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Evidence that divalent cations bind to diphtheria toxin: An ESR approach
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 997-995
September 30, 1986
EVIDENCE THAT DIVALENT CATIONS BIND TO DIPHTHERIA TOXIN: AN ESR APPROACH Riccardo Basosi+, Vladimir Kushnaryov, Tomasz Panz, and Ching-San Lai* National Biomedical ESR Center, Departments of Radiology and Microbiology Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Received July 28, 1986
Summary: We have used electron spin resonance (ESR) techniques to study the ~ o f divalent cations to diphtheria toxin (DT). Addition of DT to Mn(ll) solution at a stoichiometry of 2:1 (DT:Mn) induces a 79% loss in the i n t e n s i t y of the ESR spectrum of Mn(ll) suggesting a strong binding of Mn(ll) to DT. Inclusion of Ca(ll) at a r a t i o of 1:2:1 (Ca:DT:Mn) in the reaction mixture restores the i n t e n s i t y of the Mn(ll) signal to 64%. This indicates that Ca(ll) and Mn(ll) share same binding s i t e ( s ) in DT. The r e s u l t s presented in t h i s communication suggest that DT is a Ca(ll) binding protein. ® 1986AcademicP. . . . . Inc.
Diphtheria toxin (DT) is a protein with a molecular weight of 62,000 daltons and is extremely t o x i c to eucaryotic c e l l s (1,2). fraqments linked by d i s u l f i d e bridges.
The protein contains A and B
Fraqment B is responsible for the
binding of DT to the plasma membrane of sensitive c e l l s and fragment A is involved in i n a c t i v a t i o n of protein synthesis by ADP-ribosylation of elongation factor 2 of the protein synthesis machinery (1).
Boquet et al. proposed that DT
forms a transmembrane channel in the plasma membrane by which fragment A is transported across the membrane and appears in the cytoplasmic side of the plasma membrane (3,4).
Recent biophysical studies showed that DT forms an
ion-conducting channel in the membrane (5).
Using electron spin resonance (ESR)
spin label methods, we demonstrated previously that DT induces the leakage of acidic phospholipid membranes, which is in agreement with the concept of DT-induced channel formation (6). ~On leave from the Department of Chemistry, University of Siena, Pian dei Mantellini 44-83100, Siena, I t a l y *To whom correspondence should be addressed..
991
0006-291 X/86 $1.50 Copyright © 1986 by Academic Press, lnc. All rights of reproduction in any jorm reserved.
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The f u n c t i o n a l l y d i s t i n c t
ligand-binding s i t e s in DT molecule, namely, the
NAD s i t e and the P s i t e have been described; the NAD s i t e catalyzes the i n t r a c e l l u l a r ADP-ribosylation reaction and the P s i t e a f f e c t s DT binding to the c e l l (7).
Calcium ions have been shown to enhance the binding of DT to the c e l l
(8).
In t h i s study, by using electron spin resonance (ESR) spectroscopy we demonstrate f o r the f i r s t
time t h a t DT has Mn(II) binding s i t e ( s ) at which the binding of
Mn(II) could be displaced by addition of calcium, suggesting that DT i s a calcium binding p r o t e i n . Materials and Methods DT was obtained from Connauqht Labs, Toronto, Canada and f u r t h e r p u r i f i e d as described p r e v i o u s l y (9). P u r i f i e d DT showed a s i n g l e band of molecular weight 62,000 on 7% SDS-polyacrylamide gel e l e c t r o p h o r e s i s . The protein was dialyzed against 0.15 M NaCl, pH 7.0 p r i o r to use. ESR Measurements The ESR spectra were operating at 9.5 GHz and The f i e l d modulation and The f i e l d sweep was 1000 carried out at 22°C.
obtained with a Varian Century l i n e spectrometer, with a Varian E-9 spectrometer, oDerating at 3.4 GHz. amplitude modulation were 100 KHz and 5 G, r e s p e c t i v e l y . G and the time constant i sec. The measurements were
Results and Discussion Fig. la shows a t y p i c a l ESR spectrum of Mn(ll) in aqueous s o l u t i o n . Addition of DT to Mn(ll) s o l u t i o n at a r a t i o of 2:1 (DT to M n ( l l ) ) induces a 79% loss of the i n t e n s i t y of the Mn(ll) spectrum, Fig. l b .
The i n t e n s i t y loss may be
b
I,
q
IOOG
Effects of Diphtheria Toxin on the ESR spectra of Mn(ll) at 9.5 GHz in 0.15 M NaCI, pH 7.0. (a) 25 ~M Mn(ll) alone. (b) 25 ~M Mn(ll) plus 50 ~M Diphtheria Toxin. (c) same as in (b) except in the presence of 25 ~M Ca(ll). Note the reappearance of Mn signal due to the addition of Ca(ll).
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a t t r i b u t e d to a strong binding of Mn(II) to DT.
When Mn(II) binds to a macro-
molecule, the r o t a t i o n a l c o r r e l a t i o n time increases to the slow tumbling domain (T c ~ 10-8 sec). Therefore, the z e r o - f i e l d s p l i t t i n g values and the random o r i e n t a t i o n a l d i s t r i b u i o n of the molecules cannot be averaged out (10~11).
With
a l l allowed t r a n s i t i o n s , only the I - 1 / 2 > - - I + i / 2 > f i n e structure component can be detected, thus r e s u l t i n g in the reduction of i n t e n s i t y .
I t is of i n t e r e s t that addition of Ca(II) at a r a t i o of 1:2:1 (Ca:DT:Mn) restores the i n t e n s i t y of the Mn(II) signal to 68% of that for Mn(II) alone, Fig. l c .
This is a clear evidence that Ca(II) competes e f f e c t i v e l y with Mn(II)
for the same binding s i t e ( s ) in DT molecule.
I t is well known that Ca(II) and
Mn(II) may bind at the same sites in macromolecules (12).
I t is probable that
DT is a Ca(II) bindinq protein. However, addition of Ca(II) at a r a t i o of 10:2:1 (Ca:DT:Mn) did not t o t a l l y restore the i n t e n s i t y of the Mn(II) signal (data not shown), suggesting that there are at least two d i f f e r e n t Mn(II) binding sites in DT; one can be replaced by Ca(II) and the other cannot.
Contributions of forbidden t r a n s i t i o n to the detected ESR signal are u s u a l l y small.
However, when z e r o - f i e l d s p l i t t i n g parameters are larger than the Zeeman
energy s p l i t t i n g values, for example, due to permanent d i s t o r t i o n s of the ligand f i e l d s~metry, forbidden t r a n s i t i o n s w i l l become s i g n i f i c a n t and overlap with the allowed t r a n s i t i o n s .
The i n t e n s i t y of forbidden t r a n s i t i o n s depends upon
D2/m2, where D is the z e r o - f i e l d s p l i t t i n g parameter and m is the electron donor frequency.
I t is therefore predictable that the forbidden t r a n s i t i o n s w i l l
become more v i s i b l e at lower microwave frequencies.
This in fact is the case.
Fiq. 2a is the ESR spectrum of 14n(II) in aqueous solution at 3.4 GHz. Inclusion of DT causes 56% reduction in the i n t e n s i t y , Fiq. 2b, which is smaller compared to 79% reduction seen at 9.5 GHz, Fib. l b , as predicted from the above d i s cussion. The r e s u l t s obtained at 3.4 GHz (S-band) are consistent with the notion that Mn(II) binds strongly to DT molecule.
I t is worth noting that the l i n e -
shapes of Mn(II) signal at 3.4 GHz are d i f f e r e n t from those at 9.5 GHz (Fig. i ) , p a r t i c u l a r l y at the lower f i e l d .
The unique lineshapes of the low frequency
993
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IOOG Effects of Diphtheria Toxin on the ESR spectra of Mn(ll) at 3.4 GHz in NaCl, pH 7.0. (a) 25 pM Mn(ll) alone and (b) 25 pM Mn(ll) plus 50 pM Diphtheria Toxin.
Mn(ll) signal
imPly that they may be very sensitive to r o t a t i o n a l d i f f u s i o n of
Mn(ll) ions in solution. When Mn(ll) binds strongly to macromolecules, i t s motion is in the slow tumbling regime, namely msmc ~ 1.
Under these conditions, the i n t e n s i t y of the
ESR spectrum of Mn(ll) should be only about 9/35 or 0.26 times of the i n t e n s i t y of Mn(ll) signal in the absence of macromolecules (13). was 0.24.
In this study the value
The 9/35 r a t i o is the r e l a t i v e i n t e n s i t y of the
I-1/2>--I+i/2>
fine
structure component, which is therefore the only t r a n s i t i o n contributing to the observed signal. > 1 still
At lower frequencies, for example, S-band, the condition ms~c
holds, but t r a n s i t i o n s other than
I-1/2>--I+i/2>
become noticeable,
resultinq in a stronger i n t e n s i t y than that at X-band. The results obtained usinq a multifrequency ESR approach reported in t h i s communication demonstrate that Mn(ll) binds strongly to DT and Ca(ll) competes e f f e c t i v e l y with Mn(ll) for such binding, suggesting that DT is a Ca(ll) binding protein. (7).
Calcium is known to promote the binding of DT to the sensitive cell
Calcium binding property may be an important event in the penetration of
Df into the plasma membrane of the sensitive mammalian c e l l , thus warranting further investiqation.
Acknowledgement This research was supported in part by NIH qrants RR01008 and GM35719.
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Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13.
Papperheimer, A. M., Jr. (1977) Ann. Rev. Biochem. 46, 69-84. Yamaizumi, M., Mekeda, E., Uchida, T., and Okada, Y. (1978) Cell 15, 245-250. Boquet, P., Silverman, M. S., PaD~enheimer, A. M., and Vernon, W. B. (1976) Proc. Natl. Acad. Sci. USA 73, 4449-4453. Boquet, P. (1979) Eur. J. Biochem. 100, 483-489. Donovan, J. J., Simon, M. I . , Draper, R. K., and Montal, M. (1981) Proc. Natl. Acad. Sci. USA 78, 172-176. Lai, C.-S., Kushnaryov, V., Panz, T. and Basosi, R. (1984) Arch. Biochem. Biophys. 234, 1-6. Sandvifl, K. and Olsnes, S. (1982) J. Biol. Chem. 257, 7495-7503. Lory, S., Carroll, S. F., and C o l l i e r , R. J. (1981) J. Biol. Chem. 255, 12015-12019. Kushnaryov, V. M., Sedmak, J. J., Bendler, J. W. and Grossberg, S. E. (1982) Infect. Immun. 36, 811-821. Basosi, R., Niccolai, N., Tiezzi, E., and Valensin, G. (1978) J. Am. Chem. Soc. I00, 8047-8050. 8urlamacchi, L., Martini, G., Ottaviani, M. F., and Romanelli,M. (1978) Adv. Mol. Relax, Interact. Proc. 12, 145-186. Cohn, M. and Reed, G. H. (1982) Ann. Rev. Biochem. 51, 365-394. Meirovitch, E., Brumerger, H., and Lis, H. (1978) Biophys. Chem. 8, 215-219.
995
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 997-995
September 30, 1986
EVIDENCE THAT DIVALENT CATIONS BIND TO DIPHTHERIA TOXIN: AN ESR APPROACH Riccardo Basosi+, Vladimir Kushnaryov, Tomasz Panz, and Ching-San Lai* National Biomedical ESR Center, Departments of Radiology and Microbiology Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Received July 28, 1986
Summary: We have used electron spin resonance (ESR) techniques to study the ~ o f divalent cations to diphtheria toxin (DT). Addition of DT to Mn(ll) solution at a stoichiometry of 2:1 (DT:Mn) induces a 79% loss in the i n t e n s i t y of the ESR spectrum of Mn(ll) suggesting a strong binding of Mn(ll) to DT. Inclusion of Ca(ll) at a r a t i o of 1:2:1 (Ca:DT:Mn) in the reaction mixture restores the i n t e n s i t y of the Mn(ll) signal to 64%. This indicates that Ca(ll) and Mn(ll) share same binding s i t e ( s ) in DT. The r e s u l t s presented in t h i s communication suggest that DT is a Ca(ll) binding protein. ® 1986AcademicP. . . . . Inc.
Diphtheria toxin (DT) is a protein with a molecular weight of 62,000 daltons and is extremely t o x i c to eucaryotic c e l l s (1,2). fraqments linked by d i s u l f i d e bridges.
The protein contains A and B
Fraqment B is responsible for the
binding of DT to the plasma membrane of sensitive c e l l s and fragment A is involved in i n a c t i v a t i o n of protein synthesis by ADP-ribosylation of elongation factor 2 of the protein synthesis machinery (1).
Boquet et al. proposed that DT
forms a transmembrane channel in the plasma membrane by which fragment A is transported across the membrane and appears in the cytoplasmic side of the plasma membrane (3,4).
Recent biophysical studies showed that DT forms an
ion-conducting channel in the membrane (5).
Using electron spin resonance (ESR)
spin label methods, we demonstrated previously that DT induces the leakage of acidic phospholipid membranes, which is in agreement with the concept of DT-induced channel formation (6). ~On leave from the Department of Chemistry, University of Siena, Pian dei Mantellini 44-83100, Siena, I t a l y *To whom correspondence should be addressed..
991
0006-291 X/86 $1.50 Copyright © 1986 by Academic Press, lnc. All rights of reproduction in any jorm reserved.
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The f u n c t i o n a l l y d i s t i n c t
ligand-binding s i t e s in DT molecule, namely, the
NAD s i t e and the P s i t e have been described; the NAD s i t e catalyzes the i n t r a c e l l u l a r ADP-ribosylation reaction and the P s i t e a f f e c t s DT binding to the c e l l (7).
Calcium ions have been shown to enhance the binding of DT to the c e l l
(8).
In t h i s study, by using electron spin resonance (ESR) spectroscopy we demonstrate f o r the f i r s t
time t h a t DT has Mn(II) binding s i t e ( s ) at which the binding of
Mn(II) could be displaced by addition of calcium, suggesting that DT i s a calcium binding p r o t e i n . Materials and Methods DT was obtained from Connauqht Labs, Toronto, Canada and f u r t h e r p u r i f i e d as described p r e v i o u s l y (9). P u r i f i e d DT showed a s i n g l e band of molecular weight 62,000 on 7% SDS-polyacrylamide gel e l e c t r o p h o r e s i s . The protein was dialyzed against 0.15 M NaCl, pH 7.0 p r i o r to use. ESR Measurements The ESR spectra were operating at 9.5 GHz and The f i e l d modulation and The f i e l d sweep was 1000 carried out at 22°C.
obtained with a Varian Century l i n e spectrometer, with a Varian E-9 spectrometer, oDerating at 3.4 GHz. amplitude modulation were 100 KHz and 5 G, r e s p e c t i v e l y . G and the time constant i sec. The measurements were
Results and Discussion Fig. la shows a t y p i c a l ESR spectrum of Mn(ll) in aqueous s o l u t i o n . Addition of DT to Mn(ll) s o l u t i o n at a r a t i o of 2:1 (DT to M n ( l l ) ) induces a 79% loss of the i n t e n s i t y of the Mn(ll) spectrum, Fig. l b .
The i n t e n s i t y loss may be
b
I,
q
IOOG
Effects of Diphtheria Toxin on the ESR spectra of Mn(ll) at 9.5 GHz in 0.15 M NaCI, pH 7.0. (a) 25 ~M Mn(ll) alone. (b) 25 ~M Mn(ll) plus 50 ~M Diphtheria Toxin. (c) same as in (b) except in the presence of 25 ~M Ca(ll). Note the reappearance of Mn signal due to the addition of Ca(ll).
992
VoI, 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a t t r i b u t e d to a strong binding of Mn(II) to DT.
When Mn(II) binds to a macro-
molecule, the r o t a t i o n a l c o r r e l a t i o n time increases to the slow tumbling domain (T c ~ 10-8 sec). Therefore, the z e r o - f i e l d s p l i t t i n g values and the random o r i e n t a t i o n a l d i s t r i b u i o n of the molecules cannot be averaged out (10~11).
With
a l l allowed t r a n s i t i o n s , only the I - 1 / 2 > - - I + i / 2 > f i n e structure component can be detected, thus r e s u l t i n g in the reduction of i n t e n s i t y .
I t is of i n t e r e s t that addition of Ca(II) at a r a t i o of 1:2:1 (Ca:DT:Mn) restores the i n t e n s i t y of the Mn(II) signal to 68% of that for Mn(II) alone, Fig. l c .
This is a clear evidence that Ca(II) competes e f f e c t i v e l y with Mn(II)
for the same binding s i t e ( s ) in DT molecule.
I t is well known that Ca(II) and
Mn(II) may bind at the same sites in macromolecules (12).
I t is probable that
DT is a Ca(II) bindinq protein. However, addition of Ca(II) at a r a t i o of 10:2:1 (Ca:DT:Mn) did not t o t a l l y restore the i n t e n s i t y of the Mn(II) signal (data not shown), suggesting that there are at least two d i f f e r e n t Mn(II) binding sites in DT; one can be replaced by Ca(II) and the other cannot.
Contributions of forbidden t r a n s i t i o n to the detected ESR signal are u s u a l l y small.
However, when z e r o - f i e l d s p l i t t i n g parameters are larger than the Zeeman
energy s p l i t t i n g values, for example, due to permanent d i s t o r t i o n s of the ligand f i e l d s~metry, forbidden t r a n s i t i o n s w i l l become s i g n i f i c a n t and overlap with the allowed t r a n s i t i o n s .
The i n t e n s i t y of forbidden t r a n s i t i o n s depends upon
D2/m2, where D is the z e r o - f i e l d s p l i t t i n g parameter and m is the electron donor frequency.
I t is therefore predictable that the forbidden t r a n s i t i o n s w i l l
become more v i s i b l e at lower microwave frequencies.
This in fact is the case.
Fiq. 2a is the ESR spectrum of 14n(II) in aqueous solution at 3.4 GHz. Inclusion of DT causes 56% reduction in the i n t e n s i t y , Fiq. 2b, which is smaller compared to 79% reduction seen at 9.5 GHz, Fib. l b , as predicted from the above d i s cussion. The r e s u l t s obtained at 3.4 GHz (S-band) are consistent with the notion that Mn(II) binds strongly to DT molecule.
I t is worth noting that the l i n e -
shapes of Mn(II) signal at 3.4 GHz are d i f f e r e n t from those at 9.5 GHz (Fig. i ) , p a r t i c u l a r l y at the lower f i e l d .
The unique lineshapes of the low frequency
993
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IOOG Effects of Diphtheria Toxin on the ESR spectra of Mn(ll) at 3.4 GHz in NaCl, pH 7.0. (a) 25 pM Mn(ll) alone and (b) 25 pM Mn(ll) plus 50 pM Diphtheria Toxin.
Mn(ll) signal
imPly that they may be very sensitive to r o t a t i o n a l d i f f u s i o n of
Mn(ll) ions in solution. When Mn(ll) binds strongly to macromolecules, i t s motion is in the slow tumbling regime, namely msmc ~ 1.
Under these conditions, the i n t e n s i t y of the
ESR spectrum of Mn(ll) should be only about 9/35 or 0.26 times of the i n t e n s i t y of Mn(ll) signal in the absence of macromolecules (13). was 0.24.
In this study the value
The 9/35 r a t i o is the r e l a t i v e i n t e n s i t y of the
I-1/2>--I+i/2>
fine
structure component, which is therefore the only t r a n s i t i o n contributing to the observed signal. > 1 still
At lower frequencies, for example, S-band, the condition ms~c
holds, but t r a n s i t i o n s other than
I-1/2>--I+i/2>
become noticeable,
resultinq in a stronger i n t e n s i t y than that at X-band. The results obtained usinq a multifrequency ESR approach reported in t h i s communication demonstrate that Mn(ll) binds strongly to DT and Ca(ll) competes e f f e c t i v e l y with Mn(ll) for such binding, suggesting that DT is a Ca(ll) binding protein. (7).
Calcium is known to promote the binding of DT to the sensitive cell
Calcium binding property may be an important event in the penetration of
Df into the plasma membrane of the sensitive mammalian c e l l , thus warranting further investiqation.
Acknowledgement This research was supported in part by NIH qrants RR01008 and GM35719.
994
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13.
Papperheimer, A. M., Jr. (1977) Ann. Rev. Biochem. 46, 69-84. Yamaizumi, M., Mekeda, E., Uchida, T., and Okada, Y. (1978) Cell 15, 245-250. Boquet, P., Silverman, M. S., PaD~enheimer, A. M., and Vernon, W. B. (1976) Proc. Natl. Acad. Sci. USA 73, 4449-4453. Boquet, P. (1979) Eur. J. Biochem. 100, 483-489. Donovan, J. J., Simon, M. I . , Draper, R. K., and Montal, M. (1981) Proc. Natl. Acad. Sci. USA 78, 172-176. Lai, C.-S., Kushnaryov, V., Panz, T. and Basosi, R. (1984) Arch. Biochem. Biophys. 234, 1-6. Sandvifl, K. and Olsnes, S. (1982) J. Biol. Chem. 257, 7495-7503. Lory, S., Carroll, S. F., and C o l l i e r , R. J. (1981) J. Biol. Chem. 255, 12015-12019. Kushnaryov, V. M., Sedmak, J. J., Bendler, J. W. and Grossberg, S. E. (1982) Infect. Immun. 36, 811-821. Basosi, R., Niccolai, N., Tiezzi, E., and Valensin, G. (1978) J. Am. Chem. Soc. I00, 8047-8050. 8urlamacchi, L., Martini, G., Ottaviani, M. F., and Romanelli,M. (1978) Adv. Mol. Relax, Interact. Proc. 12, 145-186. Cohn, M. and Reed, G. H. (1982) Ann. Rev. Biochem. 51, 365-394. Meirovitch, E., Brumerger, H., and Lis, H. (1978) Biophys. Chem. 8, 215-219.
995