a five-amino acid C-terminal truncation brings about a further truncated form. N-terminal sequence analysis revealed that the product has the same N-terminal sequence as the wild type toxin. From the results of N-terminal and molecular mass analyses, we conclude that the product comprises residues 1–451 (predicted M r, 50,375.6), with a 21-amino acid truncation at the C terminus. We designate the product as Δ452 hereafter. The elution profile of Δ452 is different from those of wild type θ-toxin and two mutants, Δ471 and Δ470; the former eluted from DEAE-Sephacel column at 110 mm NaCl, whereas the latter two mutants eluted at 60 mm.Table IMolecular masses of wild type and mutant toxins measured by electrospray ionization mass spectrometryToxinObserved M rPredictedM rWild type52,671.4 ± 1.852,672.0Δ47152,394.3 ± 2.352,394.7Δ47052,295.9 ± 2.352,293.6Δ46850,384.0 ± 1852,093.4Refolded wild type52,673.6 ± 2.052,672.0Refolded Δ47152,395.7 ± 4.452,394.7 Open table in a new tab The relative hemolytic activities of purified wild type and three mutant toxins were determined (Fig. 3,upper part). No differences in hemolytic activity were detected between wild type θ-toxin and Δ471, indicating that the deletion of two amino acids from the C terminus of θ-toxin does not affect hemolytic activity. In contrast, Δ470 showed a lower hemolytic activity, 40% that of wild type, while Δ452 showed no hemolytic activity. These results indicate that truncation of the C terminus by 21 amino acids causes a loss of hemolytic activity. Hemolysis by θ-toxin involves two important steps, binding and insertion into membranes, prior to pore formation. The binding activity to cells was measured and compared among the wild type and mutant toxins (Fig. 3, lower part). Δ471 showed high-affinity binding to sheep erythrocytes similar to the wild type, but Δ470 showed only very weak binding. Δ452, which has no hemolytic activity, never bound to the cells. These results show a good correlation between cell binding activity and relative hemolytic activity. Cholesterol on plasma membranes serves as a receptor for θ-toxin. Fig. 4 a shows the cholesterol binding activity of mutant toxins on TLC plates as detected by immunostaining with anti-θ-toxin antibody. Δ471 and Δ470 were found to bind to cholesterol on TLC plates and to specifically recognize free cholesterol but not phosphatidylcholine or esterified cholesterol. Their manner of binding was the same as that of wild type toxin, although Δ470 shows weaker spots. On the other hand, Δ452 did not bind to cholesterol at all. Fig. 4 b shows the quantitative analysis of the cholesterol binding activity of the toxins by ELISA. Δ471 shows an activity comparable to the wild type toxin. The activity of Δ470 is about 40% of the wild type, while no activity could be detected for Δ452. The results show that the cholesterol binding activity of the toxins correlates well with their cell binding and hemolytic activities. We previously reported that mutants with Trp to Phe substitutions within the tryptophan-rich consensus sequence show decreased binding affinity for erythrocytes (13Sekino-Suzuki N. Nakamura M. Mitsui K. Ohno-Iwashita Y. Eur. J. Biochem. 1996; 241: 941-947Crossref PubMed Scopus (100) Google Scholar). We examined the cholesterol binding activity of two such mutants, W438F and W439F, by ELISA and compared the results with the cholesterol binding activity of the C-terminal truncation mutants (Fig. 4 b). Mutants with Trp to Phe substitutions show cholesterol binding activity similar to that of the wild type toxin (Fig. 4 b), showing that mutations of Trp in the consensus sequence has little effect on the cholesterol binding activity. The decrease in cell binding activity of the mutants should be attributable to step(s) other than cholesterol binding. This makes a district difference from the results for the mutants with C-terminal truncations. The defects in the cholesterol binding activities of Δ470 and Δ452 can be attributed to either the deletion of cholesterol-binding sites or conformational changes around the binding sites. To assess these possibilities, we first examined the susceptibility of mutant toxins to a protease (Fig.5). Digestion of wild type θ-toxin and Δ471 by subtilisin Carlsberg produced a distinctive 39-kDa fragment assigned as the C-terminal fragment (28Ohno-Iwashita Y. Iwamoto M. Mitsui K. Kawasaki H. Ando S. Biochemistry. 1986; 25: 6048-6053Crossref PubMed Scopus (44) Google Scholar); a smaller amount of this fragment was detected when Δ470 was digested. In contrast, Δ452 was digested over time into undetectable pieces showing no distinctive bands. Trypsin digestion also produced proteolytic fragments of 28 and 25 kDa (18Iwamoto M. Ohno-Iwashita Y. Ando S. Eur. J. Biochem. 1990; 194: 25-31Crossref PubMed Scopus (45) Google Scholar) from Δ471 and Δ470, but not from Δ452 (data not shown). The results indicate that the secondary or tertiary structures of Δ452 has been changed by C-terminal truncation of 21 amino acid residues. Because six out of the seven tryptophan residues in θ-toxin are located in the C-terminal region (see Fig. 1), it is reasonable to measure tryptophan fluorescence in order to monitor the conformational alterations of θ-toxin induced by truncation. When the toxins were excited at 295 nm, no differences in the peak emission wavelength at 338 nm were detected between wild type and two mutants, Δ471 and Δ470 (Table II), showing that the environmental changes around the Trp residues are not significant in those two mutants. However, environmental changes around some fluorophores other than tryptophan appear to have occurred, since the mutants exhibited a red shift in the maximal emission wavelength when excited at 280 nm (Table II). On the other hand, a distinctive red shift of the maximal emission wavelength was observed for Δ452 as compared with the wild type toxin (Table II), indicating that the environment of the tryptophan residues in these mutants is more hydrophilic than in the wild type. Simultaneously, the intensity of the tryptophan fluorescence in Δ452 excited at 295 nm was enhanced to 3.2 times that of the wild type θ-toxin. The results suggest that the inactive mutant Δ452 has significant alteration in its tertiary structure around tryptophan residues, and that this leads to the loss in hemolytic activity.Table IIMaximal emission wavelengths and the relative intensity of wild type and truncated toxinsToxinEmission maximum (λex)Relative intensity280 nm295 nm280 nm295 nmnm%Wild type332338100100Δ471334338106 ± 3127 ± 3Δ470336338115 ± 2135 ± 3Δ452340342241 ± 3319 ± 4The fluorescence measurements of wild type and truncated toxins were carried out at excitation wavelengths (λex) of 280 and 295 nm. The data represent mean ± S.E. for three independent experiments. Maximal emission wavelengths are displayed in nanometers (nm), and the maximal intensity of each mutant is shown relative to the intensity of the wild type toxin. Open table in a new tab The fluorescence measurements of wild type and truncated toxins were carried out at excitation wavelengths (λex) of 280 and 295 nm. The data represent mean ± S.E. for three independent experiments. Maximal emission wavelengths are displayed in nanometers (nm), and the maximal intensity of each mutant is shown relative to the intensity of the wild type toxin. When θ-toxin interacts with cholesterol on dioleoylphosphatidyl choline/cholesterol liposomes, there is an increase in the intensity of the tryptophan fluorescence (19Nakamura M. Sekino N. Iwamoto M. Ohno-Iwashita Y. Biochemistry. 1995; 34: 6513-6520Crossref PubMed Scopus (85) Google Scholar). The two truncated toxins, Δ471 and Δ470, also showed increases in the intensity when incubated with dioleoylphosphatidyl choline/cholesterol liposomes. In contrast, no enhancement of fluorescence intensity was detected for Δ452 (data not shown). Therefore, this mutant lacks an appropriate structure for interaction with cholesterol in membranes. In order to determine whether the deletion of C-terminal amino acids affects the secondary structure of the toxin, far ultraviolet CD spectra were measured. As shown in Fig.6 a, wild type θ-toxin has a β-sheet-rich structure and the spectra of Δ471 and Δ470 closely resemble that of the wild type toxin. On the other hand, drastic difference was detected in the spectrum of Δ452 as compared with the wild type toxin. A significant increase in negative ellipticity was observed at 208 nm and shorter wavelengths. The CD difference spectrum obtained by subtracting the wild type spectrum from the Δ452 spectrum exhibited a deep minimum at 200 nm or a shorter wavelength and a shoulder at around 225 nm. This difference spectrum resembles that usually taken to indicate an unfolded conformation (30Chang C.T. Wu C.C. Yang J.T. Anal. Biochem. 1978; 91: 13-31Crossref PubMed Scopus (1025) Google Scholar). This observation suggests a large disorder in the secondary structures of Δ452. The structural analysis suggests that several amino acids at the C terminus play essential roles in in vivo protein folding and/or the maintenance of protein conformation. We carried out in vitrorefolding experiments on the truncated mutants to define the function of C-terminal amino acids during folding. Wild type θ-toxin and mutant Δ471 (truncated by two amino acids) were denatured in 6m GdnHCl, renatured by dialysis, and their hemolytic activities were measured. As shown in Fig.7 a (upper part), wild type θ-toxin recovered 81% of full hemolytic activity while Δ471 displayed only 13% recovery, even though Δ471 has an activity comparable to the wild type before denaturation. The refolded Δ471 hardly bound to sheep erythrocytes as shown in Fig.7 a (lower part), showing a good correlation with relative hemolytic activity. To investigate whether the refolded Δ471 recognizes cholesterol, the cholesterol binding activity was measured by ELISA. The refolded Δ471 shows much less cholesterol binding activity than native Δ471, while the activity of the wild type toxin is not changed by the denaturation refolding treatment (Fig. 7 b). Although we could not judge whether all the refolded Δ471 molecules have lower binding affinities than in the native state or whether a small population of Δ471 refolds to the native form with full activity, it is clear that Δ471 easily loses its ability to bind cholesterol during the denaturation-renaturation process. A decrease in the cholesterol binding activity was also observed after denaturation-refolding of Δ470 (data not shown). To rule out the possibility that there might be a minor contaminating protease that cleaves the Δ471 protein during the refolding treatment and causes it to lose activity, the relative molecular masses of native and refolded Δ471 were determined by ESI-MS (Table I). The relative molecular masses determined for the refolded Δ471 and native Δ471 are the same and within the range of the predicted one (Table I), indicating that no proteolytic cleavage occurs during the refolding process. Wild type toxin also maintains its intact size during refolding treatment, as shown by the relative molecular masses before and after treatment (Table I). These results indicate that the loss of Δ471 activity after refolding is not caused by the action of protease. The above results show that even just two amino acid residues at the C terminus are involved in the correct folding of θ-toxin. To assess whether the inactivation of Δ471 by denaturation-refolding is accompanied by a conformational alteration, the structural properties of wild type and Δ471 after denaturation-refolding treatment were studied by CD and fluorescence analyses. Compared with native Δ471, the far-ultraviolet CD spectrum of refolded Δ471 shows a slight alteration in the secondary structure (Fig. 6 b), a 3% decrease in β-sheet content and a concomitant increase in random coil in the refolded Δ471 as estimated by the algorism program, CONTIN (31Provencher S.W. Glöckner J. Biochemistry. 1981; 20: 33-37Crossref PubMed Scopus (1886) Google Scholar). In the case of the wild type θ-toxin, no differences were observed in the CD spectra before and after refolding (data not shown). In the maximal emission wavelength of the fluorescence spectrum, no significant changes were observed in both wild type and Δ471. These results clearly show that the secondary and tertiary structural changes in the refolded Δ471 are small compared with those observed for Δ452 (Fig. 6 a and Table II), suggesting that the changes in refolded Δ471 occur in a limited region of the toxin molecule. The crystallographic study of θ-toxin showed that domain 4, the C-terminal domain supposed to contain the cholesterol-binding region, has nine β strands folded into a compact β-sheet sandwich (12Rossjohn J. Feil S.C. McKinstry W.J. Tweten R.K. Parker M.W. Cell. 1997; 89: 685-692Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Two β strands in antiparallel orientation are located within the C-terminal 20 amino residues and form a part of one β sheet (Fig. 1). In this study, focusing on the two C-terminal β strands, we constructed several C-terminal truncated θ-toxin mutants to investigate how C-terminal amino acids contribute to the folding of the protein and its toxic action. We first demonstrated that amino acids in the C-terminal β strand play an important role in correct folding of the toxin. When Δ471 was refolded after denaturation in 6 m GdnHCl, it lost its membrane binding and hemolytic activities with the reduction in cholesterol binding activity (Fig. 7), indicating the importance of the two C-terminal amino acids for correct folding in vitro into the conformation required for cholesterol binding. However, Δ471 showed essentially the same hemolytic activity and secondary structure as wild type θ-toxin. This indicates that the mutant folds into the native conformation in vivo. Taking the difference betweenin vivo and in vitro folding into consideration, probably chaperone-like molecules help to achieve correct folding in vivo (32Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3121) Google Scholar, 33Fedorov A.N. Baldwin T.O. J. Biol. Chem. 1997; 272: 32715-32718Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). As shown in Δ470, a three-amino acid truncation affects folding both in vivo andin vitro. The truncation of five amino acids from the C terminus leads to a further truncation of the protein in host E. coli, indicating that the C-terminal β strand protects the protein against proteolytic cleavage in host cells. For toxin production, we usedE. coli B strain, BL21(DE3), as a host, because it lacks both lon and ompT proteases. Some other minor protease(s) in E. coli may contribute to cleaving the product during synthesis or secretion into the periplasm (34Cook R.A. Crit. Rev. Biotechnol. 1988; 8: 159-175Crossref PubMed Scopus (5) Google Scholar). The product, Δ452, lacks the two β strands in the C terminus and completely loses its cell binding and hemolytic activities due to its inability to recognize cholesterol (Fig. 4). It is likely that the molecular structure required for the specific binding of cholesterol molecules is absent or not correctly organized in Δ452. Spectroscopic data indicate its partially unfolded secondary structure and an environmental alteration around tryptophan residues to a more hydrophilic and unrestricted state (Fig. 6 a and Table II). Since the elimination of the two β strands from the C-terminal end causes this remarkable disorder in structure, the C-terminal two β strands are suggested to play key roles in constructing the overall structure of the toxin. There are two distinct steps in cell binding by θ-toxin, cholesterol recognition and membrane insertion. In this study we demonstrated that truncation of the C terminus abolishes cholesterol-recognition ability, resulting in the loss of cell binding activity. This is in contrast to mutants with Trp to Phe substitutions within the tryptophan-rich consensus sequence (residues 430–440), which show a loss in cell binding activity despite their ability to bind cholesterol (Fig.4 b and Ref. 13Sekino-Suzuki N. Nakamura M. Mitsui K. Ohno-Iwashita Y. Eur. J. Biochem. 1996; 241: 941-947Crossref PubMed Scopus (100) Google Scholar). We previously suggested that the tryptophan-substituted mutants have some deficiency in membrane insertion activity (13Sekino-Suzuki N. Nakamura M. Mitsui K. Ohno-Iwashita Y. Eur. J. Biochem. 1996; 241: 941-947Crossref PubMed Scopus (100) Google Scholar, 20Nakamura M. Sekino-Suzuki N. Mitsui K. Ohno-Iwashita Y. J. Biochem. 1998; 123: 1145-1155Crossref PubMed Scopus (44) Google Scholar) that could cause them to lose cell binding activity. The tryptophan-rich consensus sequence locates in close proximity to one of the two β sheets in domain 4, and distant from the other β sheet to which the C-terminal β strand belongs (Fig.1 b). The crystallographic data indicate that there are some amino residues in the proximal β sheet that are possible quenchers of the fluorescence of Trp-436 and Trp-439, Trps within the consensus sequence. Thus, a change in tryptophan fluorescence intensity could be a sensitive marker for a change in three-dimensional arrangement among these Trp residues and quenchers. Since either Δ470 or refolded Δ471 shows no significant change in tryptophan fluorescence compared with the wild type toxin, the microenvironment around the Trp residues remains intact in these mutants. This strongly suggests that the C-terminal truncation does not affect the structural features around the tryptophan-rich consensus sequence despite its significant effect on cholesterol binding activity. Probably the site of cholesterol binding is in different region in domain 4 from that of membrane insertion. Since the molecular mass of Δ471 remains unchanged by the unfolding-refolding treatment (Table I), the activity loss in cholesterol binding should be ascribed to a conformational change. It is likely that the molecular structure required for the specific binding of cholesterol molecules is not correctly organized after treatment. An approximately 3% decrease in β- sheet content in Δ471 was detected after treatment. No change occurs in tryptophan fluorescence as discussed above. This implies that the change occurs within a limited region upon refolding of Δ471 and that this local change in structure directly affects the cholesterol-binding site. Since the C-terminal β strand interacts directly with the next β strand (residues 453–460) by hydrogen bonding to form part of an antiparallel β sheet (Fig. 1 and Ref. 12Rossjohn J. Feil S.C. McKinstry W.J. Tweten R.K. Parker M.W. Cell. 1997; 89: 685-692Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), it is likely that the decrease in β-sheet content produced by treatment occurs near these strands. It has been reported that C-terminal truncation of pneumolysin, another cholesterol-binding cytolysin, causes a loss of cell binding activity (17Owen R.H.G. Andrew P.W. Boulnois G.J. Michell T.J. FEMS Microbiol. Lett. 1994; 121: 217-221Crossref PubMed Scopus (38) Google Scholar). It would be interesting to know whether this loss in the cell binding activity of pneumolysin is due to the loss of cholesterol binding activity, although conformational studies and molecular mass determination of the truncated species of pneumolysin would be required to draw a conclusion. There have been several reports showing that C-terminal residues are important for the correct folding and maintenance of the native protein conformations (35Voziyan P.A. Tremblay J.M. Yarbrough L.R. Helmkamp Jr., G.M. Biochemistry. 1996; 35: 12526-12531Crossref PubMed Scopus (21) Google Scholar, 36Chen Y. Lin S. Tzeng S. Patel H.V. Lyu P. Cheng J. Proteins. 1996; 26: 465-471Crossref PubMed Scopus (29) Google Scholar). Among them, θ-toxin is a distinct example since the deletion of only two amino acids from the C terminus out of a total of 472 residues seriously affects its folding and the maintenance of the functional conformation. We have reported chemically modified θ-toxin as a new probe for cholesterol (37Iwamoto M. Morita I. Fukuda M. Murota S. Ando S. Ohno-Iwashita Y. Biochim. Biophys. Acta. 1997; 1327: 222-230Crossref PubMed Scopus (62) Google Scholar). If the relationships between binding activity and the conformation of the C-terminal region are clarified and the minimum binding unit is identified, further design of useful probes can be realized. We thank Drs. M. Inomata and M. Hayashi for technical advice and helpful discussion; Drs. S. Mizuno, H. Morisawa, and K. Shibazaki for technical advice concerning the computer analysis of the secondary structure; and Dr. H. Nakayama for technical advice in preparing the samples for ESI. We thank Drs. S. Iwashita and K. Murakami-Murofushi for critical reading of the manuscript and helpful discussion. We thank Dr. M. M. Dooley-Ohto for reading the manuscript.