Abstract
Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.
Highlights
The bacterial Cholesterol-dependent cytolysins (CDCs) and ubiquitous MACPF proteins are expressed as soluble monomers but assemble on membranes to form large, oligomeric pores
The bacterial CDCs and ubiquitous MACPF proteins are expressed as soluble monomers but assemble on membranes to form large, oligomeric pores
Cryo-electron microscopy (EM) and single particle analysis of liposome-bound CDCs led to low-resolution 3D structures of prepore and pore forms of pneumolysin, a major virulence factor of Streptococcus pneumoniae (Tilley et al, 2005)
Summary
The bacterial CDCs and ubiquitous MACPF proteins are expressed as soluble monomers but assemble on membranes to form large, oligomeric pores. Cryo-EM and single particle analysis of liposome-bound CDCs led to low-resolution 3D structures of prepore and pore forms of pneumolysin, a major virulence factor of Streptococcus pneumoniae (Tilley et al, 2005) These structures, as well as an AFM study of perfringolysin (Czajkowsky et al, 2004), established that the 11 nm high molecule must collapse to a height of 7 nm above the membrane in order to insert the TMH regions. The observed distributions of rings and arcs can be explained by a theoretical model for kinetically trapped, noncooperative assembly, fully determined by the relative kinetics of monomer binding to the membrane and monomer assembly on the membrane surface Together these studies provide substantial new understanding of the structure and dynamics of CDC pore formation
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