Analysis of CD3 sequence divergence indicates that CD3γ, CD3δ, and CD3ε genes arose from a common ancestor in a two-step process of gene duplication (38
). Mammals have three CD3 genes (γ, δ, and ε), whereas nonmammalians (birds, fish, and amphibians) have only two: a CD3γδ precursor and a CD3ε gene. Protein sequence comparison indicates that each CD3γ and CD3δ subunit evolved with highly homologous heterodimeric interfaces and membrane proximal segments for efficient and specific signaling transfer when paired with CD3ε. The compact orientation of the CD3γ FG loop and single horizontally attached glycan in the mouse is a feature of the CD3εγ heterodimer that affords lateral support of the C region domains for αβ as well as γδ TCRs. In addition, CD3εγ appears to have adapted to optimally interact with the Cβ FG loop. Our findings that the elongation of the structured Cβ FG loop coevolved with appearance of the CD3γ gene from a CD3γ/δ precursor are strong support for this notion. By contrast, the more vertical CD3δ FG loop trajectory and greater number of N-
linked glycan adducts in CD3εδ heterodimers assume a more extended geometry that cannot fit into the homologous TCRβ–CD3εγ interaction site (15
). The CD3εδ disposition on the TCRα “side” of the complex occupies intervening space between the coreceptor (CD4 or CD8) and the αβ heterodimer (44
). This bulky CD3εδ component may also be entropically advantageous to help preconfigure the coreceptor as a TCR, pMHC, and coreceptor ternary complex forms.
The crystal structure of a γδ TCR heterodimer reveals a Cγ–Cδ domain symmetry, in contradistinction to the Cα–Cβ domain asymmetry observed in αβ TCRs (16
). The γδ TCR heterodimer also differs by lacking an elongated Cβ FG loop equivalent. γδ TCR lineage commitment is associated with more robust signaling relative to that of the αβ TCR, with greater TCR copy number and/or ligand density likely affecting γδ T lineage signaling strength (22
). In contrast, αβ TCR pMHC ligands are present at low levels, mandating additional TCR modifications to compensate for weak signals promoting αβ T cell fate and function. During immune surveillance, continued cell movement following ligation of the TCR αβ clonotype by specific pMHC fosters quaternary change; the Cβ FG loop interacts with CD3εγ on one side of the TCR and the Cα domain with the bulky CD3εδ heterodimer on the other. It is the tangential rather than normal (i.e., perpendicular to the membrane) directional force that triggers TCR activation post-pMHC ligation, as shown by optical tweezer experiments (15
Our current results emphasize how the αβ TCR quaternary structure is optimal for surface expression and signaling. gives a side view of the surface-exposed TCR complex based upon existing structural information of individual components and molecular modeling (8
). The substantial N-
linked glycosylation of TCR subunits is indicated by the brown space-filling molecular representations. On the TCRβ “side,” the Cβ FG loop (17
) and the compact orientation of the CD3γ FG loop (, Supplemental Fig. 2
) are key features contributing to the asymmetry optimizing TCR signaling. Lateral movement of the TCRαβ heterodimer can apply a torque on CD3εγ via the Cβ FG loop appendage. schematically demonstrates that the extended CD3δ subunit ectodomain would sterically clash with the Cβ FG loop above, whereas that of CD3γ or the chimeric heterodimer does not. On the TCRα “side,” the bulky glycans and vertical CD3δ FG loop disposition may also likewise relay quaternary change to CD3εδ after tangential force-induced torque. Alternatively, the torque on CD3εδ could be applied through the highly conserved connecting peptide at the base of the TCRCα domain (30
). Further studies aimed at rigidifying or derigidifying segments of the TCR complex, without altering pMHC binding, should show an impact on TCR signaling, consistent with a mechanosensor mechanism of action.
The ability of the hCD3δ ectodomain to pair with mCD3ε and foster TCR complex expression signaling, as well as murine thymocyte development, might appear, at first glance, contradictory to our view that CD3γ and CD3δ ectodomains evolved to occupy a different side of the TCR complex. However, that is not the case. Isologous subunit ectodomain substitution is not permitted, whereas the heterologous hCD3δ ectodomain can replace that of mCD3γ. That functional substitution, as noted in Supplemental Fig. 4
, is possible because mCD3ε has a Thr residue in lieu of Phe89 in hCD3ε, allowing the top of the G-strand of hCD3δ, when paired with mCD3ε, to avoid steric clash with the TCRβ subunit. We emphasize that the geometry of mCD3γ and mCD3δ G-strand N-terminal residues (residues 70–75 and residues 58–63) are distinct from each other, as are the corresponding segments in the respective human orthologs.
As structural and functional analyses of these and other Ig-like domains of receptors become more sophisticated, additional subtleties and their biological implications will be revealed. The details as described in this study for CD3 heterodimers demonstrate the important functional consequences of structural evolution. Understanding these differences will help with elucidating the function of multisubunit receptors, such as the TCR.