| The alpha/beta T cell receptor (TCR) binds to a major histocompatibility complex/peptide antigen and initiates signal transduction through a set of constitutively-associated modules: CD3-epsilon/gamma, CD3-epsilon/delta, and TCR-zeta. Whereas extracellular contacts are sufficient for interactions within TCR and CD3 heterodimers, transmembrane interactions are necessary for assembly and surface expression of intact TCR/CD3 complexes. Extracellular domains of CD3 may provide additional specificity to mandatory transmembrane interactions, but specific extracellular interactions between TCR and CD3-epsilon/delta or CD3-epsilon/gamma have yet to be determined. In an effort to address the function of CD3 extracellular domains and their interaction with the T cell receptor, structural studies of CD3 dimers were conducted.; Human CD3-epsilon, CD3-delta, and CD3-gamma constructs were expressed in E. coli as insoluble inclusion bodies and refolded by rapid dilution into dimers. Stable CD3-epsilon/gamma heterodimers, but not CD3-epsilon/delta, could be refolded from several ectodomain constructs. CD3-epsilon/delta dimers could be formed by fusing a coiled-coil to the c-terminal end of each component or by refolding in the presence of a single chain fragment of the antibody UCHT1 (UCHT1-scFv), which recognizes heterodimeric CD3, but not CD3-epsilon homodimers. A 1:1:1 complex of CD3-epsilon/delta/UCHT1-scFv was purified, crystallized, and used for structure determination.; The crystal structure of a complex of CD3-epsilon/delta/UCHT1 was determined at 1.9A resolution. CD3-epsilon/delta and CD3-epsilon/gamma share a conserved interface between the immunoglobulin-fold ectodomains, with parallel packing of the two G strands. CD3-delta has a more electronegative surface and a more compact immunoglobulin fold than CD3-gamma; thus, the two CD3 heterodimers have distinctly different molecular surfaces. The UCHT1 antibody binds near an acidic region of CD3-epsilon opposite the dimer interface, occluding this region from direct interaction with the TCR.; TCR-alpha/beta ectodomain dimers were produced and used in binding experiments with CD3-epsilon/gamma and CD3-epsilon/delta ectodomains. As no measurable affinity was observed, we estimate that the affinity is weaker than 100--200 muM, the practical limit of measurable affinities for the methods used. Thus we determine that the affinity of TCR and CD3 extracellular domains in solution is very weak and conclude that future attempts to isolate TCR/CD3 complexes for structural studies will likely require inclusion of the transmembrane domains. |
