Structural Determinants in the Staphylococcus aureus-Derived Phenol-Soluble Modulin α2 Peptide Required for...

Highly pathogenic Staphylococcus aureus strains produce phenol-soluble modulins (PSMs), which are N-formylated peptides. Nanomolar concentrations of PSMα2 are recognized by formyl peptide receptor 2 (FPR2), but unlike the prototypic FPR2 agonist WKYMVM, PSMα2 is a biased signaling agonist. The truncated N-terminal PSMα2 variant, consisting of the five N-terminal residues, is no longer recognized by FPR2, showing that the C-terminal part of PSMα2 confers FPR2 selectivity, whereas the N-terminal part may interact with the FPR1 binding site. In the current study, a combined pharmacological and genetic approach involving primary human neutrophils and engineered FPR knock-in and knockout cells was used to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides as well as the receptor downstream signaling induced by these peptides. In comparison with the full-length PSMα2, we show that the peptide in which the N-terminal part of PSMα2 was replaced by fMet-Ile-Phe-Leu (an FPR1-selective peptide agonist) potently activates both FPRs for production of superoxide anions and β-arrestin recruitment. A shortened analog of PSMα2 (PSMα2_1-12), lacking the nine C-terminal residues, activated both FPR1 and FPR2 to produce reactive oxygen species, whereas β-arrestin recruitment was only mediated through FPR1. However, a single amino acid replacement (Gly-2 to Ile-2) in PSMα2_1-12 was sufficient to alter FPR2 signaling to include β-arrestin recruitment, highlighting a key role of Gly-2 in conferring FPR2-biased signaling. In conclusion, we provide structural insights into FPR1 and FPR2 recognition as well as the signaling induced by interaction with formyl peptides derived from PSMα2, originating from S. aureus bacteria.