Interaction of Escherichia coli hemolysin with biological membranes - A study using cysteine scanning mutagenesis

Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belo nging to the family of RTX-toxins. Lytic activity depends on binding of Ca2 + to the C-terminus, of the molecule. The N-terminus of HlyA harbors hydrop hobic sequences that are believed to constitute the membrane-inserting doma in. In this study, 13 HlyA cysteine-replacement mutants were constructed an d labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dim ethylaminonaphthalene (badan). The fluorescence emission of the label was e xamined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic dom ain, indicating insertion of this domain into the lipid bilayer. The emissi on shifts occurred both in the presence and absence of Ca2+, suggesting tha t Ca2+ is not required for the toxin to enter membranes. However, binding o f Ca2+ to HlyA in solution effected conformational changes in both the C-te rminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca2+ to the toxin in solution effects a conformational chan ge that is relayed to the N-terminal domain, rendering it capable of adopti ng the structure of a functional pore upon membrane binding.