Structural Investigation of the Central Components of the CD4+ T Cell Immunological Synapse - Project Summary After nearly four decades since the first crystal structure of a human MHC was published, it is still unclear how exactly T cells engage and become activated by peptide-MHC (pMHC) presented on host cells. T cells are a cornerstone of the immune system, and the interface between T cells and host cells consists of an ensemble of adhesion and signaling proteins known as the “immunological synapse.” Central to this interface are pMHC and its binding partners—the T cell receptor (TCR)-CD3 complex and coreceptor CD4 or CD8. The coreceptors can greatly amplify TCR binding and activation, and a variety of mechanisms such as cooperativity, allostery, and oligomerization have been proposed. Efforts to uncover this mystery through structural studies have resulted in atomic-resolution structures of the individual proteins, allowing for theoretical structural models of pMHCII engaging TCR-CD3 and CD4. However, the theoretical models have failed to explain how CD4 contributes to enhanced TCR binding and signal transduction; therefore, experimentally determined structures of immunological synapse are needed to shed light on this critical component of the immune system. In this proposal, established methods, as well as new developments in engineering enveloped virus-like particles (eVLPs), will be leveraged to determine novel experimental structures of the central components of the immunological synapse. The hypothesis is that the simultaneous binding of full-length TCR-CD3 and CD4 to pMHCII induces biologically relevant structural changes that are not apparent in the current model obtained by superimposing the protein complexes. In order to address this gap, in Aim (1) we will use cryo-electron microscopy (cryo-EM) to determine the structure of pMHCII engaged with full-length TCR-CD3 and CD4. While detergents are necessary to stabilize membrane proteins for cryo-EM, it would be beneficial to visualize and compare the structures in a more native environment. Therefore, Aim (2) will use cryo- EM and cryo-electron tomography (cryo-ET) to determine the structure of membrane-anchored pMHCII engaged to TCR-CD3 and CD4 using enveloped virus-like particles (eVLPs). An endosomal sorting complex required for transport (ESCRT) and ALG-2-interacting protein X (ALIX)- binding region (EABR) motif will be appended to the intracellular tail of each protein, prompting the self-assembly and budding of eVLPs containing pMHCII and separately eVLPs containing TCR-CD3 and CD4. Once initial structures are obtained for each aim, CD4 variants that affect binding affinity or kinase Lck recruitment will be introduced and structurally investigated. These data will reveal how T cells engage pMHCII and will inform future strategies of modulating T cell activation for targeted disease treatment.
