Structure

Crystallization

HLA-DR4 expresses the self-peptide MBP which is complimentary to the human autoimmune TCR MS2-3C8 which is an α/βTCR. The HLA-DR4 of the MS2-3C8–MBP–DR4 complex binds the D1 domain of the CD4 mutant to form a complex which spontaneously. 

MBP is loosely held in the HLA-DR4 complex, however MBP - TCR MS2-3C8 bind tightly due to the presence of a unique hyper-variable region (CDR3β). The optimal binding of antigen to TCR compensates for the weak interactions between the antigen and MHCII (Y. Yin, 2011).

A crystal is an array of periodically repeating identical protein molecules ordered in 3D, the molecules are linked by a variety of different interactions through a solvent. The presence of solvent is vital as the crystals must be kept in an aqueous environment. Once in excess of 20mg of the pure ternary complex protein has been isolated by flow cytometry, the protein will be concentrated to 5-20mg/ml. This concentrated sample will then have its solubility decreased until it crystalizes. This can be done easily using sparse matrix commercial random screening kits. If the crystallization procedure is successful x-ray crystallography can commence as per described method.

X-ray crystallography

Performing x-ray crystallography on the ternary complex crystal and molecular replacement using the MS2-3C8–MBP–DR4–CD4 as search models the ternary structure of TCR-pMHC-CD4 was recovered to a resolution of 4Å (Fig.5). 


The original structure obtained included carbohydrates bound to the D3 and D4 domains of CD4.The carbohydrates visualized are bound to the only N-linked glycosylation sites found in human CD4. These are Asn271 and Asn300 and are located in the D3 and D4 domains.

Figure 5: X-ray crystallographic structure of TCR-pMHC-CD4 ternary complex with CD4 
CD4 in red/orange, MHC in blue/turquoise and TCR in yellow/green

CD4 structure

The unbound conformation of CD4 had already been obtained (see introduction). CD4 has a very ridged structure. This has been determined by examining 7 different structures of unbound CD4 compared to the bound structure of CD4. A super imposition of MHC bound CD4 onto an unbound CD4 shows very few structural differences between the two conformations. These differences amount to an 11° change at the D2-D3 junction and a 4° change at the D3-D4 junction. It can therefore be assumed that any variations observed in the ternary complex are as a result of MHC.

CD4 undergoes conformational changes, different to those depicted thus far, upon the binding of its D1 domain to gp120 on the surface of HIV-1 protein. Again, due to the rigid structure of CD4 only the loop containing the Phe43 residue showed a structural change, the impact of this was a reduction in mobility. This binding of Phe43 loop to receptive cavity of gp120, evokes the onset of HIV by initiating the signals required for viral entry into targeted cell (S-T. Hsu, A. Bonvin. 2004).

Crystal structure of unbound CD4 depicts CD4 as a D4-D4 dimer, which some say contributes to T-cell activation. There are 2 theories which disprove the idea that CD4 dimerizes to assist in T-cell activation:

• The crystal structure of the ternary complex does not display CD4 as a dimer .

• The vertical distance between the D4 C terminus and the T-cell surface is too great to be spanned by the short CD4 stalk (8 residues).

 MHC and TCR structure (see introduction)