CXCR4
The C-X-C chemokine receptor 4 (CXCR4) is a Class A G-protein-coupled receptor (GPCR) that belongs to the C-X-C subfamily of chemokine receptors. It is known to stimulate cell migration, hematopoiesis, and angiogenesis by binding to its unique chemokine ligand – C-X-C chemokine 12 (CXCL12). CXCR4 is involved in various pathological processes, including cancer, HIV infection and immune disorders, making it a prominent drug target.
Numerous CXCR4-targeting therapeutics have been developed, such as the small-molecule antagonist AMD3100 (plerixafor), monoclonal antibodies (mAbs) and peptides. However, detailed structural insights into the receptor’s regulation are still missing, and more 3D structures could significantly enhance our knowledge of the target and consequently the discovery of novel drugs.
To deepen structural understanding of the CXCR4 protein, Regeneron scientists determined several Cryo-EM structures of CXCR4-ligand complexes for the first time. Additionally, they resolved and analyzed multiple oligomeric structures (trimeric and tetrameric assemblies), providing previously unknown insights into higher order GPCR oligomerization, crucial for rational drug design.
Structural basis for various modes of CXCR4 regulation and oligomerization - Key structural insights
CXCR4-ligand complexes analysis
Structural studies elucidated the binding mode of the protein with its native ligand, a small molecule inhibitor, and an antibody. Below are the key findings of each complex.
CXCR4 activation by its native ligand CXCL12: The CryoEM structure of CXCR14/CXCL12 complex (3.3 Å) shows that CXCL12 N-terminus is responsible for receptor activation. CXCL12 N-terminal tail extends deep into the receptor orthosteric pocket, forming extensive hydrogen bonds and electrostatic interactions with the transmembrane (TM) core. Ligand binding triggers conformational changes in key receptor residues - E288, Y255, F292, and W252 - causing structural rearrangements that stabilize the active conformation of ligand-bound CXCR4.
Binding mode of the small-molecule antagonist (AMD310): AMD3100 (plerixafor) is the only FDA-approved small-molecule CXCR4 antagonist. From the CryoEM structure (3.2 Å), it is evident that AMD3100 is oriented diagonally in the receptor orthosteric pocket, directly blocking the ligand-binding site. Both AMD3100 cyclam rings form electrostatic interactions with the key receptor signaling residues - E288 and D262 - stabilizing the antagonist binding mode and preventing native ligand binding.
CXCR4 antagonism by monoclonal antibody REGN7663: This highly potent mAb binds onto the extracellular face of the receptor sterically blocking the native ligand binding. It induces distinct conformations of the extracellular N-terminal region and ECL2 compared to those seen in native ligand or antagonist binding. Notably, CDR-H3 loop of REGN7663 partially inserts into the orthosteric pocket, with residue R105 forming an electrostatic interaction with the pocket-facing E288 of CXCR4.
CXCR4 oligomerization
Scientists observed that CXCR4 existed in different oligomeric states in vitro, and so they isolated and solved the CryoEM structures of trimeric and tetrameric CXCR4-REGN7663 assemblies, revealing previously unknown structural arrangements of the receptor:
Trimer and tetramer interprotomer interfaces are generally similar, with TM5, TM6 and TM7 of one protomer interacting with TM1 and TM7 of the neighboring one. This interface arrangement differs from the one observed in the crystal structures of CXCR4 dimers.
Although similar, a notable difference between the two assemblies’ conformations has been found: tetrameric structures display a sterol shaped density (likely cholesterol) between protomers (TM5 and TM6 of one and TM1 and TM7 of the other), which is absent in the trimeric form. These findings suggest that the presence or absence of lipid molecules regulates receptor oligomerization.
In summary, this detailed structural study provides valuable insights into CXCR4 structures, enhancing our understanding of its ligand-binding and oligomerization processes. Additionally, the first-ever visualization of CXCR4 higher-order oligomeric assemblies highlights how oligomerization could influence allosteric modulation. Further studies are needed to better understand the contribution of higher-order oligomerization to receptor activation. This would create opportunities for novel drug discovery and development.
Reference
Saotome K, McGoldrick LL, Ho JH, Ramlall TF, Shah S, Moore MJ, Kim JH, Leidich R, Olson WC, Franklin MC. Structural insights into CXCR4 modulation and oligomerization. Nat Struct Mol Biol. 2025 Feb;32(2):315-325. doi: 10.1038/s41594-024-01397-1. Epub 2024 Sep 23. PMID: 39313635; PMCID: PMC11832422.