Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from seaanemone - Topology in lipid membranes

Equinatoxin II is a cysteineless pen-forming protein from the sea anemone A ctinia equina, It readily creates pens in membranes containing sphingomyeli n. Its topology when bound in lipid membranes has been studied using cystei ne-scanning mutagenesis. At approximately every tenth residue, a cysteine w as introduced. Nineteen single cysteine mutants were produced in Escherichi a coli and purified. The accessibility of the thiol groups in lipid-embedde d cysteine mutants was studied by reaction with biotin maleimide. Most of t he mutants were modified, except those with cysteines at positions 105 and 114, Mutants R144C and S160C were modified only at high concentrations of t he probe. Similar results were obtained if membrane-bound biotinylated muta nts were tested for avidin binding, but in this case three more mutants gav e a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K2 0C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurrin g upon membrane binding. These results were further confirmed by labeling t he mutants with acrylodan, a polarity-sensitive fluorescent probe. When lab eled mutants were combined with vesicles, the following mutants exhibited b lue-shifts, indicating the transfer of acrylodan into a hydrophobic environ ment: S13C, K20C S105C, S114C, R120C, R120C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: t he N-terminal 13-20 region. probably forming an amphiphilic helix, and the tryptophan-rich 105-120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with th e membrane appears to be unique and different from that of bacterial port-f orming proteins and therefore equinatoxin II may serve as a model for eukar yotic channel-forming toxins.