Structural characterization of Staphylococcal γ-hemolysin in the presence of a lipid environment using single particle cryo-EM

Abstract

γ-Hemolysin (γ-HL) is a quintessential bicomponent β-pore forming toxin (β-PFT) belonging to the arsenal of “superbug” Staphylococcus aureus. Here, we performed negative staining-TEM and single particle cryo-EM-based studies on the bicomponent γ-HL from Newman strain in a lipid environment. We purified the two functionally active protein components, HlgA and HlgB, and obtained transmembrane pore complexes in presence of liposomes constituting equimolar phosphatidylcholine and cholesterol. Strikingly, for the first time, we observed the presence of octahedral supramolecular assemblies of HlgAB octameric pore complexes under both room temperature NS-TEM and cryo-EM conditions. Using our in-house 200kV Talos Arctica at IISc Bangalore, we resolved the cryo-EM structure of these assemblies to near-atomic global resolution of 3.5 Å. Our atomic model not only elucidates the structural basis of the phenomenon of PFT super-assemblies, but also throws light on the critical lipid-binding residues for both the components based on the extra densities in our high resolution cryo-EM map. We further modelled the previously missing NTR through our study which also illuminates important steric aspects of γ-HL oligomerization and pore formation. Finally, our cryo-EM structure of octameric HlgAB also represents the first wild-type pre-pore structure for any Staphylococcal β-PFT. Thus, this study provides significantly novel understanding of these bicomponent β-PFTs in a near-native environment.