Research On Structural And Assembly Of The Human TCR-CD3 Complex

T cells are a critical component of the human adaptive immune system and have a critical role in pathogen infections,cancer and autoimmune diseases.The T cell receptor TCR is an antigen receptor expressed on the surface of T cells,and initiates T cell-mediated immune response by recognition of antigen peptides bound to major histocompatibility complex(p MHC).The TCR does not have the function of signaling,TCR carry out downstream signaling through its co-receptor CD3 signalling apparatus,TCR-CD3 complex is formed through noncovalent association between TCR and CD3.The TCR-CD3 complex comprises an antigen-recognizing module of the TCR?? heterodimer and a CD3-signalling apparatus containing the dimeric subunits CD3??,CD3?? and CD3??.Engagement of TCR by p MHC induces TCR signaling activation.Through a series of signal transmission,then activates several signalling pathways involving transcription factors such as activator protein 1(AP-1),nuclear factor-?B(NF-?B)and nuclear factor of activated T cells(NFAT).Signalling events downstream of these pathways include T cell activation,proliferation,cytokine production and effector functions.In the last two decades,several models of TCR-CD3 triggering have been proposed,but the underlying mechanism is unknown.Many structural studies have been conducted on the ECDs of the TCR-CD3 complex,but the organization and structure of the entire TCR-CD3 complex remain unknown.Such structural information is important for better understanding of T cell activation and the development of effective immunotherapy directed against the complex.In this study,the problem that the TCR-CD3 complex is difficult to express and purify to obtain a uniform stable complex is solved,genes encoding the full-length sequences of the TCR chains were amplified from a human c DNA library,sub-cloned into the p CAG vector and separated by E2A-peptide consensus motifs.The full-length sequences of CD3 subunits,in which each individual chain was separated by P2 Apeptide consensus motifs,was sub-cloned into the pc DNA3.4 vector.The six subunits constituting the TCR-CD3 complex were co-expressed by mammalian cell expression system,the TCR-CD3 complex with high expression and biological activity was screened,and each subunit was verified by immunoblotting.Further the TCR-CD3 complex were crosslinked by addition of chemical crosslinking agent glutaraldehyde,the glutaraldehyde crosslinking has no effect on the thermal stability of the TCR-CD3 complex and the affinity with the specific antibody,after the glutaraldehyde crosslinking the biological activity of the TCRCD3 complex did not change.The results of negative staining showed that the TCR-CD3 complex particles after glutaraldehyde crosslinking were evenly distributed,no obviously aggregation,and the particles of the same size,while the uncrosslinked TCR-CD3 complex had smaller particles,so the cryo-electron microscopy data was collected using the crosslinked TCR-CD3 complex.We report a cryo-electron microscopy of human TCR?? incomplex with the CD3 octameric at 3.7 ? resolution.The structure contains the complete extracellular domains and all the transmembrane helices of TCR-CD3,the overall structure of the TCR-CD3 complex is shaped like an ice cream cone.The octameric TCR-CD3 complex is assembled with 1:1:1:1 stoichiometry of TCR??:CD3??:CD3??:CD3??.Assembly of the extracellular domains of TCR-CD3 is mediated by the constant domains and connecting peptides of TCR?? that pack against CD3??-CD3??,forming a trimer-like structure proximal to the plasma membrane and the TCR? subunit is at the center.Moreover,the formation of disulfide bonds between the linker peptides of the subunit membrane regions of each subunit plays an important role in maintaining the stability of the conformation of the TCR-CD3 complex.The transmembrane region of the TCR-CD3 complex forms a barrel-like structure composed of eight transmembrane helices.The transmembrane helices of TCR?? subunit is located at the center of the barrel-like structure,and TCR-CD3 complex is stabilized by hydrophobic interaction and electrostatic interaction.Comparing the structure of TCR-CD3 complex with TCR??-p MHC and OKT3-CD3??,the binding of p MHC and OKT3 induces no marked conformational changes in TCR-CD3 complex.The comparison also shows that p MHC and OKT3 occupy completely different positions when interacting with TCR-CD3,and we find the potential interactions sites of TCRCD3 with the OKT3.In summary,this study reports the high-resolution structure of TCR-CD3 complex,reveals the structural basis for TCR-CD3 complex assembly,and provides a key to explain the molecular mechanism of initiating immune response after ligands bind to TCR,providing clues to TCR triggering and a foundation for rational design of immunotherapies that target the complex.