Retrieving Biological Activity from LukF-PV Mutants Combined with Different S Components Implies Compatibility between the Stem Domains of These Staphylococcal Bicomponent Leucotoxins

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Abstract
Bicomponent leucotoxins, such as Panton-Valentine leucocidin, are composed of two classes of proteins, a class S protein such as LukS-PV, which bears the cell membrane binding function, and a class F protein such as LukF-PV, which interacts to form a bipartite hexameric pore. These leucotoxins induce cell activation, linked to a Ca2+ influx, and pore formation as two consecutive and independently inhibitable events. Knowledge of the LukF-PV monomer structure has indicated that the stem domain is folded into three antiparallel β-strands in the water-soluble form and has to refold into a transmembrane β-hairpin during pore formation. To investigate the requirements for the cooperative assembly of the stems of the S and F components to produce biological activity, we introduced multiple deletions or single point mutations into the stem domains of LukF-PV and HlgB. While the binding of the mutated proteins was weakly dependent on these changes, Ca2+ influx and pore formation were affected differently, confirming that they are independent events. Ca2+ entry into human polymorphonuclear cells requires oligomerization and may follow the formation of a prepore. The activity of some of the LukF-PV mutants, carrying the shorter deletions, was actually improved. This demonstrated that a crucial event in the action of these toxins is the transition of the prefolded stem into the extended β-hairpins and that this step may be facilitated by small deletions that remove some of the interactions stabilizing the folded structure.

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