Molecular architecture of the αβ T cell receptor-CD3 complex

Abstract

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.

Keywords: T-cell receptor; electron microscopy; small-angle X-ray scattering.

Fig. 1.

Generation of a stable TCR–CD3εδ complex and analysis via SAXS. (A) Cartoon depicting the TM domain-based assembly of TCRα with CD3εδ. The polar Asp (marked as “D”) and Lys (marked as “K”) residues that mediate assembly are depicted. Transmembrane α-helices are represented by circles. (B) The parallel heterotrimeric coiled coil used to link TCR and CD3εδ viewed down the long axis of the helices. (C) The ab initio model of LC13 TCR–CD3εδ shown in two orientations. (D) Overlay of the LC13 TCR–CD3εδ ab initio model (light gray spheres) with a representative CORAL model in which the TCR (α, blue; β, cyan), CD3 (ε, red; δ, orange) and coiled coil (black) are represented as dots. (E) Independent LC13 TCR–CD3εδ models predicted via CORAL. (F) CORAL-constrained model for the LC13 TCR–CD3εδ complex bound to OKT3 Fab and HLA-B44 pMHC. In E and F, potential N-linked glycosylation sites in the TCR and CD3 subunits are indicated by asterisks.

Fig. 2.

Production and characterization of full-length 1G4 TCR–CD3 complex. (A) Schematic for expression of 1G4 TCR–CD3 complex in HEK-293 cells via coinfection of baculoviruses. The baculoviruses respectively encode for TCRα/β and CD3ε/γ/δ/ζ, with each polypeptide chain separated by a viral 2A peptide (P2A). Each CD3 subunit contains a Rhinovirus 3C protease cleavage site (dashed yellow line) to remove intracellular domains after protein expression. (B) Expression of folded TCR–CD3 complex on HEK-293 cells as demonstrated by an anti-TCR antibody (Left), anti-CD3ε antibody (Center), and cognate pMHC (Right). A high-affinity TCR allows for staining of monomeric pMHC. (C) Equal staining for orthogonal epitope tags on the N termini of CD3γ and CD3γδ indicate 1:1 incorporation of CD3εγ and CD3εδ into the TCR–CD3 complex. (D) Size-exclusion chromatography for the TCR–CD3 complex bound by pMHC. (E) Western blot of size-exclusion chromatography fractions of the pMHC–TCR–CD3 complex peak shows staining for pMHC (via anti-β2m antibody) and CD3γ/δ (via anti-His antibody). The blot was simultaneously treated with both primary antibodies. (F) SDS-PAGE gel of final 1G4 TCR–CD3–ESO–A2 material showing presence of TCR, CD3, and MHC. CD3ζ is not visible because of its small size after protease cleavage (5 kDa).

Fig. 3.

Negative-stain EM of soluble pMHC–1G4–TCR complex (A), membrane-bound pMHC–1G4–TCR–CD3 complex (B), and pMHC-1G4–TCR–CD3 complex decorated with anti-CD3 Fab (C) indicates a dimeric membrane-bound TCR–CD3 complex. (Left) Cartoon representation of the domain structure of each complex. Representative 2D class averages for each complex (Top Right) are oriented to a model based on the solved crystal structure of the pMHC–TCR and CD3εδ–UCHT1 complexes (Bottom Right). Red circles (B and C) are density noted in the class averages not accounted for by the TCR or pMHC ECDs, likely consisting of the CD3 ECDs and the TCR/CD3 transmembrane helices. The side length of the class averages in A is 25.6 nm, and the side length of the class averages in B and C is 42.6 nm.

Fig. 4.

Proposed mechanism for TCR–CD3 signaling. TCR binding of pMHC ligand may induce a reorientation of the TCR–CD3 subunits, which would propagate through the TCR–CD3 ECDs and TM domains, exposing the CD3 ICDs for activation. It is also possible that pMHC (or other Ag) itself induces dimerization of the TCR–CD3 complex.