On the structure and assembly of staphylococcal leukocidin: a study of the molecular architecture of beta-barrel pore-forming toxins

Abstract

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homooligomeric pores. For example, the staphylococcal α- hemolysin is a homoheptamer. Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore. In this work, biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC) components encoded by Staphylococcus aureus. I demonstrate that LukF and LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In addition, the pore-forming properties of recombinant leukocidin were investigated with planar lipid bilayers. Although leukocidins and staphylococcal α-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5 mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin measured under the same conditions. The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods, gel shift electrophoresis and sitespecific chemical modification during single channel recording. Four LukF and four LukS subunits were shown to co-assemble into an octameric transmembrane structure. The existence of an additional subunit in part explains properties of the leukocidin pore, such as its high conductance. Additionally, this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein. It is also relevant to understanding the mechanism of assembly of β-PFT pores, and suggests new possibilities for engineering these proteins. In additional studies, the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of β-PFTs. HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology, such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials.