Structural insight into the Staphylococcus aureus ATP-driven exporter of virulent peptide toxins

Abstract

Staphylococcus aureus is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5′-triphosphate (ATP)–binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an “open” conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

Two transporters cooperate to secrete amphipathic peptides from the cytoplasmic and membranous milieus

“…We previously reported that chromosomal deletion of the entire pmtABCD operon, which additionally encodes for the distinct PmtAB transporter that has no role in α-type PSM secretion ( 19 ), is lethal in a wild-type background due to accumulation of α-type PSMs in the cellular membrane that led to membrane defects ( 17 ). However, pmtABCD deletion mutants were readily obtained in a PSM null background.…”

“…Because of their amphipathic nature, newly synthesized PSMs before export can spontaneously partition between the cytoplasm and the inner leaflet of the cellular membrane, with which PSMs likely interact interfacially and parallel to the plane of the membrane ( 16 ). Two distinct ABC transporters, AbcA and PmtCD, that secrete α-type PSMs have been identified ( 17 – 19 ). Intriguingly, only the pmtABCD operon (which also encodes for the PmtAB transporter that does not transport α-type PSMs) and not the abcA gene provide self-immunity against α-type PSMs, which indicates that export via each transporter occurs through distinct mechanisms and that each transporter may perform specialized roles in secreting PSMs.…”

An analgesic peptide H-20 attenuates chronic pain via the PD-1 pathway with few adverse effects

“…The calculation result was −42.61 kcal/mol, suggesting that H-20 has favorable and stable interactions with PD-1 ( SI Appendix , Table 2 ). Microscale thermography (MST) is a new method to verify ligand–protein binding ( 17 – 19 ). Here, we performed a MST assay to further confirm the binding of H-20 with PD-1.…”

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6

“…The eluted proteins were further purified with a Superdex 200 GL 10/300 column (GE Healthcare, USA) in a buffer containing 20 mM HEPES pH 7.0, 200 mM NaCl, 0.056% (w/v) 6-cyclohexyl-1-hexyl-β-D-maltoside (Cymal-6; Anatrace) and 0.0056% (w/v) CHS. The ABCB6-∆TMD0 protein was also reconstituted into peptidisc by the on-beads method as previously described ( Angiulli et al, 2020 ; Carlson et al, 2018 ; Zeytuni et al, 2020 ). Briefly, after DDM-solubilization, the supernatant was loaded onto anti-GFP DARPin resin, followed by 10 column volumes of wash buffer containing 20 mM HEPES pH 7.0, 200 mM NaCl and 0.5 mg/ml peptidisc (PEPTIDISC BIOTECH, Canada).…”