Structural basis for recognition of N-formyl peptides as pathogen-associated molecular patterns

Abstract

The formyl peptide receptor 1 (FPR1) is primarily responsible for detection of short peptides bearing N-formylated methionine (fMet) that are characteristic of protein synthesis in bacteria and mitochondria. As a result, FPR1 is critical to phagocyte migration and activation in bacterial infection, tissue injury and inflammation. How FPR1 distinguishes between formyl peptides and non-formyl peptides remains elusive. Here we report cryo-EM structures of human FPR1-Gi protein complex bound to S. aureus-derived peptide fMet-Ile-Phe-Leu (fMIFL) and E. coli-derived peptide fMet-Leu-Phe (fMLF). Both structures of FPR1 adopt an active conformation and exhibit a binding pocket containing the R201^5.38XXXR205^5.42 (RGIIR) motif for formyl group interaction and receptor activation. This motif works together with D106^3.33 for hydrogen bond formation with the N-formyl group and with fMet, a model supported by MD simulation and functional assays of mutant receptors with key residues for recognition substituted by alanine. The cryo-EM model of agonist-bound FPR1 provides a structural basis for recognition of bacteria-derived chemotactic peptides with potential applications in developing FPR1-targeting agents.

Fig. 1. EM density map and overall structure of FPR1-Gi-scFv16 bound to N-formyl peptides.

a, b Side view and extracellular view of the 3D cryo-EM density map of FPR1-Gi-scFv16 bound to fMIFL (a) and fMLF (b). The position of the N-terminal fMet is marked (insets). c, d Side view and extracellular view of the overall structure in cartoon representations. FPR1 bound to formyl peptide is colored in cyan (c, fMIFL) or green (d, fMLF), respectively. Gαi, Gβ1, Gγ2, scFv16 are colored in marine blue, yellow, magenta, and gray, respectively.

Fig. 2. Ligand binding mode of FPR1 to fMIFL.

a Side view (left) and extracellular view (right) of the FPR1-fMIFL structure. The receptor is shown as surface and cartoon, colored in cyan. The ligand fMIFL is shown as sphere with carbons in pink. b Slab view (light gray) of the binding cavity of fMIFL in FPR1. fMIFL assumes an N-terminus-in pose circled in red dashed line. c Side view of the binding pocket of FPR1-fMIFL structure. The receptor is shown as cartoon and colored in marine blue. The ligand fMIFL is shown as licorice with carbons in pink. Hydrogen bonds formed of R201^5.38XXXR205^5.42 motif with the N-formyl group, carbonyl groups of fMet in fMIFL, indicated in dash line. The residues of FPR1 within 4.5 Å to the atoms of fMIFL are shown in cyan licorice. d Extracellular (top) view of the FPR1-fMIFL structure. Red dashed lines indicate polar interactions between D106^3.33, the R201^5.38XXXR205^5.42 motif, and fMet in fMIFL. e, f Local density map of the ligand fMIFL and residues of FPR1 nearby the formyl group (CHO), viewed from two different angles.

Fig. 3. MD simulation of the Cryo-EM models.

Two representative hydrogen-bonds networks characterized by either minimizing the average donor-acceptor (D–A) distance or maximizing the number of concurrent hydrogen-bonds from the conformation ensemble of the whole 3-µs trajectories of FPR1-Gi-scFv16 bound with fMIFL (a) and fMLF (b). Both representatives show that the formyl group is recognized by the D106^3.33-R201^5.38-R205^5.42 motif, in particular the salt bridge between D106^3.33 and R201^5.38 stabilized the side-chain orientation of R201^5.38. The residues D106^3.33-R201^5.38 directly recognizes the formyl-O of fMLF (or fMIFL), as both the short-range H-bond (left) and the long-range electrostatic attraction (right) could be offered by the positively charged guanidino group of R201^5.38 during the thermodynamics fluctuation. The yellow dashed lines indicate distance shorter than 3 Å. Source data are provided as a Source data file.

Fig. 4. Binding poses of fMLF, non-formyl analogs and small molecule ligands to FPR1.

a Chemical structure of fMLF and its non-formyl analogs. b Slab views of the binding pocket of fMLF (left, cryo-EM model), MLF (middle, docking model), and tBOC-MLF (right, docking model) in FPR1, respectively. The ligands are displayed in licorice with carbon in orange. The binding pocket is highlighted in white. c Molecular interaction of bound fMLF (left), MLF (middle), and tBOC-MLF (right) with the FPR1 binding pocket. d Chemical structures of WKYMVm, AG-14 and Compound 17b (Cpd 17b). e Slab views of the binding pocket of WKYMVm, AG-14 and Cpd 17b, all from docking models. f molecular interaction of bound WKYMVm (left), AG-14 (middle), and Cpd 17b (right) with the FPR1 binding pocket, respectively. The residues of FPR1 within 4.5 Å to the atoms of the ligands are shown as green licorice.

Fig. 5. Dose-response curves of FPR1 and its mutants in cAMP inhibition assays.

FPR1 and selected mutants were expressed in transiently transfected cells. The receptors were stimulated with different concentrations of the indicated agonists, fMIFL (a), WKYMVm (b), AG-14 (c) or Compound 17b (d) plus forskolin for 30 min. Changes in cytoplasmic cAMP concentrations were measured and data were plotted with the maximal cAMP concentrations set as 100%. Data are shown as mean ± SEM of three independent experiments, each in duplicates. Source data are provided as a Source Data file.

Fig. 6. Interface of G_αi with FPR1 and FPR2.

a Overview of the Gαi (pink) interacting with FPR1 (green, left) and FPR2 (blue, middle, PDB ID: 6OMM). The structures are front view from the intracellular side, showing in cartoon overlap with surface in 50% transparency. The crystal structure of FPR2 (right) is colored in olive and showed in cartoon and surface. b Slab view showing the interactions between FPR1 and Gαi protein (left). The region TM2, TM3, TM6, and ICL3 of FPR1 form direct contact with α5 helix of Gαi protein (middle). C126 in TM3, Y64 in TM2 forms hydrogen bond to N347, N351 in α5 helix of Gαi protein, respectively. ICL2 of FPR1 has polar interactions with αN helix and β1-β2 loop of Gαi protein (right). c Slab view showing the interactions between FPR2 and α5 helix of Gαi protein (left), as well as interactions of ICL2 of FPR2 with αN helix and β1-β2 loop of Gαi protein (right).