Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: Implications for pore formation

Mark G. Hinds, Wei Zhang, Gregor Anderluh, Poul Erik Hansen, Raymond S. Norton

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128 Citations (Scopus)

Abstract

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 actinoporin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a β-sandwich structure formed by two five-stranded β-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two β-sheets. 15N 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 disulfide 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 globule. Equinatoxin II has two significant 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.

Original languageEnglish
Pages (from-to)1219-1229
Number of pages11
JournalJournal of Molecular Biology
Volume315
Issue number5
DOIs
Publication statusPublished - 2002
Externally publishedYes

Keywords

  • Cytolysin
  • Pore formation
  • Sea anemone
  • Toxin
  • β-sheet

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