Instability and mutagenesis

Instability

The TCR-pMHC-CD4 ternary complex is largely unstable which has proven problematic for solving the structure by x-ray crystallography.

The stability of an interaction is determined by obtaining K_D values for the interacting molecules.

• pMHC-CD4, Kd = 200µM – 2mM
• TCR-pMHC, Kd = 1µM – 100µM

These high K_D values are indicative of low affinity binding.

Mutagenesis of CD4

There are 4 domains of the CD4 co-receptor (D1-D4). The distal domain, D1, is responsible for the low affinity interactions with MHCII. By increasing the affinity of the CD4-pMHC interactions x-ray crystallography may be possible, this can be done by in-vitro random mutagenesis.

The CD4 D1 domain was subjected to in-vitro random mutagenesis, to induce point mutations. Fluorescently labelled mutants were used to perform flow cytometry with HLA-DR1 and allow the mutants with highest affinity for HLA-DR1 to be isolated. HLA-DR1 is a serotype of HLA-DR, hence displays the same affinity for CD4 as HLA-DR MHCII.

Choosing the mutant - SPR

A mutant containing two substitutions from the wild type was isolated: Gln 40 was substituted for Tyr and Thr 45 was substituted for Trp. This CD4 mutant had a marked increase in affinity for HLA-DR1 and HLA-DR4. The affinity increase was measured by Surface Plasmon Resonance (SPR). Calculating the root-mean-square of HLA-DR4 – CD4 superposition onto a mouse and wild type complex proved that affinity maturation did not affect CD4 – MHC binding.

Mutant has low K_D:

• CD4 mutant-HLA-DR1, Kd = 8.8µM
• CD4 mutant-HLA-DR4, Kd = 10.1µM

The HLA-DR1/4 is immobilized onto a gold film and above the gold film is a grating. A beam of light is shone through the grating onto the gold film and reflects back out of the grating onto a detector. The angle of incidence of the light beam is altered until the resonance angle is reached, where by the plasmons in the gold film absorb some of the light energy and resonate to form an evanescent electric field. This leads to a decrease in wavelength of the reflected light that will be detected. The high affinity CD4 mutant can be flowed past the immobilized HLA-D1/4 molecules and bind, this alters the resonance angle. A buffer is then washed past to dissociate the bound CD4 mutant. This is repeated at eight different concentrations of the CD4 mutant. The change in resonance angles in the association and dissociation of each mutant concentration can be used to calculate k_on and k_off and ultimately K_D. The results from the SPR experiment for the characterisation of the mutant HLA-DR4 is shown in Figure 4 below.

 

Figure 4: Surface Plasmon Resonance results 
Surface plasmon resonance of increasing concentrations of the CD4 mutant
(0.3, 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, and 37.5 μM) binding to immobilized HLA-DR4.