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Investigations into the relationships between structure and function of diphtheria toxin fragment b
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INVE :iTIGATIONS INTO TII's RELATIGATSHIfS BETI~'EEN STRUCTURE AND FUNCTION OF DIPHTFIERIA TOIiIN FRAGbIEATT B P . Falmagne, P, Lambotte, C, Capiau, J .-M, Ruyaschaert (I) and J . Dirkx Laboratoire de Chimie biologique et de Biophysique, Université de l'État à Mons, bions and (I) Laboratoire de Chimie physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Bruxelles, Belgium
Fragment B (40 OGO daltons) of diphtheria toxin (62 000 daltons) is responsible for the binding of the whole molecule to the membranes of sensitive eukaryotic cells and for the penetration of the enzymically active fragment A (22 000 daltons) into the cytoaol where it inhibits protein synthesis . Liko membrane proteins, fragment H possesses a hydrophobic domain, located in its 23 000 daltons Iv-terminal region (BOQUET et al,, I976) . The Cterminal part (I7 000 daltons) is involved in the recognition of the cell receptors . In a previous work we isolated and characterized the CNßr peptides of fragment B (FALI"'.AGNE et el ., I97?) . In this report we present the complete amino acid sequences of CB4a, CB4b and C1:$, and the N-terminal sequences of CB2, CBI and CB3, obtained by automated Edman and manual enzymic degradati~ns . CD4a : SVGSSLKCISLDS1DVhIDhM ; Cß4b : F(TKIESLKEI1GPIhNDiGKT:~,A1 ; Cß2 : SESPhî~TV SEEKAKQYhE1;FT:?TALESPEIENIKAV ; CB5 s GIADGAVIiF-IN'fE~IV .1r).SIALáSLM ; CBI : YA!? :1IPL VGELVDIGFAAYtTFVßSIIhZFQVYIINNYI~; ; CB3 : RCitAIDG~1VTi'CRPKSPVYVCL Sequences of CB4a, CB4b, CB2 on ono hand, and CB5 and CDI on the other hand provide respectively the N- and C-terminal covalent structures of tl~e re ;;ion of fragment H containing the hydrophobic domain . Structural analyses, circular dichroism measurements and liposomes studies reveal that both structures are amphipatic and exhibit the properties of the lipid-associating domains of lipoproteins, as defined by SEGREST and JACKSON (I977) ; however, the hydrophilic hT-terminal aide would belong to the surface type dr~main specific to the plasma lipoproteins while the hydrophobic C-terminal aide would be of the transverse type domain found in the I~iN-glycoprotein . Therefore, diphtheria toxin might first interact with the phoapholipid bilayer by reversible surface associations and, then, anchor itself by more stable transverse associations . 7f, as postulated by BOQUET et al . (I976), the insertion of fragment B into the cell membrane requires the preliminary release of a segment at its COOH-terminus, the clustering of basic residues in the vicinity of the two cysteines of CB3 (positions 2 and I2), i .e ., at about 8 000 daltons from the C-terminal end of fragment B, could find a physiological significance in providing an increased sensitivity to cellular proteases to that region of fragment B . BOQUET, P,, SILVERIIAN, bI .S,, PAPPENHEIMER, A,1~f ., Jr . and VERNON, W .B . (I976) Proc . Natl . Aca . Sci . US A ~, 4449-4453 . FALt"IAGNE, P,, L14~SBOTTE, P, and DIRKX, J, (I97ß) Biochim. Biophys . Acts 54-65 . SEGREST, J .P . and JACKSON, R .L . (I977) in "Membrane Proteine and their Inter actions with Lipids ", ed . by R,A . CAPALDI, hTew-York and Basel, b1 . DEKKER, Inc ., pp . 2I-45 .
INVE :iTIGATIONS INTO TII's RELATIGATSHIfS BETI~'EEN STRUCTURE AND FUNCTION OF DIPHTFIERIA TOIiIN FRAGbIEATT B P . Falmagne, P, Lambotte, C, Capiau, J .-M, Ruyaschaert (I) and J . Dirkx Laboratoire de Chimie biologique et de Biophysique, Université de l'État à Mons, bions and (I) Laboratoire de Chimie physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Bruxelles, Belgium
Fragment B (40 OGO daltons) of diphtheria toxin (62 000 daltons) is responsible for the binding of the whole molecule to the membranes of sensitive eukaryotic cells and for the penetration of the enzymically active fragment A (22 000 daltons) into the cytoaol where it inhibits protein synthesis . Liko membrane proteins, fragment H possesses a hydrophobic domain, located in its 23 000 daltons Iv-terminal region (BOQUET et al,, I976) . The Cterminal part (I7 000 daltons) is involved in the recognition of the cell receptors . In a previous work we isolated and characterized the CNßr peptides of fragment B (FALI"'.AGNE et el ., I97?) . In this report we present the complete amino acid sequences of CB4a, CB4b and C1:$, and the N-terminal sequences of CB2, CBI and CB3, obtained by automated Edman and manual enzymic degradati~ns . CD4a : SVGSSLKCISLDS1DVhIDhM ; Cß4b : F(TKIESLKEI1GPIhNDiGKT:~,A1 ; Cß2 : SESPhî~TV SEEKAKQYhE1;FT:?TALESPEIENIKAV ; CB5 s GIADGAVIiF-IN'fE~IV .1r).SIALáSLM ; CBI : YA!? :1IPL VGELVDIGFAAYtTFVßSIIhZFQVYIINNYI~; ; CB3 : RCitAIDG~1VTi'CRPKSPVYVCL Sequences of CB4a, CB4b, CB2 on ono hand, and CB5 and CDI on the other hand provide respectively the N- and C-terminal covalent structures of tl~e re ;;ion of fragment H containing the hydrophobic domain . Structural analyses, circular dichroism measurements and liposomes studies reveal that both structures are amphipatic and exhibit the properties of the lipid-associating domains of lipoproteins, as defined by SEGREST and JACKSON (I977) ; however, the hydrophilic hT-terminal aide would belong to the surface type dr~main specific to the plasma lipoproteins while the hydrophobic C-terminal aide would be of the transverse type domain found in the I~iN-glycoprotein . Therefore, diphtheria toxin might first interact with the phoapholipid bilayer by reversible surface associations and, then, anchor itself by more stable transverse associations . 7f, as postulated by BOQUET et al . (I976), the insertion of fragment B into the cell membrane requires the preliminary release of a segment at its COOH-terminus, the clustering of basic residues in the vicinity of the two cysteines of CB3 (positions 2 and I2), i .e ., at about 8 000 daltons from the C-terminal end of fragment B, could find a physiological significance in providing an increased sensitivity to cellular proteases to that region of fragment B . BOQUET, P,, SILVERIIAN, bI .S,, PAPPENHEIMER, A,1~f ., Jr . and VERNON, W .B . (I976) Proc . Natl . Aca . Sci . US A ~, 4449-4453 . FALt"IAGNE, P,, L14~SBOTTE, P, and DIRKX, J, (I97ß) Biochim. Biophys . Acts 54-65 . SEGREST, J .P . and JACKSON, R .L . (I977) in "Membrane Proteine and their Inter actions with Lipids ", ed . by R,A . CAPALDI, hTew-York and Basel, b1 . DEKKER, Inc ., pp . 2I-45 .