Structural Determination

• The TCR-pMHC-CD4 complex has an intrinsic instability because of the very weak affinity of the pMHC-CD4 interaction (even weaker than TCR-pMHC). Therefore, crystallizing the complex proved to be a challenge
• This difficulty was overcome by using in vitro random mutagenesis of CD4. The resulting mutant CD4 library was sorted by flow cytometry with MHC (HLA-DR1) to isolate CD4 mutant variants with higher affinity for MHC and produced from insect cells vector.
• Unlike the wild-type CD4 which was not detected to bind to HLA-DR1, the mutant CD4 bound to HLA-DR1 with  K_D = 8.8 µM and with similar affinity (measured by surface plasmon resonance, see Materials and Methods) The mutant CD4 variant was used for cocrystallization with HLA-DR4 bearing a self-peptide from myelin basic protein (MBP), and human autoimmune TCR (MS2-3C8).
• MS2-3C8 binds to its ligand by canonical docking mode of αβ mode. The affinity of TCR for MHC is at the high end of the range for TCR-pMHC interactions (see the SPR experiment) , so is used.

Figure: SPR analysis of binding affinity of CD4 mutant to HLA-DR4 , Yin Weng et. al 2012

• Remarkbably,  mutant CD4 containing TCR-pMHC-CD4 complex crystallized spontaneously without the aid of precipitants.
• The structure was determined to 4 A^o resolution by molecular replacement using the MS2-3C8-MBP-DR4-CD4 complex and despite the moderate resolution, it was sufficient to interpret out the electron density and map out this clear entire TCR-pMHC-CD4 complex. In addition, for the carbohydrates attached to CD4 residues (Asn271 and Asn 300 in domains D3 and D4), the electron density was visible. these Asn271 and Asn300 domains are the only two N-linked glycosylation sites in human CD4 as shown in the figure below.

Figure: The structure of TCR-pMHC-CD4 complex  (click to zoom in) .  Yin Weng et. al 2012

Figure: Pymol Structure of TCR-pMHC with visible ASn271 and Asn300 in CD4 D3 and D4