Calorimetric scrutiny of lipid binding by sticholysin II toxin mutants.
The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7x10(8) M(-1)). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.
Alegre-Cebollada
Jorge
J
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Cunietti
Michela
M
Herrero-Galán
Elías
E
Gavilanes
José G
JG
Martínez-del-Pozo
Alvaro
A
D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry / Q000379:methods*; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006461:Hemolysis; D008055:Lipids / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012615:Sea Anemones / Q000737:chemistry; D013816:Thermodynamics
D003064:Cnidarian Venoms; D008055:Lipids; D052899:Pore Forming Cytotoxic Proteins; C404794:sticholysin II