Structural determinants in the Staphylococcus aureus derived phenol-soluble modulin α2 peptide required for neutrophil formyl peptide receptor activation

Highly pathogenic Staphylococcus aureus strains produce phenol-soluble modulins (PSMs), peptides which are formylated N-terminally. Nanomolar concentrations of PSM-alpha2 are recognized by formyl peptide receptor 2 (FPR2), but unlike the prototypic FPR2 agonist WKYMVM, PSM-alpha2 is a biased signaling agonist. A shortened N-terminal PSM-alpha2 variant, consisting of the five N-terminal residues, is selectively recognized by the closely related FPR1, showing that the C-terminal part of PSM-alpha2 confers FPR2 selectivity, while the N-terminal part may interact with the FPR1 binding site. In the present study, a combined pharmacological and genetic approach, involving primary neutrophils and engineered FPR knock-in and knock-out cells, was used to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides and the receptor downstream signaling induced by these peptides. In comparison to the full-length PSM-alpha2, we show that the peptide in which the N-terminal part of PSM-alpha2 was replaced by fMIFL (an FPR1-selective peptide agonist) potently activates both FPRs for production of superoxide anions and beta-arrestin recruitment. A shortened analogue of PSM-alpha2 (PSM-alpha2,1-12), lacking the nine C-terminal residues activated both FPR1 and FPR2 to produce ROS, whereas beta-arrestin recruitment was only mediated through FPR1. However, a single amino acid replacement (Gly-2 to Ile-2) in PSM-alpha2,1-12 was sufficient to alter FPR2 signaling to include beta-arrestin recruitment, highlighting a key role of Gly-2 in conferring FPR2 biased signaling. In conclusion, we provide novel structural insights into FPR1 and FPR2 recognition as well as the signaling induced by interaction with formyl peptides derived from PSM-alpha2, originating from Staphylococcus aureus bacteria.