Interaction of the Eukaryotic Pore-forming Cytolysin Equinatoxin II with Model Membranes: 19 F NMR Studies Gregor Anderluh 1 , Andrej Razpotnik 1 , Zdravko Podlesek 1 , Peter Mac ˇek 1 Frances Separovic 2 and Raymond S. Norton 3 * 1 Department of Biology Biotechnical Faculty, University of Ljubljana, Vec ˇna pot 111 1000 Ljubljana, Slovenia 2 School of Chemistry University of Melbourne VIC 3010, Australia 3 Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050 Australia Sea anemones produce a family of 18–20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19 F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site- directed mutagenesis. The 19 F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingo- myelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the b-sandwich, which forms the bulk of the protein. q 2005 Elsevier Ltd. All rights reserved. Keywords: cytolysin; actinoporin; pore formation; membrane; NMR *Corresponding author Introduction The actinoporins are a family of sea anemone toxins that function by forming pores in cell membranes. 1–3 These highly basic proteins, of mass 18–20 kDa, display permeabilising activity in model lipid and cell membranes that is markedly enhanced by the presence of sphingomyelin (SM). The actinoporins differ from the various classes of bacterial pore-forming toxins 4–7 in several respects: they are more potent, the pore they form does not have a stable structure and has not yet been visualized directly, and they are smaller and extremely stable towards proteolytic degradation. Indeed, the potency and properties of these cytoly- sins have prompted their evaluation as the toxic component of chimeric proteins targeted at tumour cells 8,9 and human parasites. 10 The nature of their interaction with lipids in bilayer membranes and the specific role of sphingomyelin in pore formation are not understood at the molecular level. In common with many pore-forming toxins, the actinoporins are highly water-soluble, stable proteins, and yet their only known activity is the formation of oligomeric pores in membranes, consisting of three or, more likely, four monomers. 11–15 Moreover, tetramers of the 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: DHPC, dihexanoyl- phosphatidylcholine; DMPC, dimyristoyl- phosphatidylcholine; DPC, dodecylphosphocholine- 2 H 38 ; EqTII, equinatoxin II; F-Trp-EqTII, equinatoxin II with all five Trp replaced with 5-F-Trp; IPTG, isopropyl-1-thio-b- D-galactopyranoside; PC, phosphatidylcholine; POC, phosphocholine; SM, sphingomyelin; SUV, small unilamellar vesicles. E-mail address of the corresponding author: ray.norton@wehi.edu.au doi:10.1016/j.jmb.2004.12.058 J. Mol. Biol. (2005) 347, 27–39