Structural Biology Of Human Chemokine Receptor CCR8

Cancer has been a serious threat to human health and life safety.With the rapid development of biomedical technology,tumor immunotherapy has been successfully applied in a variety of cancers,but a large number of tumor-infiltrating Treg cells may hinder the effect of tumor immunotherapy.Chemokine receptor CCR8 is specifically expressed on tumor-infiltrating Treg cells,and targeting CCR8 can selectively depletes tumor-infiltrating Treg cells,thus enhancing the immune response.Therefore,CCR8 is a potential tumor therapeutic target,and structural studies on CCR8 can accelerate the development of relevant anti-tumor drugs.In this study,X-ray crystal diffraction technique will be used to explore the structural biology of the complex of CCR8 and small molecule inhibitor(inactive state)to reveal the binding mode between them.On the other hand,the structure of CCR8-Gi complex(active state)will be analyzed by single particle cryo-electron microscopy to elucidate the activation mechanism of CCR8.By constructing different clonal vectors,using insect cell expression system and metal ion affinity chromatography,we have successfully expressed and purified CCR8 protein with high stability and good homogeneity for crystal screening of CCR8 and small molecule inhibitor complex.In addition,CCR8-the endogenous ligand CCL1-Gi protein complex and CCR8-the small molecule agonist LMD-009-Gi protein complex have been purified.At the same time,we have assembled the two CCR8-Gi complexes in nanodiscs.It is expected to obtain high resolution structures of CCR8 in the near future.In conclusion,we have successfully screened and optimized the clons of CCR8.CCR8-CCL1-Gi protein complex and CCR8-LMD-009-Gi protein complex have been successfully expressed and purified.This study is helpful to pave the way for subsequent structural analysis of inactive state and active state of CCR8.