The Cholesterol-Dependent Cytolysin Signature Motif: A Critical Element in the Allosteric Pathway that Couples Membrane Binding to Pore Assembly
Figure 7
ILY binding and pore formation on cholesterol-rich liposomes.
(A) Binding of PFO, ILY and ILYDM to cholesterol-rich POPC liposomes was measured by SPR. The data is representative of 3 experiments. (B) Pore formation on liposomes was measured as the emission intensity of CF increased upon dilution as pores are formed in the liposomes. The change in the emission intensity of CF over time in an untreated sample was subtracted from the experimental data. The data are representative of at least 3 analyses. ILYDM contains glycine substitutions for the ILY CRM residues Thr-517 and Leu-518, which knocks out CRM-dependent binding to cholesterol-rich membranes [22].(C) To measure the insertion of the β-barrel pore a cysteine was substitutedand modified with NBDfor TMH1 residue Ala-215 of PFO or its analog, His-242 in ILY. Each derivative was incubated in the presence (dashed line) and absence (solid line) of cholesterol-rich liposomes. As the soluble monomer binds to and forms a pore in the membrane the NBD probe positioned in TMH1 makes the transition from a polar environment in the soluble monomer (solid line) to the nonpolar environment of the membrane (dashed line), which is reflected by an increase in the NBD fluorescence emission [27].