Intramembrane organization of T cell receptor-CD3 complex assembly

The T cell receptor (TCR) recognizes peptides presented by MHC molecules and delivers signals that are crucial at all stages of T cell development and function. The disulfide-linked TCR[alpha][beta] heterodimer binds ligands and delivers signals via cytoplasmic tyrosine-based sequence motifs contributed by the associated CD3[delta][varepsilon], CD3[gamma][varepsilon] and [zeta][zeta] dimers. While the structures of soluble fragments representing all globular ectodomains are known, the stoichiometry and structural organization of subunits within the intact complex had not been determined. Persisting uncertainties on these points led to the proposal of multiple models of TCR-CD3 complex composition that are mutually exclusive and have distinct implications for the mechanism of signal transduction. We addressed these issues using a modified in vitro translation system that provides distinct technical advantages over the cellular systems utilized in previous studies. Combining this flexible experimental platform with novel techniques for isolating intact membrane protein complexes, we made the first direct measurements of receptor composition and found that the receptor is monovalent with a stoichiometry of TCR[alpha][beta]:CD3[delta][varepsilon]:CD3[gamma][varepsilon]:[zeta][zeta]. Conserved basic and acidic residues in TCR-CD3 TM regions had been proposed to contribute to assembly through formation of intramembrane salt bridges. Our in vitro mutagenesis demonstrated that instead these residues organize TCR-CD3 assembly through formation of triad groupings consisting of one basic residue from the TCR and a pair of acidic residues from a particular signaling dimer. This motif strictly required a specific steric arrangement, and mutagenesis experiments indicated that the acidic pair represented a single functional unit. We subsequently found that this mechanism of intramembrane assembly was utilized by a broad array of modular activating receptors expressed by hematopoietic lineage cells, and in all cases "assembly potential" was focused at the intramembrane site of the basic-acidic-acidic triad. The high-resolution structure of the [zeta][zeta] TM homodimer confirmed that the acidic pair packs closely within the dimer interface and creates the binding site for the receptor through a complex intermolecular hydrogen bond network which includes one or more structured water molecule(s). These studies provided novel insights into the architecture of the intact TCR-CD3 complex and the first atomic-resolution structural information for the membrane-embedded domains that organize TCR-CD3 assembly.