Effects of the MR1 E76Q E149Q salt bridge mutations on the binding affinities of AF-7 TCR, E8 TCR, and E8 TRBV6-1 TCR
| . | MR1 Wt . | MR1 E76Q E149Q . | ||||
|---|---|---|---|---|---|---|
| TCR . | 5-OP-RU . | 6-FP . | 5-FSA . | 5-OP-RU . | 6-FP . | 5-FSA . |
| AF-7 | 1 ± 0.02 μM | >150 μM | >150 μM | 0.6 ± 0.03 μM | >150 μM | >150 μM |
| E8 | 1.3 ± 0.1 nM | 0.6 ± 0.07 μM | 0.2 ± 0.03 μM | 11.2 ± 1.5 nM | 3.5 ± 0.2 μM | 1.8 ± 0.2 μM |
| E8 TRBV6-1 | 80.3 ± 8.3 nM | 10.8 ± 0.7 μM | 6.9 ± 0.9 μM | 0.6 ± 0.05 μM | 42.1 ± 2.6 μM | 32.1 ± 3.7 μM |
| . | MR1 Wt . | MR1 E76Q E149Q . | ||||
|---|---|---|---|---|---|---|
| TCR . | 5-OP-RU . | 6-FP . | 5-FSA . | 5-OP-RU . | 6-FP . | 5-FSA . |
| AF-7 | 1 ± 0.02 μM | >150 μM | >150 μM | 0.6 ± 0.03 μM | >150 μM | >150 μM |
| E8 | 1.3 ± 0.1 nM | 0.6 ± 0.07 μM | 0.2 ± 0.03 μM | 11.2 ± 1.5 nM | 3.5 ± 0.2 μM | 1.8 ± 0.2 μM |
| E8 TRBV6-1 | 80.3 ± 8.3 nM | 10.8 ± 0.7 μM | 6.9 ± 0.9 μM | 0.6 ± 0.05 μM | 42.1 ± 2.6 μM | 32.1 ± 3.7 μM |
Binding affinities, as measured by surface plasmon resonance, of the AF-7, E8 TCR, and E8 TRBV6-1 TCR interacting with wildtype MR1 and MR1 E76Q E149Q refolded with 5-OP-RU, 6-FP, and 5-FSA. >150 μM: the measured KD of the TCR MR1 interaction was >150 μM and therefore is unlikely to generate a MAIT cell response. KD are indicated with the standard error in brackets. The binding affinity of the E8 TCR to MR1 5-OP-RU and MR1 E76Q E149Q 5-OP-RU was measured using the BIAcore8K using single-cycle kinetic analysis. The remining measurements were completed on a BiacoreT200 and the KDs were calculated using steady-state analysis.