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Structure-activity relationships of the B fragment of diphtheria toxin: The lipid-binding domains
Tozicon, Vo1 .20, No .l, pp .243-246, 1982 Printed in Great Britain.
0041-0101/82/010243-04$02 .00/0 Pergamon Press Ltd .
STRUCTURE-ACTIVITY RELATIONSHIPS OF THE B FRAGMENT OF DIPHTHERIA TOXIN : THE LIPID-BINDING DOMAINS P . Falmagne l , C . Capiau l , J . Zane~1 , G . Kayser 2 and J .-M . Ruysschaert 1Laboratoire de Chimie Biologique, Université de l'État à Mons, B-7000 Mons, Belgium 2Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, B-1050 Bruxelles, Belgium
ABSTRACT Two different lipid-associating domains have previously been identified in diphtheria toxin fragment B : one of the surface type in the N-terminus of B and one of the transverse type in its middle region . We have now determined about 85$ of the primary structure of fragment B and show, here, that the middle part of fragment B contains a highly hydrophobic region of 72 amino acid residues (polarity index : 29$) which includes the transverse lipidassociating domain . That this domain is actually involved in a process of membrane penetration is suggested by lipid bilayer conductance measurements of the CNBr peptides of fragment B and trypsin treatment of fragment Bmultilamellar liposome complexes . KEYWORDS Diphtheria toxin ; fragment B structure and function ; protein sequencing ; protein-lipid interactions . INTRODUCTION Fragment B (40 700 daltons) of diphtheria toxin (62 000 daltons) is responsible for the receptor-mediated internalization of the toxin enzymically active fragment A (21 150 daltons) into the cytoplasm of sensitive eukaryotie cells where A inhibits the protein synthesis by ADP-ribosylation of elongation factor 2 (for rev ., see Pappenheimer, 1977) . Fragment B has been shown to contain at least two functionally different regions . One, hydrophobic, is located in the 23 000 daltons N-terminal moiety and would participate to the interaction of B with the phospholipid bilayer of the cell membrane (Boquet and co-workers, 1976) ; in the toxin molecule, this hydrophobic region is not accessible at neutral pH but becomes exposed at pH below 5 .0 (Sandvig and Olsnes, 1981) . The other region, hydrophilic, is located in the 17 000 daltons C-terminal part and belongs to the cell-receptors recognition site (Lory and Collier, 1980 ; Lory and co-workers, 1980 ; Proia and co-workers, 1980 ; Uchida and co-workers, 1972) . Fragment B not only interacts with eukaryotic cell membranes, but also with synthetic model membranes (Boquet, 1979) . To investigate the relationships between the structure and the lipid-binding functions of fragment B, we are determining its primary structure . By automated Edman degradation (Brauer and co-workers, 1975 ; Hunkapiller and Hood, 1978) of peptides produced by chemical and enzymatic cleavage procedures, we have presently aligned about 320 amino acid residues on a total of approximately 380 (Fig . 1) . 243
244
p, FAT .xer_xu
Btal.
39 S V G S S L K C I S L D W D V I R D K T K T K I E S L K E Ii G P I K N(K G N T CB 4 77 Q)M S E S P N K T V S E E K A K Q Y L E E F T Q T A L E S P E I E N I K A V( . CB 2 161 . . . . .)A V A A L S I L P G I G S V M G I A D G A V H H N T E E I V A Q S I CB 5 200 A L S S L M V A Q A I P L V G E L V D I G F A A Y N F V E S I I N L F Q V V H 239 N N Y N(S P A Y)R T V D N L H D G Y A V S W N T V E D S I I R T G F Q G E S G CB 1 281 H D I K I T A E N T P L P I A G V L L P T I P G K L D V N K(K I . .)R W A V N 321 V A Q V I D S E T A D N L E K P T A(A L . .)M R C R A I D G D V T F C R P K S 360 P V Y V G S N(N H A V P H A . .)R S S S E K I H S N E I S S N S I G V L G Y Q CB 3 K T V D H T K V N S K L K L F F E T K S
Fig . 1 . Partial amino acid sequence of diphtheria toxin fragment B . One letter amino acid abbreviations used are : A, alanine ; C, cysteine ; D, aspartic acid ; E, glutamio acid ; F, phenylalanine ; G, glycine ; H, histidine ; I, isoleucine ; K, lysine ; L, leucine ; M, methionine ; N, asparagine ; P, proline ; Q, glutamine ; R, arginine ; S, serine ; T, threonine ; V, valine ; W, tryptophane ; Y, tyrosine . Numbering of the amino acid residues is based on the molecular weights and the amino acid compositions of the cyanogen bromide peptides (CB1-5) .
Diphtheria Toxin Lipid-Binding Domains
245
By comparing this covalent structure and its predicted secondary structure with the primary and secondary structures of proteins known to interact with phospholipids (Sogrest and Jackson, 1977), we were able to identify two different lipid-binding domains in fragment B, located in its 23 000 daltons N-terminal moiety (Falmagne and co-workers, 1980 ; Lambotte and co-workers 1980) . Both have predicted amphiphilic helical structures but widely different polarity indexes . The first one (residues 1-77, N-terminal region of B ; includes CNBr peptide CB4 and the N-terminal amino acid sequence of CNBr peptide CB2) is highly hydrophilic and designed to interact with the surface of lipid bilayers, like the surface lipid-associating domains of apolipoprotein AI . In contrast, the second one (residues 168-199, middle region of B ; Nterminal amino acid sequence of CNBr peptide CB1) is highly hydrophobic and built to span a lipid bilayer, like the transverse lipid-associating domains of intrinsic membrane proteins . Study of the interaction of fragment B and its CNBr peptides with synthetic model membranes has provided experimental support to the above structurefunction relationships . INTERACTION WITH SYNTHETIC MODEL MEMBRANES Fragment B and four of its CNBr peptides (CB4, 2,5 and 1, Fig .1) form stable complexes with multilamellar vesicles of L-a-dimyristoylphosphatidyl choline (DMPC) . One mg of fragment B incubated with 10 mg of DMPC at 37°C in 0 .1 M citric acid-0 .2 M phosphate buffer pH 7 .0 incorporated 0 .026 mg per mg of DMPC . CB3, the C-terminal CNBr peptide of fragment B, does not interact with the DMPC liposomes . CB1, which contains the predicted transverse lipid-binding domain in its Nterminal region, is the sole CNBr peptide of fragment B to modify the conductance of a planar bilayer of glycerol monooleate formed in 0 .1 M NH4HC0 3 pH 7 .8 containing 0 .15 M NaCl (16-fold increase in conductance ; Kayser and coworkers, 1981) ; the strong change in permeability supposes the penetration of CB1 into the planar membrane, which induces a destabilization of the lipid bilayer . Fragment B showed only a slight increase in conductance (3-folà) while no effect was observed with the whole diphtheria toxin molecule . Therefore, at pH 7 .8, the region of CB1 responsible for its penetration appears to be poorly accessible in fragment B and not at all in diphtheria toxin ; this region should be the predicted transverse lipid-associating domain since no other regions of CB1 contain structures susceptible to penetrate into a lipid bilayer . That the middle region of fragment B is actually inserted into the lipid bilayer was further shown by enzymatic cleavage of complexes of fragment B with multilamellar DMPC vesicles . These complexes, formed in 0 .1 M NH 4HC0 3 pH 7 .8 containing 0 .2 M NaCl, were digested with TPCK-treated trypsin (fragment B : trypsin 20 :1, w/w) in 0 .1 M NH 4 HC0 3 pH 7 .8 during 4 hrs at 37°C . Automated Edman degradation of the carefully washed liposome-peptide complexes gave a unique amino acid sequence starting at residue 128 in Fig . 1 . The size of the segment inserted into the liposomes is about 100 residues ; its amino acid composition has a low polarity index (40$) and correlates well with the known sequence of the middle region of fragment B . This segment contains the hydrophobic predicted transverse lipid-associating domain (residues 168-199 ; polarity index : 25$) which is preceded by another highly hydrophobic amino acid sequence (residues 128-167 ; polarity index : 32 .5$) but devoid of amphiphilic helical structures . The N- and C-terminal regions of fragment B are removed from the liposomes by the trypsin treatment and should therefore be outside the DMPC vesicles .
246
P . FAIMAGNE et al.
CONCLUSIONS The middle part of diphtheria toxin fragment B contains a highly hydrophobic region of 72 amino acid residues (128-199) with a very low polarity index equal to 29$ . Part of this hydrophobic region (residues 168-199), which covers the N-terminal sequence of CNBr peptide CB1, has the characteristic structural properties of intramembranous segments of intrinsic membrane proteins like glycophorin and probably the same function since only CB1 is able to strongly destabilize a lipid membrane as the expected consequence of its penetration into the bilayer . At pH 7 .8, this hydrophobic region is not exposed in diphtheria toxin and only partly accessible in fragment B . It may become exposed at lower pH (below 5 .0) and, therefore, could be responsible for the reported properties of diphtheria toxin at low pH, such as that found within lysosomes and which would be needed for the penetration of diphtheria toxin into the cytoplasm (Donovan and co-workers, 1981 ; Kagan and co-workers 1981 ; Sandvig and Olsnes, 1981) . On the other hand, the N-terminal region of fragment B, in which a surface lipid-binding domain has been identified, is apparently able to associate with multilamellar liposomes but not to penetrate into the lipid bilayer . This region may be responsible for preliminary interactions of fragment B with the cytoplasmic membrane during the intoxication process, before its anchoring in the membrane through its middle hydrophobic region . REFERENCES Boquet, P . (1979) . Eur . J . Biochem . 100, 483-489 . Boquet, P ., M .S . Si verman, . Pappenheimer, Jr ., and W .B . Vernon (1976) . Proc . Natl . Acad . Sci . USA 73, 4449-4453 . Brauer, A . ., . . argolies, an E . Haber (1975) . Biochemistry, 14 , 30293035 . Donovan, J .J ., M .I . Simon, R .K . Draper, and M . Montal (1981) . Proc . Natl . Acad . Sci . US A 78, 172-176 . Falmagne, ., . Lambotte, C . Capiau, J .-M. Ruysschaert, and J . Dirkx (1980) . In D . Eaker and T . Wadström (Eds .), Natural Toxins , Pergamon Press, Oxford and New York, pp . 433-437 . Hunkapiller, M .W ., and L .E . Hood (1978) . Biochemistr 17, 2124-2133 . Kagan, B .L ., A . Finkelstein, and M. Colom ini roc . Natl . Acad . Sci . USA, in press . Kayser,G ., P . Lambotte, P . Falmagne, C . Capiau, J . Zanen, and J .-M . Ruysschaert (1981) . Biochem. Bio h s . Res . Commun . 99, 358-363 . Lambotte, P ., P . Falmagne, C . apiau, J . anen, J .-M . Ruysschaert, and J . Dirkx (1980) . J . Cell . Biol . 87, 837-840 . Lory, S ., and R .J . Co lier 1 roc . Natl . Acad . Sci . USA 77, 267-271 . Lory, S ., S .F . Carroll, and R .J . Col ier 1 . Biol . hem . , 255 , 12016-12019 . Pappenheimer, A .M .,Jr . (1977) . Annu . Rev . Biochem . 46, 69-94 . Proia, R .L ., S .K . Wray, D .A . Hart, an . Ei e s ) . J . Biol . Chem ., 255 12025-12033 . Sandvig, K., and S . Olsnes (1981) . J . Biol . Chem ., in press . Segrest, J .P ., and R .L . Jackson (197 n R . . Capaldi (Ed .), Membrane Proteins and Their Interactions with " Li ids, M. Dekker, Inc ., ew orc and üase , pp . Uchida, T ., A.M . Pappenheimer, Jr ., and A .A . Harper (1972) . Science (Gush . D . C .) 175, 901-903 .
0041-0101/82/010243-04$02 .00/0 Pergamon Press Ltd .
STRUCTURE-ACTIVITY RELATIONSHIPS OF THE B FRAGMENT OF DIPHTHERIA TOXIN : THE LIPID-BINDING DOMAINS P . Falmagne l , C . Capiau l , J . Zane~1 , G . Kayser 2 and J .-M . Ruysschaert 1Laboratoire de Chimie Biologique, Université de l'État à Mons, B-7000 Mons, Belgium 2Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, B-1050 Bruxelles, Belgium
ABSTRACT Two different lipid-associating domains have previously been identified in diphtheria toxin fragment B : one of the surface type in the N-terminus of B and one of the transverse type in its middle region . We have now determined about 85$ of the primary structure of fragment B and show, here, that the middle part of fragment B contains a highly hydrophobic region of 72 amino acid residues (polarity index : 29$) which includes the transverse lipidassociating domain . That this domain is actually involved in a process of membrane penetration is suggested by lipid bilayer conductance measurements of the CNBr peptides of fragment B and trypsin treatment of fragment Bmultilamellar liposome complexes . KEYWORDS Diphtheria toxin ; fragment B structure and function ; protein sequencing ; protein-lipid interactions . INTRODUCTION Fragment B (40 700 daltons) of diphtheria toxin (62 000 daltons) is responsible for the receptor-mediated internalization of the toxin enzymically active fragment A (21 150 daltons) into the cytoplasm of sensitive eukaryotie cells where A inhibits the protein synthesis by ADP-ribosylation of elongation factor 2 (for rev ., see Pappenheimer, 1977) . Fragment B has been shown to contain at least two functionally different regions . One, hydrophobic, is located in the 23 000 daltons N-terminal moiety and would participate to the interaction of B with the phospholipid bilayer of the cell membrane (Boquet and co-workers, 1976) ; in the toxin molecule, this hydrophobic region is not accessible at neutral pH but becomes exposed at pH below 5 .0 (Sandvig and Olsnes, 1981) . The other region, hydrophilic, is located in the 17 000 daltons C-terminal part and belongs to the cell-receptors recognition site (Lory and Collier, 1980 ; Lory and co-workers, 1980 ; Proia and co-workers, 1980 ; Uchida and co-workers, 1972) . Fragment B not only interacts with eukaryotic cell membranes, but also with synthetic model membranes (Boquet, 1979) . To investigate the relationships between the structure and the lipid-binding functions of fragment B, we are determining its primary structure . By automated Edman degradation (Brauer and co-workers, 1975 ; Hunkapiller and Hood, 1978) of peptides produced by chemical and enzymatic cleavage procedures, we have presently aligned about 320 amino acid residues on a total of approximately 380 (Fig . 1) . 243
244
p, FAT .xer_xu
Btal.
39 S V G S S L K C I S L D W D V I R D K T K T K I E S L K E Ii G P I K N(K G N T CB 4 77 Q)M S E S P N K T V S E E K A K Q Y L E E F T Q T A L E S P E I E N I K A V( . CB 2 161 . . . . .)A V A A L S I L P G I G S V M G I A D G A V H H N T E E I V A Q S I CB 5 200 A L S S L M V A Q A I P L V G E L V D I G F A A Y N F V E S I I N L F Q V V H 239 N N Y N(S P A Y)R T V D N L H D G Y A V S W N T V E D S I I R T G F Q G E S G CB 1 281 H D I K I T A E N T P L P I A G V L L P T I P G K L D V N K(K I . .)R W A V N 321 V A Q V I D S E T A D N L E K P T A(A L . .)M R C R A I D G D V T F C R P K S 360 P V Y V G S N(N H A V P H A . .)R S S S E K I H S N E I S S N S I G V L G Y Q CB 3 K T V D H T K V N S K L K L F F E T K S
Fig . 1 . Partial amino acid sequence of diphtheria toxin fragment B . One letter amino acid abbreviations used are : A, alanine ; C, cysteine ; D, aspartic acid ; E, glutamio acid ; F, phenylalanine ; G, glycine ; H, histidine ; I, isoleucine ; K, lysine ; L, leucine ; M, methionine ; N, asparagine ; P, proline ; Q, glutamine ; R, arginine ; S, serine ; T, threonine ; V, valine ; W, tryptophane ; Y, tyrosine . Numbering of the amino acid residues is based on the molecular weights and the amino acid compositions of the cyanogen bromide peptides (CB1-5) .
Diphtheria Toxin Lipid-Binding Domains
245
By comparing this covalent structure and its predicted secondary structure with the primary and secondary structures of proteins known to interact with phospholipids (Sogrest and Jackson, 1977), we were able to identify two different lipid-binding domains in fragment B, located in its 23 000 daltons N-terminal moiety (Falmagne and co-workers, 1980 ; Lambotte and co-workers 1980) . Both have predicted amphiphilic helical structures but widely different polarity indexes . The first one (residues 1-77, N-terminal region of B ; includes CNBr peptide CB4 and the N-terminal amino acid sequence of CNBr peptide CB2) is highly hydrophilic and designed to interact with the surface of lipid bilayers, like the surface lipid-associating domains of apolipoprotein AI . In contrast, the second one (residues 168-199, middle region of B ; Nterminal amino acid sequence of CNBr peptide CB1) is highly hydrophobic and built to span a lipid bilayer, like the transverse lipid-associating domains of intrinsic membrane proteins . Study of the interaction of fragment B and its CNBr peptides with synthetic model membranes has provided experimental support to the above structurefunction relationships . INTERACTION WITH SYNTHETIC MODEL MEMBRANES Fragment B and four of its CNBr peptides (CB4, 2,5 and 1, Fig .1) form stable complexes with multilamellar vesicles of L-a-dimyristoylphosphatidyl choline (DMPC) . One mg of fragment B incubated with 10 mg of DMPC at 37°C in 0 .1 M citric acid-0 .2 M phosphate buffer pH 7 .0 incorporated 0 .026 mg per mg of DMPC . CB3, the C-terminal CNBr peptide of fragment B, does not interact with the DMPC liposomes . CB1, which contains the predicted transverse lipid-binding domain in its Nterminal region, is the sole CNBr peptide of fragment B to modify the conductance of a planar bilayer of glycerol monooleate formed in 0 .1 M NH4HC0 3 pH 7 .8 containing 0 .15 M NaCl (16-fold increase in conductance ; Kayser and coworkers, 1981) ; the strong change in permeability supposes the penetration of CB1 into the planar membrane, which induces a destabilization of the lipid bilayer . Fragment B showed only a slight increase in conductance (3-folà) while no effect was observed with the whole diphtheria toxin molecule . Therefore, at pH 7 .8, the region of CB1 responsible for its penetration appears to be poorly accessible in fragment B and not at all in diphtheria toxin ; this region should be the predicted transverse lipid-associating domain since no other regions of CB1 contain structures susceptible to penetrate into a lipid bilayer . That the middle region of fragment B is actually inserted into the lipid bilayer was further shown by enzymatic cleavage of complexes of fragment B with multilamellar DMPC vesicles . These complexes, formed in 0 .1 M NH 4HC0 3 pH 7 .8 containing 0 .2 M NaCl, were digested with TPCK-treated trypsin (fragment B : trypsin 20 :1, w/w) in 0 .1 M NH 4 HC0 3 pH 7 .8 during 4 hrs at 37°C . Automated Edman degradation of the carefully washed liposome-peptide complexes gave a unique amino acid sequence starting at residue 128 in Fig . 1 . The size of the segment inserted into the liposomes is about 100 residues ; its amino acid composition has a low polarity index (40$) and correlates well with the known sequence of the middle region of fragment B . This segment contains the hydrophobic predicted transverse lipid-associating domain (residues 168-199 ; polarity index : 25$) which is preceded by another highly hydrophobic amino acid sequence (residues 128-167 ; polarity index : 32 .5$) but devoid of amphiphilic helical structures . The N- and C-terminal regions of fragment B are removed from the liposomes by the trypsin treatment and should therefore be outside the DMPC vesicles .
246
P . FAIMAGNE et al.
CONCLUSIONS The middle part of diphtheria toxin fragment B contains a highly hydrophobic region of 72 amino acid residues (128-199) with a very low polarity index equal to 29$ . Part of this hydrophobic region (residues 168-199), which covers the N-terminal sequence of CNBr peptide CB1, has the characteristic structural properties of intramembranous segments of intrinsic membrane proteins like glycophorin and probably the same function since only CB1 is able to strongly destabilize a lipid membrane as the expected consequence of its penetration into the bilayer . At pH 7 .8, this hydrophobic region is not exposed in diphtheria toxin and only partly accessible in fragment B . It may become exposed at lower pH (below 5 .0) and, therefore, could be responsible for the reported properties of diphtheria toxin at low pH, such as that found within lysosomes and which would be needed for the penetration of diphtheria toxin into the cytoplasm (Donovan and co-workers, 1981 ; Kagan and co-workers 1981 ; Sandvig and Olsnes, 1981) . On the other hand, the N-terminal region of fragment B, in which a surface lipid-binding domain has been identified, is apparently able to associate with multilamellar liposomes but not to penetrate into the lipid bilayer . This region may be responsible for preliminary interactions of fragment B with the cytoplasmic membrane during the intoxication process, before its anchoring in the membrane through its middle hydrophobic region . REFERENCES Boquet, P . (1979) . Eur . J . Biochem . 100, 483-489 . Boquet, P ., M .S . Si verman, . Pappenheimer, Jr ., and W .B . Vernon (1976) . Proc . Natl . Acad . Sci . USA 73, 4449-4453 . Brauer, A . ., . . argolies, an E . Haber (1975) . Biochemistry, 14 , 30293035 . Donovan, J .J ., M .I . Simon, R .K . Draper, and M . Montal (1981) . Proc . Natl . Acad . Sci . US A 78, 172-176 . Falmagne, ., . Lambotte, C . Capiau, J .-M. Ruysschaert, and J . Dirkx (1980) . In D . Eaker and T . Wadström (Eds .), Natural Toxins , Pergamon Press, Oxford and New York, pp . 433-437 . Hunkapiller, M .W ., and L .E . Hood (1978) . Biochemistr 17, 2124-2133 . Kagan, B .L ., A . Finkelstein, and M. Colom ini roc . Natl . Acad . Sci . USA, in press . Kayser,G ., P . Lambotte, P . Falmagne, C . Capiau, J . Zanen, and J .-M . Ruysschaert (1981) . Biochem. Bio h s . Res . Commun . 99, 358-363 . Lambotte, P ., P . Falmagne, C . apiau, J . anen, J .-M . Ruysschaert, and J . Dirkx (1980) . J . Cell . Biol . 87, 837-840 . Lory, S ., and R .J . Co lier 1 roc . Natl . Acad . Sci . USA 77, 267-271 . Lory, S ., S .F . Carroll, and R .J . Col ier 1 . Biol . hem . , 255 , 12016-12019 . Pappenheimer, A .M .,Jr . (1977) . Annu . Rev . Biochem . 46, 69-94 . Proia, R .L ., S .K . Wray, D .A . Hart, an . Ei e s ) . J . Biol . Chem ., 255 12025-12033 . Sandvig, K., and S . Olsnes (1981) . J . Biol . Chem ., in press . Segrest, J .P ., and R .L . Jackson (197 n R . . Capaldi (Ed .), Membrane Proteins and Their Interactions with " Li ids, M. Dekker, Inc ., ew orc and üase , pp . Uchida, T ., A.M . Pappenheimer, Jr ., and A .A . Harper (1972) . Science (Gush . D . C .) 175, 901-903 .