Structural Analysis of the Protein/Lipid Complexes Associated with Pore Formation by the Bacterial Toxin Pneumolysin

Boyan B Bonev,Robertj.C Gilbert,Peter W Andrew,Olwyn Byron,Anthony Watts

doi:10.1074/jbc.m005126200

Boyan B Bonev, Robertj.C Gilbert + Show 3 more

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https://doi.org/10.1074/jbc.m005126200

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Abstract

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.

Highlights

Pneumolysin is a major virulence factor from the human pathogen Steptococcus pneumoniae and is a member of a family of cholesterol-binding toxins (CBTs),1 which includes per
Changes in protein-induced phospholipid phase behavior can be observed through characteristic intensity distribution in the chemical shift anisotropy (CSA)-dominated lineshapes of wideline 31P NMR spectra
In addition to the inversion of spectral intensity distribution, the overall width of the CSA-dominated intensity distribution is expected to decrease by a factor of 2

Summary

Introduction

Pneumolysin is a major virulence factor from the human pathogen Steptococcus pneumoniae and is a member of a family of cholesterol-binding toxins (CBTs), which includes per-. Solid state NMR provides a direct and quantitative way for investigating lipid-protein interactions in membranes (10 –12). Changes in protein-induced phospholipid phase behavior can be observed through characteristic intensity distribution in the chemical shift anisotropy (CSA)-dominated lineshapes of wideline 31P NMR spectra. Using 31P magic angle spinning (MAS) NMR, the individual lipid components in the bilayer can be resolved [11], and changes in their isotropic chemical shift, CSA and full-width at half-height (FWHH) in response to modulation of the phosphate motions in the presence of proteins determined [11, 12, 16]. Results from solid-state NMR and electron microscopy are presented that provide novel insights into the mechanism of CBTs and complement data already acquired using other or environments; ␶RϪ1, rate of rotational motion; PC, phosphatidylcholine; DCP, dicetylphosphate

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