Structural Studies Of Chemokine Receptor CCR5 In Complex With Leukocidin E

G protein-coupled receptors（GPCRs）are the largest membrane protein family of eukaryotes,which trigger different downstream signaling pathways and regulate a wide range of cellular functions by recognizing multiple signaling molecules.Chemokines are a family of proteins with chemotactic function,they form a concentration gradient in vivo and bind chemokine receptors to induce cellular directional migration.The chemokine receptor CCR5 belongs to Class A GPCR family,which is mainly expressed on the surface of T cells,macrophages and dendritic cells,etc.CCR5 is involved in regulating biological processes such as migration,proliferation and inflammatory response of immune cells by binding a variety of endogenous chemokine ligands.In addition,CCR5 can also bind various pathogen-related proteins,such as the surface glycoprotein gp120 of human immunodeficiency virus-1（HIV-1）and leukocidin E（LukE）secreted by Staphylococcus aureus,thereby promoting the infection of pathogens to human bodies.Staphylococcus aureus is a Gram-positive bacterium that causes extremely high rates of morbidity and mortality worldwide.The pathogenesis is related to various virulence factors,some of which can help bacteria to escape from the host immune system and increase their virulence.Previous studies have shown that LukE,a toxin secreted by S.aureus,can specifically bind the chemokine receptor CCR5 on the surface of immune cells to promote the pore formation on the cell membrane,ultimately lead to cell rupture and death.At present,drug resistance of S.aureus is becoming increasingly serious,the development of drugs targeting CCR5 can improve the ability of the host immune system to resist bacterial infection,which provides a new antimicrobial intervention for solving antibiotic resistance.This study aims to determine the complex structure of LukE and its receptor CCR5by using X-ray crystallography,and clarify their fine interaction mode,which provides structural basis for the development of antibacterial drugs targeting CCR5.By screening different fusion proteins inserted into the third intracellular loop of CCR5 and optimizing junction sites,we obtained complex samples with high quality.Subsequently,crystallization trials of the complex were carried out using the lipid cubic phase（LCP）method.Crystals of the complex were obtained by extensively screening and optimizing crystallization conditions.Currently,the optimal X-ray diffraction resolution is 6（?）,which lays a good foundation for structure determination of the CCR5-LukE complex.Further efforts will continue to be made to solve the structure of the complex,obtain their interaction information and elucidate the immune escape mechanism of S.aureus mediated by CCR5.Another study in this article is aimed at the ligand recognition and activation mechanism of the chemokine receptor CX3CR1.CX3CR1 is the only member of the CX3C subfamily of chemokine receptors and binds its only endogenous ligand,CX3CL1.CX3CL1-CX3CR1 axis maintains tissue homeostasis and also plays a key role in pathological conditions such as atherosclerosis,neurodegenerative diseases and cancer.Different from the redundancy of other chemokine ligand-receptor binding,the unique ligand recognition mechanism of CX3CR1 gives it significant drug development potential.In recent years,researchers have reported a number of drugs that target CX3CL1-CX3CR1 axis,but no drugs have yet been approved for marketing.Based on the complex structures of CX3CR1-G_i1and CX3CL1-CX3CR1-G_i1,we designed and constructed a series of CX3CR1 mutants for verifying the role of key amino acids in cholesterol binding sites and in the interaction interface between the receptor and G protein.The expression data show that all mutants had high expression levels in mammalian cells.These results will provide the necessary functional research information for elucidating the ligand recognition and activation mechanisms of CX3CR1.