The role of water in activation mechanism of human N-formyl peptide receptor 1 (FPR1) based on molecular dynamics simulations

Abstract

The Formyl Peptide Receptor 1 (FPR1) is an important chemotaxis receptor involved in various aspects of host defense and inflammatory processes. We constructed a model of FPR1 using as a novel template the chemokine receptor CXCR4 from the same branch of the phylogenetic tree of G-protein-coupled receptors. The previously employed template of rhodopsin contained a bulge at the extracellular part of TM2 which directly influenced binding of ligands. We also conducted molecular dynamics (MD) simulations of FPR1 in the apo form as well as in a form complexed with the agonist fMLF and the antagonist tBocMLF in the model membrane. During all MD simulation of the fMLF-FPR1 complex a water molecule transiently bridged the hydrogen bond between W254(6.48) and N108(3.35) in the middle of the receptor. We also observed a change in the cytoplasmic part of FPR1 of a rotamer of the Y301(7.53) residue (tyrosine rotamer switch). This effect facilitated movement of more water molecules toward the receptor center. Such rotamer of Y301(7.53) was not observed in any crystal structures of GPCRs which can suggest that this state is temporarily formed to pass the water molecules during the activation process. The presence of a distance between agonist and residues R201(5.38) and R205(5.42) on helix TM5 may suggest that the activation of FPR1 is similar to the activation of β-adrenergic receptors since their agonists are separated from serine residues on helix TM5. The removal of water molecules bridging these interactions in FPR1 can result in shrinking of the binding site during activation similarly to the shrinking observed in β-ARs. The number of GPCR crystal structures with agonists is still scarce so the designing of new ligands with agonistic properties is hampered, therefore homology modeling and docking can provide suitable models. Additionally, the MD simulations can be beneficial to outline the mechanisms of receptor activation and the agonist/antagonist sensing.

Figure 1. The structure of homology model of FPR1 and its binding pocket.

(A) overall view of FPR1 model; (B) alternative view of FPR1 model from extracellular side; (C) important residues in binding site of FPR1. The whole pocket was visually divided into two zones: the anchor region (on the left - in blue) and the activation region (on the right – in green).

Figure 2. Extracellular surface of FPR1 model after equilibration period.

The structure is mapped with electrostatic potential (positive in blue, negative in red) and a position of agonist is shown. (A) The whole structure of the model. (B) The model without all extracellular loops. Selected important and visible on molecular surface residues are labeled.

Figure 3. The chemical formulas of fMLF (agonist) and tBocMLF (antagonist).

Both ligands share most of the structure so only differences in N-termini are shown in detail and colored in blue.

Figure 4. The structure of agonist fMLF interacting with FPR1 after equilibration period.

(A) Interactions between C-terminus of fMLF with FPR1. The residues R84^2.63 and K85^2.64 were found to form direct salt bridges with carboxyl terminus of ligand while D284^7.38 interacts with the same group of agonist through a water molecule. Hydrophobic side chain of fMLF is surrounded by F81^2.60, V101^3.28, F102^3.29 and F291^7.43. (B) Interactions between N-terminus of fMLF and FPR1. The carbonyl group in peptide bond in residue M1 forms a direct hydrogen bond with Y257^6.51 while the formyl group can interact with both R205^5.42 and D106^3.33 through water molecules.

Figure 5. The structure of antagonist tBocMLF interacting with FPR1 after equilibration period.

(A) Interactions between C-terminus of tBocMLF with FPR1. The residues R84^2.63, K85^2.64 and D284^7.38 form hydrogen bonds with tBocMLF directly. Hydrophobic side chains of antagonist are surrounded by F81^2.60, V101^3.28, F102^3.29 and F291^7.43. (B) Interactions between N-terminus of tBocMLF and FPR1.

Figure 6. The ligand-receptor interactions after 100 ns MD simulation.

View from extracellular side. (A) The agonist fMLF (in orange). (B) The antagonist tBocMLF (in cyan). The M1 residue of agonist went down toward W254^6.48 while that of antagonist went up toward EC2 loop.

Figure 7. A hydrogen bond network in the structure of the agonist-FPR1 complex.

A side view of initial equilibrated structure. (A) The binding site showing the hydrogen bond network involving water molecules. (B) A continuation of the hydrogen bond network of the same complex at the intracellular side.

Figure 8. Mechanism of partial activation of FPR1.

(A) Movement of helices due to agonist binding. Apo structure in gray and with agonist bound in green; (B) structure of cytoplasmic part of FPR1 in Apo form and with antagonist bound; (C) structure of cytoplasmic part of FPR1 in agonist bound complex. The hydrogen bond between Y301^7.53 and N298^7.49 was found bridged by a water molecule and the residue Y301^7.53 was switched.

Figure 9. A scheme of interactions in the final structure of the agonist-FPR1 complex after MD simulation.

A movement of two water molecules during MD simulation is shown. These molecules can bridge the hydrogen bonds between some residues. A water molecule transiently bridges a hydrogen bond between W254^6.48 and N108^3.35.

Figure 10. Superimposed models of FPRs constructed using CXCR4 template structure.

(A) Superimposition of FPR1 (gray) and FPR2 (cyan). (B) Superimposition of FPR1 (gray) and FPR3 (green).

Figure 11. Comparison of FPR1 models constructed on different templates.

A model based on rhodopsin is colored in cyan while that based on CXCR4 in green. Some residues in TM2 are shown in red dashed ellipses to exemplify differences between both models. A change of a template from rhodopsin to CXCR4 leads to the rotation about 100° of extracellular part of TM2 starting from S76^2.55 and removal of a bulge at T77^2.56.

Figure 12. A comparison of rotamers of residue Y7.53 from NPxxY motif in different receptor structures.

FPR1 with agonist bound in yellow (Y301), inactive rhodopsin (PDB id 1GZM) in light blue (Y306), activated rhodopsin (PDB id 2X72) is dark blue (Y306). Dashed red line encircles the three abovementioned tyrosines.