<i>In Silico</i> Analysis Reveals Sequential Interactions and Protein Conformational Changes during the Binding of Chemokine CXCL-8 to Its Receptor CXCR1

<div><p>Chemokine CXCL-8 plays a central role in human immune response by binding to and activate its cognate receptor CXCR1, a member of the G-protein coupled receptor (GPCR) family. The full-length structure of CXCR1 is modeled by combining the structures of previous NMR experiments with those from homology modeling. Molecular docking is performed to search favorable binding sites of monomeric and dimeric CXCL-8 with CXCR1 and a mutated form of it. The receptor-ligand complex is embedded into a lipid bilayer and used in multi ns molecular dynamics (MD) simulations. A multi-steps binding mode is proposed: (i) the N-loop of CXCL-8 initially binds to the N-terminal domain of receptor CXCR1 driven predominantly by electrostatic interactions; (ii) hydrophobic interactions allow the N-terminal Glu-Leu-Arg (ELR) motif of CXCL-8 to move closer to the extracellular loops of CXCR1; (iii) electrostatic interactions finally dominate the interaction between the N-terminal ELR motif of CXCL-8 and the EC-loops of CXCR1. Mutation of CXCR1 abrogates this mode of binding. The detailed binding process may help to facilitate the discovery of agonists and antagonists for rational drug design.</p></div

The surface lipophilicity distribution for ligand binding with receptor.

<p>The complex structure is represented as ribbon structure with the N-loop of the ligand colored green, the N-terminus of receptor colored pink, and the EC-loops colored yellow. Blue color represents the hydrophilic part while green color represents hydrophobic part. Residues around the binding interface are labeled and shown as sticks; black font is for receptor, while red font is for ligand. (A): Monomeric CXCL-8 binding with CXCR1 at the initial time. Hydrophobic pocket of ligand CXCL-8 is also marked. (B): Monomeric CXCL-8 binding with CXCR1 at the final simulation time. (C): CXCL-8 binding with CXCR1_mut at the initial time. (D): CXCL-8 binding with CXCR1_mut after the 300 ns runs.</p

The surface charge distribution of the complex structure based on Poisson-Boltzmann equation.

<p>The complex structure is represented as ribbon structure with the N-loop of the ligand colored green, the N-terminus of receptor colored pink, and the EC-loops colored yellow. Blue color corresponds to positive and red color to negative electrostatic potential. Residues around the binding interface are labeled and shown as sticks, black is for receptor, while red is for ligand. (A): Monomeric CXCL-8 binding with CXCR1 at the initial time. Binding groove of CXCR1 is also marked. (B): Monomeric CXCL-8 binding with CXCR1 at the final simulation time. (C): CXCL-8 binding with CXCR1_mut at the initial time. (D): CXCL-8 binding with CXCR1_mut after the 300 ns runs.</p

The binding orientation of ligand for various systems at different MD simulation time.

<p>Due to the similar final binding orientations of the three replicates of each system and the figure clarity, only one representative simulation run of each system is shown herein. (A) and (B): For monomeric CXCL-8 system at initial and final simulation time; (C) and (D): For mutated receptor CXCR1_mut system at initial and final simulation time. In all figures, ligands are colored with green, receptors are colored with red, and phosphorous and nitrogen atoms are colored with pink and blue, respectively. The direction of dipole moment of ligand is represented as blue arrow. The distance between the two layers is represented as the thickness of the membrane.</p

<b>Summary of all simulation systems.</b>

<p>Detailed simulation systems are listed herein including ligands, receptor, complex atoms, POPC lipids, and total atoms of the simulation box.</p

The distances between the charged groups of ligand and receptor forming electrostatic interactions.

<p>(A) Some positively charged residues of the N-loop of CXCL-8 gradually approach to the negatively charged residues of the N-terminus of CXCR1 by electrostatic interactions (K3^CXCL-8-D194^CXCR1 (black), K11^CXCL-8-D14^CXCR1 (red), K15^CXCL-8-D13^CXCR1 (green)) during 300 ns MD simulations. (B) Other positively charged residues of CXCL-8 interact with the negatively charged residues of CXCR1 by electrostatic interactions (R47^CXCL-8-D14^CXCR1 (pink), K64^CXCL-8-E35^CXCR1 (blue), R60^CXCL-8-E275^CXCR1 (yellow)) during 300 ns MD simulations. Distances are the average values with the function of time for three replicates of the system monomeric CXCL-8 binding to CXCR1. Error bars of the curves are omitted for figure clarity.</p

Modeled full-length CXCR1 structure and RMSD values during MD simulations.

<p>(A) Ribbon representations of the modeled full-length receptor CXCR1 (residues 2∼347) after embedded into a POPC lipid bilayer for 50 ns MD simulations. The CXCR1 is composed of the structure from the NMR experiment (residues 29∼324, red color), the N-terminal (residues 2∼28) and C-terminal (residues 325∼347) domains from homology modeling results (green color). (B) Plot of the RMSD for the backbone atoms of CXCR1 embedded into POPC lipid bilayers throughout 50 ns MD trajectory.</p

The orientation angle distribution of ligands binding with the receptor throughout the MD simulations.

<p>The orientation angle of the bound ligand is defined as the angle between the unit vector normal to the membrane and the unit vector along the dipole of ligand. The averaged curves for three replicates of each system are shown for monomeric CXCL-8 in black, dimeric CXCL-8 in red, and CXCR1_mut in blue, respectively. Error bars of the curves are omitted for figure clarity.</p

<b>The changes of orientation angles with time for all simulation systems.</b>

<p>The changes of orientation angles are clustered every 50 ns during 300 ns MD simulations.</p