Structural Determinants in the Staphylococcus aureus–Derived Phenol-Soluble Modulin α2 Peptide Required for Neutrophil Formyl Peptide Receptor Activation

Abstract

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 (PSMα21-12), 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 PSMα21-12 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.

G protein coupled pattern recognition receptors expressed in neutrophils: Recognition, activation/modulation, signaling and receptor regulated functions

“…The two receptors have different recognition profiles, and it seems that FPR1 preferentially interacts with shorter peptides whereas FPR2 exhibits a preference for longer peptides. Data obtained with chimeric fMIFL‐PSMα2 peptides show that even though longer formyl peptides often possess FPR2 selectivity, this does not exclude recognition by FPR1 51 . Clearly, recognition of formyl peptides by both FPRs is not determined solely by the N‐terminal 4–5 amino acids, suggested to fit directly into the binding pocket.…”

“…Data obtained with chimeric fMIFL-PSMα2 peptides show that even though longer formyl peptides often possess FPR2 selectivity, this does not exclude recognition by FPR1. 51 Clearly, recognition of formyl peptides by both FPRs is not determined solely by the Nterminal 4-5 amino acids, suggested to fit directly into the binding pocket. The importance of the C-terminal part in conferring FPR subtype selectivity is in fact supported by studies showing that a peptide in which the hydrophobic Ile 11 positioned close to the C terminus of a dual FPR agonist was removed (or replaced with Ala), no longer was recognized by FPR2, whereas its ability to be recognized by FPR1 was retained.…”

“…The five amino acid long peptide, corresponding to the N‐terminus of PSMα2 no longer interact with FPR2 but is instead recognized by FPR1. A combined pharmacological and genetic approach to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides, show that the peptide in which the N‐terminal part of a 12 amino acid long PSMα2 peptide was replaced by the FPR1 selective peptide fMet‐Ile‐Phe‐Leu, potently activates both FPRs 51 . Comparison of several modified PSMα2 analogs that differ in length and amino acid composition in the N‐terminal part show that FPR1 as well as FPR2 recognize formyl peptides.…”

“…A combined pharmacological and genetic approach to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides, show that the peptide in which the N-terminal part of a 12 amino acid long PSMα2 peptide was replaced by the FPR1 selective peptide fMet-Ile-Phe-Leu, potently activates both FPRs. 51 Comparison of several modified PSMα2 analogs that differ in length and amino acid composition in the N-terminal part show that FPR1 as well as FPR2 recognize formyl peptides. The two receptors have different recognition profiles, and it seems that FPR1 preferentially interacts with shorter peptides whereas FPR2 exhibits a preference for longer peptides.…”