Abstract
Pneumolysin is a cholesterol-dependent cytolysin (CDC) and virulence factor of Streptococcus pneumoniae. It kills cells by forming pores assembled from oligomeric rings in cholesterol-containing membranes. Cryo-EM has revealed the structures of the membrane-surface bound pre-pore and inserted-pore oligomers, however the molecular contacts that mediate these oligomers are unknown because high-resolution information is not available. Here we have determined the crystal structure of full-length pneumolysin at 1.98 Å resolution. In the structure, crystal contacts demonstrate the likely interactions that enable polymerisation on the cell membrane and the molecular packing of the pre-pore complex. The hemolytic activity is abrogated in mutants that disrupt these intermolecular contacts, highlighting their importance during pore formation. An additional crystal structure of the membrane-binding domain alone suggests that changes in the conformation of a tryptophan rich-loop at the base of the toxin promote monomer-monomer interactions upon membrane binding by creating new contacts. Notably, residues at the interface are conserved in other members of the CDC family, suggesting a common mechanism for pore and pre-pore assembly.
Highlights
cholesterol-dependent cytolysin (CDC), suilysin, show that that pore formation follows the vertical collapse of the pre-pore structure with two extended beta-hairpins penetrating the membrane to form the large beta-barrel pore[7,15]
Additional structural and mutagenesis data has identified residues that are likely to contribute towards oligomerisation during pre-pore formation
It has been shown that pneumolysin oligomerises spontaneously in solution but only at high protein concentrations, revealing weak interactions between monomers, even in the absence of a cholesterol-containing lipid bilayer[21,22]
Summary
CDC, suilysin, show that that pore formation follows the vertical collapse of the pre-pore structure with two extended beta-hairpins penetrating the membrane to form the large beta-barrel pore[7,15]. High-resolution X-ray structures have been determined for several CDCs, pneumolysin has proved to be elusive. Structure-function analysis has relied on models based on the structures of the other CDC toxins. The lack of high-resolution data means that the interactions enabling pneumolysin monomers to pack together to form the pore and pre-pore complexes is not known. We have determined the structure of pneumolysin at 1.98 Å resolution. The side-by-side packing arrangement in the crystal demonstrates the packing of monomers in the pre-pore complex. Additional structural and mutagenesis data has identified residues that are likely to contribute towards oligomerisation during pre-pore formation
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