Specific Protein-Membrane Contacts Are Required for Prepore and Pore Assembly by a Cholesterol-dependent Cytolysin

Eileen M. Hotze,Rodney K. Tweten,Arthur E. Johnson,Casie E. Soltani

doi:10.1074/jbc.m701173200

Abstract

Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. This observation suggested that the D4 loops, which include loops L1-L3 and the undecapeptide, of ILY were no longer required for its cell binding. However, we show here that membrane insertion of the D4 loops is required for the cytolysis by ILY. Receptor binding triggers changes in the structure of ILY that are necessary for oligomerization, but membrane insertion of the D4 loops is critical for oligomer assembly and pore formation. Defects that prevent membrane insertion of the undecapeptide also block assembly of the prepore oligomer, while defects in the membrane insertion of the L1-L3 loops prevent the conversion of the prepore oligomer to the pore complex. These studies reveal that pore formation by ILY, and probably other CDCs, is affected by an intricate and coupled sequence of interactions between domain 4 and the membrane.

Highlights

Monomers bind to the membrane and oligomerize into a prepore complex (4 – 6) of 34 –50 monomers [7,8,9], which is converted to the ␤-barrel pore complex by the cooperative membrane insertion [10] of two amphipathic ␤-hairpins per monomer [11, 12]
Our studies revealed that the domain 4 (D4) loops of ILY are critical for pore formation and that each polypeptide segment is required for the proper progression of ILY through specific stages of oligomer assembly and pore formation
Most cholesterol-dependent cytolysins (CDCs) are thought to bind to the cell surface via cholesterol-rich domains (reviewed in [30]), and this interaction is mediated by the short hydrophobic loops and the undecapeptide at the tip of domain 4 [6, 15, 17, 20]

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Chemicals—The gene for ILY was cloned into pTrcHisA (Invitrogen) as described previously [25]. The HD50 is defined as the concentration of toxin required to lyse 50% of the hRBCs. Modification of ILY with Fluorescent Probes—The labeling of ILY cysteine-containing mutants with the environmentally sensitive fluorescent probe iodoacetamido-N,NЈ-dimethyl-N-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylene diamine (NBD; Molecular Probes) via the sulfhydryl was carried out as described previously [25]. Samples containing 180 pmol of total toxin were incubated with hRBC ghost membranes (equivalent to 303.25 ␮g of membrane protein) or hRBC ghost membranes incorporated with doxyl-stearic acid in PBS (10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 3 mM KCl (pH 7.5)) at 37 °C for 5–10 min before making spectral measurements. SDS-Agarose Gel Electrophoresis (AGE) and Immunoblot quenched the NBD fluorescence (data not shown) This result is Analyses—Denaturing agarose gel electrophoresis was carried similar to that previously observed for PFO [15].

RESULTS

Hemolytic activity

Findings

DISCUSSION