Cholesterol dependent cytolysins (CDCs) are bacterial pore-forming proteins that form part of the major membrane-attack-complex/perforin (MACPF)/CDC superfamily. Upon binding to the membrane, they undergo a major refolding transition from soluble monomers to oligomeric pores inserted into the target membrane. To understand the pathways of assembly and conformational changes in this transformation, we have studied suilysin from Streptococcus suis, a bacterial CDC, using real-time atomic force microscopy, electron microscopy and atomic structure fitting.
We established that the suilysin assembly is a noncooperative process that is terminated in the prepore intermediate state before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are determined by the respective rates of monomer binding to the membrane and prepore oligomerization. Both complete and incomplete rings perforate the membrane in the latter case forming unsealed edges of lipid and β-barrels and causing the visible ejection of lipids. Using cryo-electron microscopy and atomic structure fitting, we discovered large changes in subunit conformation and packing in the prepore-to-pore transition, including a radial expansion of the assembly and a sideways tilt by which each subunit extends a domain to contact its nearest neighbor.
We have thus visualized the various stages of membrane pore formation by a CDC in unprecedented detail and accuracy, to provide new insights in domain movements and pathways of assembly for a major superfamily of pore-forming proteins.