Dual Mode of Action for Plusbacin A₃ in Staphylococcus aureus

We have used C{F}, N{F}, and N{P} rotational-echo double resonance NMR to determine the location and conformation of ¹⁹F and ¹⁵N double-labeled plusbacin A₃ and of double-labeled deslipo-plusbacin A₃, each bound to the cell walls of whole cells of Staphyloccocus aureus grown in media containing [1-¹³C]glycine. The ³¹P is primarily in wall teichoic acid. Approximately 25% of plusbacin headgroups (the cyclic depsipeptide backbone) are in a closed conformation (N-F separation of 6 Å), while 75% are in a more open conformation (N-F separation of 12 Å). The closed headgroups have no contact with wall teichoic acid, whereas the open headgroups have a strong contact. This places the closed headgroups in hydrophobic regions of the cell wall and the open headgroups in hydrophilic regions. None of the plusbacin tails have contact with the ³¹P of either wall teichoic acid or the cell membrane and thus are in hydrophobic regions of the cell wall. In addition, both heads and tails of plusbacin A₃ have contact with the glycyl ¹³C incorporated in cell-wall peptidoglycan pentaglycyl bridges and with ¹³C-labeled purines near the membrane surface. We interpret these results in terms of a dual mode of action for plusbacin A₃: first, disruption of the peptidoglycan layer nearest to the membrane surface by closed-conformation plusbacin A₃ leading to an inhibition of chain extension by transglycosylation; second, thinning and disruption of the membrane (possibly including disruption of ATP-binding cassette transporters embedded in the membrane) by open-conformation plusbacin A₃, thereby leading to release of ATP to the hydrophilic regions of the cell wall and subsequent binding by plusbacin A₃.