Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Structure-function relationships of a novel bacterial toxin, hemolysin E. The role of alpha G
AU - Atkins, Angela
AU - Wyborn, Neil R.
AU - Wallace, Alistair J.
AU - Stillman, Timothy J.
AU - Black, Lance K.
AU - Fielding, Andrew B.
AU - Hisakado, Masataka
AU - Artymiuk, Peter J.
AU - Green, Jeffrey
PY - 2000/12/29
Y1 - 2000/12/29
N2 - The novel pore-forming toxin hemolysin E (HlyE, ClyA, or SheA) consists of a long four-helix bundle with a subdomain (beta tongue) that interacts with target membranes at one pole and an additional helix (alpha(G)) that, with the four long helices, forms a five-helix bundle (tail domain) at the other pole. Random amino acid substitutions that impair hemolytic activity were clustered mostly, but not exclusively, within the tail domain, specifically amino acids within, adjacent to, or interacting with alpha(G). Deletion of amino acids downstream of alpha(G) did not affect activity, but deletions encompassing alpha(G) yielded insoluble and inactive proteins. In the periplasm Cys-285 (alpha(G)) is linked to Cys-87 (alpha(B)) of the four-helix bundle via an intramolecular disulfide. Oxidized HlyE did not form spontaneously in vitro but could be generated by addition of Cu(II) or mimicked by treatment with Hg(II) salts to yield inactive proteins. Such treatments did not affect binding to target membranes nor assembly into non-covalently linked octameric complexes once associated with a membrane. However, gel filtration analyses suggested that immobilizing alpha(G) inhibits oligomerization in solution. Thus once associated with a membrane, immobilizing alpha(G) inhibits HlyE activity at a late stage of pore formation, whereas in solution it prevents aggregation and consequent inactivation.
AB - The novel pore-forming toxin hemolysin E (HlyE, ClyA, or SheA) consists of a long four-helix bundle with a subdomain (beta tongue) that interacts with target membranes at one pole and an additional helix (alpha(G)) that, with the four long helices, forms a five-helix bundle (tail domain) at the other pole. Random amino acid substitutions that impair hemolytic activity were clustered mostly, but not exclusively, within the tail domain, specifically amino acids within, adjacent to, or interacting with alpha(G). Deletion of amino acids downstream of alpha(G) did not affect activity, but deletions encompassing alpha(G) yielded insoluble and inactive proteins. In the periplasm Cys-285 (alpha(G)) is linked to Cys-87 (alpha(B)) of the four-helix bundle via an intramolecular disulfide. Oxidized HlyE did not form spontaneously in vitro but could be generated by addition of Cu(II) or mimicked by treatment with Hg(II) salts to yield inactive proteins. Such treatments did not affect binding to target membranes nor assembly into non-covalently linked octameric complexes once associated with a membrane. However, gel filtration analyses suggested that immobilizing alpha(G) inhibits oligomerization in solution. Thus once associated with a membrane, immobilizing alpha(G) inhibits HlyE activity at a late stage of pore formation, whereas in solution it prevents aggregation and consequent inactivation.
KW - Amino Acid Sequence
KW - Hemolysin Proteins
KW - Molecular Sequence Data
KW - Molecular Weight
KW - Protein Structure, Secondary
KW - Structure-Activity Relationship
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1074/jbc.M005420200
DO - 10.1074/jbc.M005420200
M3 - Journal article
C2 - 11006277
VL - 275
SP - 41150
EP - 41155
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 52
ER -