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Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation.

Wacklin, Hanna LU ; Bremec, Biserka Bakrač; Moulin, Martina; Rojko, Nejc; Haertlein, Michael; Forsyth, Trevor; Anderluh, Gregor and Norton, Raymond S (2015) In Biochimica et Biophysica Acta
Abstract
Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII-membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to... (More)
Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII-membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of EqtII, but Chol is required for insertion into the core of the membrane. Cooling the EqtII-lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC-SM-Chol membranes in a manner identical to the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein-membrane interactions available from neutron reflection measurements. (Less)
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Biochimica et Biophysica Acta
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Elsevier
external identifiers
  • pmid:26706098
  • scopus:84957585840
  • wos:000372564500004
ISSN
0006-3002
DOI
10.1016/j.bbamem.2015.12.019
language
English
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yes
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d314b75d-35dd-43de-a080-9c1bddf8c3aa (old id 8503444)
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2016-01-07 13:29:56
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2017-10-29 03:56:53
@article{d314b75d-35dd-43de-a080-9c1bddf8c3aa,
  abstract     = {Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII-membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of EqtII, but Chol is required for insertion into the core of the membrane. Cooling the EqtII-lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC-SM-Chol membranes in a manner identical to the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein-membrane interactions available from neutron reflection measurements.},
  author       = {Wacklin, Hanna and Bremec, Biserka Bakrač and Moulin, Martina and Rojko, Nejc and Haertlein, Michael and Forsyth, Trevor and Anderluh, Gregor and Norton, Raymond S},
  issn         = {0006-3002},
  language     = {eng},
  month        = {12},
  publisher    = {Elsevier},
  series       = {Biochimica et Biophysica Acta},
  title        = {Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation.},
  url          = {http://dx.doi.org/10.1016/j.bbamem.2015.12.019},
  year         = {2015},
}