Solution Structure of the Eukaryotic Pore-forming Cytolysin Equinatoxin II: Implications for Pore Formation Mark G. Hinds 1 , Wei Zhang 2 , Gregor Anderluh 3 , Poul Erik Hansen 2 and Raymond S. Norton 1 * 1 Biomolecular Research Institute, 343 Royal Parade Parkville 3052, Australia 2 Institute of Life Sciences and Chemistry, Roskilde University Denmark 3 Biotechnical Faculty Department of Biology University of Ljubljana 1000 Ljubljana, Slovenia Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, that 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. The structure of the actino- porin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a b-sand- wich structure formed by two ®ve-stranded b-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two b-sheets. 15 N relaxation data show uniform backbone dynamics, implying that equinatoxin II in solution is relatively rigid, except at the N terminus; its inferred rotational correlation time is consistent with values for monomeric proteins of similar mass. Backbone amide exchange rate data also support the view of a stable structure, even though equinatoxin II lacks disul®de bonds. As monitored by NMR, it unfolds at around 70  C at pH 5.5. At 25  C the structure is stable over the pH range 2.5-7.3 but below pH 2.5 it undergoes a slow transition to an incompletely unfolded structure resembling a molten glo- bule. Equinatoxin II has two signi®cant patches of positive electrostatic potential formed by surface-exposed Lys and Arg residues, which may assist its interaction with charged regions of the lipid head groups. Tyr and Trp residues on the surface may also contribute by interacting with the carbonyl groups of the acyl chains of target membranes. Data from mutational studies and truncated analogues identify two regions of the protein involved in membrane interactions, the N-terminal helix and the Trp-rich region. Once the protein is anchored, the N-terminal helix may penetrate the membrane, with up to four helices lining the pore, although other mechanisms of pore formation cannot be ruled out. # 2002 Elsevier Science Ltd. Keywords: cytolysin; sea anemone; toxin; b-sheet; pore formation *Corresponding author Introduction Sea anemones produce a family of toxins, the actinoporins, that function by forming pores in cell membranes. 1±3 These highly basic proteins, with molecular masses in the range 18-20 kDa, display permeabilising activity in model lipid and cell membranes that is markedly enhanced by the pre- sence of sphingomyelin. In contrast to bacterial pore-forming toxins such as staphylococcal a-toxin and the leukocidin family, 4 aerolysin and related toxins from Aeromonas, 5 colicins 6 and cholesterol- dependent cytolysins, 7 there is little detailed infor- mation on the mechanism of action of these toxins. Actinoporins differ from these toxins 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 of smaller Present addresses: M. G. Hinds and R. S. Norton, The Walter and Eliza Hall Institute of Medical Research, P. O. Royal Melbourne, Parkville, VIC 3050, Australia. Abbreviations used: EqTII, equinatoxin II; ANS, 2-anilinonaphthalene-6-sulfonic acid; CD, circular dichroism; IR, infrared; NOE, nuclear Overhauser effect; RMS, root mean square; FTIR, Fourier transform infrared; NOESY, NOE spectroscopy. E-mail address of the corresponding author: rnorton@wehi.edu.au doi:10.1006/jmbi.2001.5321 available online at http://www.idealibrary.com on J. Mol. Biol. (2002) 315, 1219±1229 0022-2836/02/051219±11 $35.00/0 # 2002 Elsevier Science Ltd.