The chemokine receptor CXCR4 belonging to rhodopsin superfamily of GPCRs is commonly expressed in immune cells and central nervous system. Engagement of CXCR4 in regulating the cell migration and developmental process makes CXCR4 an important drug target. Upon binding CXCL12 activates CXCR4 which is associated with disparate types of cancer, HIV and inflammatory diseases. Initial binding of the ligand leads to the opening of the cytoplasmic cervices for the G-protein binding. For more than a decade the chemokine receptor CXCR4 has been in the spotlight and yet it still lacks detailed information about activation and signaling mechanisms. Here we unraveled the signaling mechanism of CXCR4 bound with its ligands, agonist CXCL12, antagonist peptide CXCL12p, and Gαi protein by using MD simulations and in vitro cellular assays for confirmation. The initial binding interactions between a modelled full-length CXCR4 and CXCL12 were performed by a 1.8 µs MD simulations to determine an antagonist peptide CXCL12p and MM/PBSA binding free energy calculations. The results revealed that electrostatic interactions dominate the binding. The cell experiments also confirmed that the designed peptide can inhibit CXCL12-induced monocytes adhesion, transmigration, wound healing and downstream signal transmission of A549 cells. The further simulations showed a hydrophobic layer HL1 diverts the internal water channel pathway in the activated CXCL12/CXCR4 complex system and formed a continuous internal water channel as Gαi protein bound to the cytoplasmic region of CXCR4. The α5 helix of Gαi with GDP bound approached CXCR4 to lead to the dissociation of GDP. Accelerated MD simulations (flooding) were performed to offer conformational changes of G-protein and potential signaling transmission pathway for the CXCR4 activation mechanism. This study provides atomic-level insights into the structural and functional importance of CXCR4 and can be applied in the design of the anti-cancer peptide drugs.

In silico and in vitro studies on the activation and signaling mechanisms of CXCL12 binding to its cognate receptor CXCR4

256th National Meeting (2018)

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