domain located on the portion of the B fragment that is linked to A. This region is masked .... is maximum binding, c is the concentration of free Triton X-100 and.
Proc. Nati. Acad. Sci. USA
Vol. 73, No. 12, pp. 4449-4453, December 1976
Biochemistry
Binding of Triton X-100 to diphtheria toxin, crossreacting material 45, and their fragments (hydrophobicity/plasma membrane)
PATRICE BOQUET*, MITCHELL S. SILVERMAN, A. M. PAPPENHEIMER, JR.t, AND WALTER B. VERNON Biological Laboritories, Harvard University, Cambridge, Massachusetts 02138
Contributed by A. M. Pappenheimer, Jr., September 24, 1976
Binding of the nonionic detergent [3HrTriton ABSTRACT X-100 by diphtheria toxin, by the nontoxic serologically related protein crossreacting material (CRM) 45, and by their respective A and B fragments has been studied. If first denatured in 0.1% sodium dodecyl sulfate, all of the proteins with the exception of fragment A bind increasing amounts of Triton X-100, reaching a maximum of more than 40 mol bound per mol of protein when the detergent concentration exceeds its critical micelle concentration. No measurable amount of Triton X-100 is bound by native toxin or its A fragment at any concentration of the detergent. Undenatured CRM45 or its B45 fragment, on the other hand, readily become inserted into Triton X-100 micelles when the detergent reaches its critical micelle concentration. The results show that the toxin molecule contains a hydrophobic domain located on the portion of the B fragment that is linked to A. This region is masked in native toxin. Based on these findings, a model is proposed to describe how fragment B facilitates the transport of the enzymically active hydrophilic fragment A across the plasma membrane to reach the cytoplasm.
Diphtheria toxin exerts its lethal effect on sensitive mammalian cells by inhibition of protein synthesis. In order to intoxicate a cell, the 62,000 dalton toxin molecule must first interact with a plasma membrane surface receptor and then an NH-terminal 21,150 dalton polypeptide (fragment A) must be split off and transported across the lipid bilayer to reach the cytoplasm. Fragment A then catalyzes the transfer of the ADP-ribosyl group from NAD+ to elongation factor 2 (EF-2), thereby causing its inactivation (1, 2). Previous studies (3, 4) with mammalian cell cultures have shown that each sensitive cell carries about 4000 specific surface membrane receptors that initially react reversibly with groups located near the COOH-terminus of the toxin B fragment. This initial rapid reaction is followed by a slow irreversible process involving a major conformational change, during which the molecule enters the plasma membrane. Finally, fragment A is split off to reach the cytoplasm while B apparently remains behind in the membrane. If this model is indeed correct, we would expect that fragment B should behave like other membrane proteins and should contain a hydrophobic "domain" (5) which becomes inserted into the lipid bilayer during the entry process. Membrane proteins may be extracted into aqueous solvents that contain a nonionic detergent such as Lubrol or Triton X100. During this process, protein-bound phospholipid molecules are replaced by molecules of the detergent. By measuring the quantity of detergent that it can bind, it is possible to estimate the fraction of a protein molecule's surface capable of hydrophobic interaction (6, 7). We are now reporting studies on the Abbreviations: CRM, crossreacting material; CMC, critical micelle concentration; NaDodSO4, sodium dodecyl sulfate. * Present address: Pasteur Institute, Paris, France. t To whom reprint requests should be addressed. 4449
binding-of Triton X-100 to diphtheria toxin, to its A and B fragments, and to the nontoxic, serologically related tox gene product, crossreacting material (CRM) 45, which lacks the 17,000 dalton COOH-terminal amino acid sequence of the intact toxin molecule (8). Based on our findings, we are proposing a model to describe the process by which the diphtheria toxin A fragment is transported across the plasma membrane. MATERIALS AND METHODS Detergent Preparation. Triton X-100 (polyoxyethylene octyl phenol, averaging 9.6 ethylene oxide units per monomer) was obtained from Rohm & Haas Co., Philadelphia. An aqueous solution of ring-labeled [3H]Triton X-100 (0.93 mg/ml) was a gift from Steven Clarke. The concentration of this stock solution was calculated from the absorbance at 274 nm of dilutions in 1 mM Tris-HCI buffer containing 0.1 M Na2SO4 at pH 7.5, assuming A (1%) = 23.2. The specific activity was 125 cpm/,gg of [3H]Triton X-100 as determined in 3 ml of Aquasol (New England Nuclear) in a Beckman LS230 liquid scintillation counter. The critical micelle concentration (CMG) of Triton X-100 was determined using methyl orange by the method of Benzonana (9) to be 0.13 mg/ml in the 10 mM phosphate buffer at pH 7.2 used for binding studies. Labile tritium was estimated by passing a small volume of the stock [3H]Triton X-100 solution Icolumn. Almost 99% of the radioacthrough a Sephadex G-0 tivity emerged in the void volume. Proteins. Partially purified diphtheria toxin (30-5% nicked) was obtained from Connaught Laboratories, Toronto, and was purified further by DE52 DEAE-cellulose chromatography (10). After treatment with trypsin in the presence of thiol (11), fragments A and B were separated from one another by gel filtration through a Sephadex G-100 column equilibrated with 10 mM sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (NaDodSO4) and 0.01% 2-mercaptoethanol at pH 7.2. Pooled fractions containing fragment B were maintained in the 0. 1% NaDodSO4-containing buffer in order to prevent precipitation of the protein. The NaDodSO4 was removed from the pooled fragment A fractions by dialysis. The CRM45 protein was isolated from culture filtrates of Corynebacterium diphtheriae C7(#45). The details of its purification by salt fractionation and DE52 chromatography will be described elsewhere. After treatment with trypsin under reducing conditions, fragment B45 was separated from fragment A by two or more precipitations at pH 4.4 and resolution in 10 mM sodium phosphate buffer, pH 7.2. Fig. 1 shows the patterns obtained on NaDodSO4/10% polyacrylamide gel electrophoresis of the various proteins used in the binding studies to be described. Triton X-100 Binding. The method described by Clarke (7) was followed. Sucrose gradients (5-20%, wt/vol) were prepared
4450
Biochemistry: Boquet et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
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in 10 mM sodium phosphate buffer, pH 7.2, containing increasing concentrations of [3H]Triton X-100. Gradients were poured into thin-walled polyallomer tubes of 4 ml capacity and stored for at least 4 hr at 40 before use. Samples of 100-200 AI (3 mg of protein per ml in 10 mM phosphate buffer, pH 7.2, containing an excess of [3H]Triton X-100) were layered on top of the gradients, which were then centrifuged in a SB405 rotor using an IE60B ultracentrifuge. Speed, temperature, and duration of each run are given in the legend accompanying each figure. In each experiment, a control gradient containing no protein was run to determine the radioactivity of the base line. After centrifugation, 25 to 30 fractions of three to five drops each were collected from the bottom of the tubes and 25 pI aliquots were counted by liquid scintillation. Protein was determined by the Lowry et al. method (12) using 40 ,.d aliquots diluted to 200 Al in the phosphate buffer to avoid precipitation of detergent. Binding was calculated as follows: for each point associated with the protein peak, the base line counts were subtracted from the total counts and the differences were divided by the specific activity of the [3H]Triton X-100 (125 cpm/Ag) and by the amount of protein in the aliquot. For calculation of molar ratios the following molecular weights were assumed: for Triton X-100, 636; toxin, 62,000; fragment A, 22,000; fragment B, 40,000; CRM45, 45,000; and fragment B45, 23,000.
RESULTS Triton X-100 Binding to Diphtheria Toxin and Its Frag-
ments. Fig. 2A shows that in the absence of NaDodSO4, there is no measurable binding of Triton X-100 to diphtheria toxin above the CMC, nor does isolated fragment A, under similar conditions, bind a measurable amount of the detergent (Fig. 2B). Even after "nicking" with trypsin and/or reduction before centrifugation through gradients containing 0.3% 2-mercaptoethanol, no binding of Triton X-100 to toxin could be detected. From the scatter of the base-line data, binding in excess
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FRACTION NUMBER FIC. 2. Binding of [3H]Triton X-100 by toxin and its fragments. Proteins were centrifuged into sucrose gradients containing the detergent above its CMC. Samples (200 Ml) in 10 mM phosphate buffer, pH 7.2, containing 5 mg/ml of [3H]Triton X-100 and 0.8 mg of protein were layered on 3.7 ml of 5-20% sucrose gradients containing 0.5 mg/ml of I:HJTriton X-100 and centrifuged at 4°. Panel A, toxin, 55,000 rpm for 16 hr; Panel B, fragment A, 60,000 rpm for 20 hr; Panel C, fragment B, 60,000 rpm for 20 hr.
of 1 or 2 mol of detergent per mol of protein would have been easily detectable. Thus, in this respect, both toxin and its A fragment behave as do other soluble proteins that have been studied (6, 7). Fragment B, on the other hand, binds an appreciable amount of nonionic detergent, as shown in Fig. 2C. A large peak of radioactivity is associated with the protein peak, equivalent to at least 0.7 mg of Triton X-100 bound per mg of fragment B (about 44 mol/mole of protein). Fig. 3 plots the Triton X-100 bound to fragment B as a function of increasing detergent concentration, both below and above CMC. It is clear that, under these particular conditions, fragment B is able to bind Triton X-100 monomers. The curve shown in Fig. 3 is drawn to fit a theoretical equation describing a system in which a maximum of 52 mol of Triton X-100 is bound per mol of protein (0.83 mg/mg of protein) with a dissociation constant KD = 0.2 mM. The same data are shown in the insert as a double reciprocal plot. It should be stressed at this point that because of its insolubility in ordinary buffers, the fragment B solutions layered on
Biochemistry: Boquet et al. 0.7
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Proc. Natl. Acad. Sci. USA 73 (1976) n7 v.,1
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Fi(c. 3. Binding of Triton X-100 to fragment B as a function of detergent concentration. Samples of 100 ,l, each containing 0.5 mg of protein in 10 mM phosphate buffer containing 0.1% NaDodSO4 and 1-5 mg/ml of [:'H]Triton X-100, were layered on 5-20% sucrose gradients made in 10 mM phosphate containing increasing concentrations of the tritiated detergent. Gradients were centrifuged at 40 for 20 hr at 60,000 rpm. The curve is drawn to fit the equation B = (Bo X C)/KD + C) where B is mg of detergent bound per mg of protein, Bo is maximum binding, c is the concentration of free Triton X-100 and K11 is the dissociation constant in mg/ml. Bo and KD were estimated from the reciprocal plot shown in the insert.
the gradients shown in Figs. 2C and 3 contained 0.1% NaDodSO4 and therefore B was not in its native conformation. However, from our own experiments and those of Clarke (7), when a protein dissolved in NaDoda5SO4 is centrifuged through a gradient containing Triton X-100, all or almost all of the radioactive NaDodSO4 remains behind at the top of the gradient. After replacement of NaDodSO4 by Triton X-100 in the gradient during centrifugation, B remains fully soluble. When intact toxin dissolved in 0. 1% NaDodSO4-containing buffer was centrifuged into a gradient containing 0.11 mg/ml of [3H]Triton X-100 (i.e., just below CMC) 0.33 mg of detergent were bound per mg of toxin (32 mol/mol), a figure that is close to that expected from its B content. Even when treated with NaDodSO4, however, fragment A binds no detergent. Triton X-100 Binding to CRM45 and to Its B Fragment, B45. As seen in Fig. 4, CRM45 binds no Triton X-100 until the detergent concentration reaches its CMC. Above CMC, binding abruptly becomes maximal whether CRM45 is nicked or unnicked, whether it is reduced or unreduced. Therefore, in contrast to toxin itself, CRM45 readily enters into the detergent micelles, even in the absence of NaDodSO4. When CRM45 solutions are dialyzed against 0.1% NaDodSO4-containing buffers before centrifugation into gradients containing increasing concentrations of Triton X-100, binding of monomers does occur and a typical binding curve similar to that shown for fragment B in Fig. 3 is obtained. The curve shown in Fig. 4 was drawn to fit a theoretical system with maximal binding of 51 mol of detergent per mol of CRM45 (0.71 mg/mg of protein) and a KD = 0.15 mM. The maximum observed number of Triton X-100 molecules bound per molecule CRM45 was reached at a detergent concentration well above CMC and was about 42 mol/mol of CRM45 whether or not the protein had been pretreated with NaDodSO4. Assuming that there are 120 Triton X-100 monomers per micelle (13), it may be calculated that 3 molecules of CRM45 are inserted per micelle. The density of such a micelle-CRM45 complex is high enough that the complex passes into the gradient. On the other hand, isolated fragment B45 appears to bind so much [3H]Triton X-100 at
ih
0.3 0.4 0.5 Free Triton X-100, mg/mI FIG. 4. Binding of [3H]Triton X-100 to CRM45 as a function of detergent concentration. Samples of 200 ul, each containing 0.8 mg of protein dialyzed against 10 mM phosphate buffer at pH 7.2, either with 0.1% NaDodSO4 (0) or without NaDodSO4 (0) were layered on 5-20% sucrose gradients containing increasing concentrations of ['H]Triton X-100. Centrifugation was at 40 for 20 hr at 57,000 rpm. 0.1
0.2
In the insert the data for CRM45 previously treated with NaDodSO4 are shown as a double reciprocal plot.
concentrations above CMC that it remains on top of the gradient even after 24 hr centrifugation at 60,000 rpm. Below CMC there is no binding of detergent (data not shown). However, just as in the case of toxin itself and CRM45, when B45 was dialyzed against 0.1% NaDodSO4-containing buffer before centrifuging, a typical binding curve for monomeric [3H]Triton X-100 was obtained. A reciprocal plot of the data is shown in Fig. 5. The data fit a system with a maximum of 55 mol of detergent bound per mole of B45 and KD = 0.2 mM. These constants do not differ significantly from those found for toxin itself or for CRM45. DISCUSSION We have studied the hydrophobicity of diphtheria toxin, CRM45, and their isolated A and B fragments by measuring Bound Triton X- I00
Free Triton X-IO0 FIG. 5. Double reciprocal plot of [3H]Triton X-100 binding to fragment B45 as a function of detergent concentration. Samples of 200 Ml containing 0.6 mg of protein previously dialyzed against 10 mM phosphate buffer, pH 7.2, containing 0.1% NaDodSO4 and 1-1.5 mg/ml of [3H]Triton X-100 were layered on 5-20% sucrose gradients containing increasing concentration of the tritiated detergent below critical micelle concentration. Centrifugation was at 70 for 24 hr at 60,000 rpm. Concentrations of bound Triton X-100 are in terms of mg/mg of protein; free detergent is in mg/ml.
4452
Biochemistry: Boquet et at.
binding of tritiated Triton X-100 according to the method described by Clarke (7). When these proteins were centrifuged through sucrose gradients containing [3H]Triton X-100 at concentrations below its critical micelle concentration, none of them bound measurable amounts of the detergent. On the other hand, when the same proteins were first dialyzed against the ionic detergent sodium dodecyl sulfate (0.1% solution in 10 mM buffer) before the binding assay, toxin, CRM45, and their isolated B fragments (but not fragment A) each bound a maximum of about 40 mol of monomeric Triton X-100 per mol of protein. The fact that under these conditions B45 binds as much detergent as CRM45 or as the entire B fragment of toxin clearly demonstrates that the 23,000 amino acid sequence linking fragment A to the remainder of B carries a hydrophobic "domain." Within this domain, in the presence of NaDodSO4, it seems likely that the amino acids become coiled in such a manner that their hydrophobic side chains are clustered and thus able to bind monomeric Triton X-100 molecules (14). As mentioned above, without prior dialysis against NaDodS04, none of the toxin-related proteins binds Triton X-100 below its CMC. Above CMC, however, the proteins behave very differently from one another. Toxin itself, whether nicked or unnicked, whether reduced or unreduced, does not bind Triton X-100 or enter into micelles. Nor does fragment A bind the detergent at any concentration tested. In contrast, when the concentration of Triton X-100 in the gradient reaches its CMC, both CRM45 and B45 abruptly become inserted into micelles (see Fig. 4). We have calculated that three molecules CRM45 are bound for each micelle containing 120 molecules of the detergent (13). Because of the fragment B's insolubility, in aqueous buffers, it has not been feasible to measure the Triton X-100 binding of the toxin B fragment without prior treatment with NaDodSO4. It is not possible to redissolve precipitated B in Triton X-100containing buffers, although after solution with the aid of NaDodSO4, the NaDodSO4 may be replaced by Triton X-100 without causing B to precipitate. The instability of fragment B in aqueous solution is clearly not due to its hydrophobicity, since B45, which contains the hydrophobic domain, is soluble and stable in neutral buffers. Its relatively high hydrophobicity as compared to fragment A may account for the fact that in NaDodSO4 polyacrylamide gel electrophoresis, B45 travels faster than A even though its molecular weight is almost certainly greater (15). Fragment A travels slower than would be expected from its molecular weight of 21,150 calculated from the sequence data (16), probably because it binds less NaDodS04 than most proteins. B45, because of its hydrophobicity, probably binds more. That a large segment of the CRM45 actually enters into the micelles and is not merely adsorbed to their surface is shown by the following experiment involving two gradients. In the first, CRM45 was dialyzed against 10 mM buffer containing 0.1% NaDodSO4. At this low ionic strength, NaDodSO4 does not form micelles (17). just before centrifugation, 1 mg/ml of Triton X-100 was added (about seven times its CMG) and the protein was then centrifuged through a sucrose gradient containing 0.01% [3H]Triton X-100 (i.e., below the CMC). The expected amount of labeled detergent was found associated with the protein peak (see Fig. 4, closed circles). In the second gradient, the order of addition of the two detergents was reversed, so that CRM45 had already become inserted into Triton X-100 micelles before NaDodSO4 was added. Under these circumstances, no significant radioactivity was associated with the protein peak, presumably because NaDodSO4 could not readily penetrate the Triton X-100 micelles to interact with the
Proc. Natl. Acad. Sci. USA 73 (1976)
hydrophobic region of CRM45. Upon entering the gradient, with detergent below CMC, the micelles dissociate and release native CRM45 while the NaDodSO4 remains behind at the top of the gradient. Although the toxin-specific receptors on growing HeLa cell membranes fail to react with CRM45, preliminary experiments have shown that unpurified membrane ghost preparations from HeLa cells do bind 125I-labeled CRM45. In fact, such preparations can bind at least 30-40 times as much 125I-CRM45 as 125I-toxin. In this respect at least, a lipid membrane preparation resembles the detergent micelles in its behavior. Our earlier studies (3) have shown that the initial reaction between diphtheria toxin and specific surface receptor is followed by irreversible entry of the molecule into the plasma membrane. As a result, fragment A reaches the cytoplasm and B remains, temporarily at least (4), within the lipid bilayer. The fact that CRM45 can enter detergent micelles and is taken up avidly by cell membrane ghosts, whereas the toxin molecule itself is not, poses some interesting questions. Is a COOH-terminal polypeptide split off after toxin becomes bound to the cell surface so that the rest of the molecule, now resembling CRM45 and being in close proximity to the membrane, can insert itself into the lipid bilayer? Does the receptor itself play an active role in this process? Is it possible that the receptor might be a specific protease? We propose the following hypothetical model to describe how the fragment A of toxin traverses the plasma membrane. As a result of the initial reaction of membrane receptor with groups mainly located on the COOH-terminal portion of the toxin molecule, the hydrophobic domain of B is brought into close proximity with the phospholipid bilayer. Before this region can enter into the bilayer, either a COOH-terminal hydrophilic polypeptide must be split off so as to produce a molecule resembling CRM45 or a major conformational change must be brought about by some other mechanism. In any case, the hydrophobic domain becomes inserted into the bilayer, where it may form a channel either by itself or in association with that part of the receptor molecule already lying within the mem' brane. Since fragment A is attached to that part of the B polypeptide in which the hydrophobic region is located, A may be drawn through the channel as it is being formed until the disulfide bridge and the short exposed loop that link the two fragments together reach the inner surface of the membrane. There, "nicking" and reduction take place and A enters the cytoplasm. Because fragment A readily renatures even after being subjected to drastic denaturing conditions, any channel through the membrane need only be large enough to accommodate A in its unfolded form. We are indebted to Dr. Steven Clarke and to Dr. Eva Neer for helpful discussion and suggestions and to Dr. Guido Guidotti for critical reading of the manuscript. This work was supported by Grant PCM75-15314 from the National Science Foundation. P.B. is a Postdoctoral Fellow supported in part by the French government and in part by the Institut National de la Sante et de la Recherche Medicale (INSERM). 1. Pappenheimer, A. M., Jr. & Gill, D. M. (1973) Science 182, 353-358. 2. Collier, R. J. (1975) Bacteriol. Rev. 39,54-82. 3. Boquet, P. & Pappenheimer, A. M., Jr. (1976) J. Biol. Chem., 251,
5770-5778. Boquet, P., Silverman, M. & Pappenheimer, A. M., Jr. (1976) in Proc. of the 1976 UCLA Winter Symposium on Cell Shape and Surface Architecture (Alan R. Liss, New York), in press. 5. Bretscher, M. S. & Raff M. C. (1975) Nature 258,43-49. 6. Helenius, A. & Simon, K. (1972) J. Biol. Chem. 247, 36564.
3661.
Biochemistry: Boquet et al. 7. Clarke, S. (1975) J. Biol. Chem. 250,5459-5469. 8. Uchida, T., Gill, D. M. & Pappenheimer, A. M., Jr. (1971) Nature New Biol. 233, 8-11. 9. Benzonana, G. (1969) Biochim. Biophys. Acta 176, 836-848. 10. Pappenheimer, A. M., Jr., Uchida, T. & Harper, A. A. (1972) Immunochemistry 9,891-906. 11. Gill, D. M. & Dinius, L. L. (1971) J. Biol. Chem. 246, 14851491. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275.
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13. Becher, P. (1967) in Nonionic Surfactants, ed. Schick, M. J. (Marcel Dekker, New York), p. 495. 14. Visser, L., Robinson, N. C. & Tanford, C. (1975) Biochemistry 14, 1194-1199. 15. Grefrath, S. P. & Reynolds, J. A. (1974) Proc. NatI. Acad. Sci. USA 71,3913-3916. 16. DeLange, R. J., Drazin, R. E. & Collier, R. J. (1976) Proc. NatI. Acad. Sci. USA 73, 69-72. 17. Reynolds, J. A. & Tanford, C. (1970) Proc. NatI. Acad. Sci. USA 66, 1002-1007.
