Abstract
A major unanswered question is how a TCR discriminates between foreign and self-peptides presented on the APC surface. Here, we used in situ fluorescence resonance energy transfer (FRET) to measure the distances of single TCR–pMHC bonds and the conformations of individual TCR–CD3ζ receptors at the membranes of live primary T cells. We found that a TCR discriminates between closely related peptides by forming single TCR–pMHC bonds with different conformations, and the most potent pMHC forms the shortest bond. The bond conformation is an intrinsic property that is independent of the binding affinity and kinetics, TCR microcluster formation, and CD4 binding. The bond conformation dictates the degree of CD3ζ dissociation from the inner leaflet of the plasma membrane via a positive calcium signaling feedback loop to precisely control the accessibility of CD3ζ ITAMs for phosphorylation. Our data revealed the mechanism by which a TCR deciphers the structural differences among peptides via the TCR–pMHC bond conformation.
Highlights
A TCR consists of a variable TCRαβ heterodimer and a nonvariable transmembrane signal transduction CD3 complex containing CD3γε and CD3δε heterodimers and a CD3ζζ homodimer
fluorescence resonance energy transfer (FRET) design To determine the conformation of a single TCR–pMHC bond, a TCR and a pMHC were site- labeled with the FRET acceptor Cy5 and the FRET donor Cy3, respectively.[8]
SmFRET only measured the conformational dynamics of single TCR–pMHC bonds during the bound state, which are independent of those of unbound molecules and bond association/dissociation
Summary
A TCR consists of a variable TCRαβ heterodimer and a nonvariable transmembrane signal transduction CD3 complex containing CD3γε and CD3δε heterodimers and a CD3ζζ homodimer. A typical example of this is that the in situ binding kinetics and affinities of TCR–pMHC interactions measured at the T-cell membrane are dramatically different from those measured in vitro in solution.[3,8] It has long been speculated that the TCR at the cell membrane undergoes conformational changes upon pMHC binding This hypothesis is attractive, but it has never been experimentally proven at the membranes of live primary. S1–4 and Movies S1–3 measure the intermolecular distance of a TCR–pMHC bond and the intramolecular conformation of a TCR–CD3ζ complex at the immunological synapse of a live primary CD4+ T cell in real time with high spatiotemporal resolution These experiments enabled us to critically test the TCR conformational change model and probe the molecular mechanism underlying TCR ligand discrimination
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