Helical crystallization on nickel-lipid nanotubes: perfringolysin O as a model protein.
To facilitate purification and subsequent structural studies of recombinant proteins the most widely used genetically encoded tag is the histidine tag (His-tag) which specifically binds to N-nitrilotriacetic-acid-chelated nickel ions. Lipids derivatized with a nickel-chelating head group can be mixed with galactosylceramide glycolipids to prepare lipid nanotubes that bind His-tagged proteins. In this study, we use His-tagged perfringolysin O (PFO), a soluble toxin secreted by the bacterial pathogen Clostridium perfringens, as a model protein to test the utility of nickel-lipid nanotubes as a tool for structural studies of His-tagged proteins. PFO is a member of the cholesterol dependent cytolysin family (CDC) of oligomerizing, pore-forming toxins found in a variety of Gram-positive bacterial pathogens. CDC pores have been difficult to study by X-ray crystallography because they are membrane associated and vary in size. We demonstrate that both a wild-type and a mutant form of PFO form helical arrays on nickel-lipid containing nanotubes. Cryo-electron microscopy and image analysis of the helical arrays were used to reconstruct a 3D density map of wild-type PFO. This study suggests that the use of nickel-lipid nanotubes may offer a general approach for structural studies of recombinant proteins and may provide insights into the molecular interactions of proteins that have a natural affinity for a membrane surface.
Dang
Thanh X
TX
The Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Milligan
Ronald A
RA
Tweten
Rodney K
RK
Wilson-Kubalek
Elizabeth M
EM
D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D003016:Clostridium perfringens / Q000378:metabolism; D020285:Cryoelectron Microscopy; D003460:Crystallization; D006460:Hemolysin Proteins; D006639:Histidine / Q000737:chemistry; D021621:Imaging, Three-Dimensional; D008055:Lipids / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D036103:Nanotechnology; D043942:Nanotubes / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure; D014961:X-Ray Diffraction
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008055:Lipids; D011506:Proteins; D006639:Histidine; C020452:Clostridium perfringens theta-toxin; D009532:Nickel