Solution NMR analysis of the interaction between the actinoporin sticholysin I and DHPC micelles--correlation with backbone dynamics.
Sticholysin I (StI), an actinoporin expressed as a water-soluble protein by the sea anemone Stichodactyla helianthus, binds to natural and model membranes, forming oligomeric pores. It is proposed that the first event of a multistep pore formation mechanism consists of the monomeric protein attachment to the lipid bilayer. To date there is no high-resolution structure of the actinoporin pore or other membrane-bound form available. Here we evaluated StI:micelle complexes of variable lipid composition to look for a suitable model for NMR studies. Micelles of pure or mixed lysophospholipids and of dihexanoyl phosphatidylcholine (DHPC) were examined. The StI:DHPC micelle was found to be the best system, yielding a stable sample and good quality spectra. A comprehensive chemical shift perturbation analysis was performed to map the StI membrane recognition site in the presence of DHPC micelles. The region mapped (residues F(51), R(52), S(53) in loop 3; F(107), D(108), Y(109), W(111), Y(112), W(115) in loop 7; Q(129), Y(132), D(134), M(135), Y(136), Y(137), G(138) in helix-α2) is in agreement with previously reported data, but additional residues were found to interact, especially residues V(81), A(82), T(83), G(84) in loop 5, and A(85), A(87) in strand-β5. Backbone dynamics measurements of StI free in solution and bound to micelles highlighted the relevance of protein flexibility for membrane binding and suggested that a conformer selection process may take place during protein-membrane interaction. We conclude that the StI:DHPC micelles system is a suitable model for further characterization of an actinoporin membrane-bound form by solution NMR.
Centro de Estudio de Proteinas, Facultad de Biologia, Universidad de la Habana, Habana, Cuba; Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, RJ, Brazil.
Aracelys
A
López-Castilla
Fabiola
F
Pazos
Shirley
S
Schreier
José Ricardo
JR
Pires
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009930:Organic Chemicals / Q000737:chemistry; D010742:Phospholipid Ethers / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012615:Sea Anemones; D012996:Solutions; D013499:Surface Properties
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D008823:Micelles; D009930:Organic Chemicals; D010742:Phospholipid Ethers; D012996:Solutions; C121348:stycholysin I
actinoporin; model membrane; phospholipids; protein-lipid interaction; toxin