Isoleucine102 and tryptophan104 of the Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FLIPr) are critical for mouse Fcγ receptor binding and inhibition of neutrophil phagocytosis.

Staphylococcus aureus employs a diverse repertoire of immune evasion proteins to escape host defenses. Formyl peptide receptor-like 1 inhibitory protein (FLIPr) is a secreted staphylococcal factor that targets both chemoattractant receptors and Fc gamma receptors (FcγRs) to suppress leukocyte activation and antibody-dependent phagocytosis. However, the specific molecular determinants governing FLIPr engagement with FcγRs remain undefined. Here, we systematically mapped the FcγR-binding interface of FLIPr using N- and C-terminal truncation analyses, alanine-scanning mutagenesis, biolayer interferometry (BLI), circular dichroism (CD) spectroscopy, and primary neutrophil phagocytosis assays. Truncation of the N-terminal 20 amino acids (FLIPr21-105) had no significant impact on FcγR recognition or downstream functional inhibition. In contrast, deletion of the C-terminal five amino acids (FLIPr1-100) completely abolished binding to all major mouse FcγR isoforms (FcγR1, FcγR2b, FcγR3, and FcγR4). Site-directed alanine scanning of the C-terminus identified isoleucine102 (Ile102) and tryptophan104 (Trp104) as indispensible functional hot spots; individual substitution of either residue (F_I102A or F_W104A) led to a complete loss of high-affinity interaction with mouse FcγR1 (KD = 6.26 nM for wild-type FLIPr) and completely abrogated FLIPr-mediated inhibition of mouse neutrophil phagocytosis. CD spectra confirmed that while these hydrophobic C-terminal residues contribute to local secondary structure stability, direct receptor contact drives the loss of function. Together, our findings demonstrate a structural division of labor within FLIPr, establishing the C-terminal Ile102/Trp104 motif as critical molecular determinants of FcγR engagement and providing new mechanistic insights into how S. aureus targets FcγRs to evade antibody-mediated immune clearance.