Extended Data Table 1 |. Ligand affinity data for MT2 mutants.
Data were acquired with MT2 wild-type (WT) and mutants expressed in HEK293T cells by radioligand competition binding using [3H]-melatonin (0.2–0.8 nM, unless otherwise indicated) to yield Kd or Ki affinity estimates. Data represent mean ± s.e.m. for n independent experiments as indicated in square brackets. Crystal constructs (CC) were expressed in Sf9 cells. ND, not determined. Binding isotherms are shown in Supplementary Figure 2. For determining the effect of NaCl, binding assays were performed in the presence of 147 nM NaCl (binding isotherms in Supplementary Figure 3).
| Mutant | melatonin | 2-pmt | ramelteon | agomelatine |
|---|---|---|---|---|
| Kd, nM [n] (pKd ±s.e.m.) |
Ki, nM [n] (pKi ±s.e.m.) |
Ki, nM [n] (pKi ±s.e.m.) |
Ki, nM [n] (pKi ±s.e.m.) |
|
| WT | 0.54
[10] (9.27±0.12) |
0.17
[10] (9.78±0.11) |
0.23
[4] (9.66±0.10) |
0.24
[4] (9.63±0.08) |
| WT + NaCI | 1.56 [6] (8.81 ± 0.18) |
ND | ND | ND |
| MT2-CC (Sf9) | 63.10
[3] (7.20±0.06) |
3.14
[4] (8.50±0.06) |
2.60
[4] (8.59±0.02) |
6.88 [4] (8.16± 0.05) |
| MT2-CC (sf9) + NaCI | 48.23
[3] (7.32±0.03) |
ND | ND | ND |
| MT2-CC-N862.50D (Sf9) | 29.40
[6] (7.53±0.34) |
6.46
[4] (8.19±0.15) |
7.37
[4] (8.13±0.09) |
26.76
[4] (7.57±0.05) |
| MT2-CC-H2085.46A (Sf9) | 10.81
[6] (7.97±0.16) |
3.57
[6] (8.45±0.03) |
2.03
[6] (8.69±0.13) |
4.98
[6] (8.30±0.04) |
| D86N (2.50) | 5.80
[3] (8.24±0.04) |
0.33
[3] (9.48±0.12) |
ND | ND |
| D86N (2.50) + NaCI | 3.26
[3] (8.49±0.09) |
ND | ND | ND |
| L108F (ECL1) | 0.94 [3] (9.03±0.11) |
0.13 [3] (9.87±0.15) |
ND | ND |
| F129W (3.41) | 2.84 [3] (8.55±0.04) |
0.22 [3] (9.65±0.08) |
ND | ND |
| N137D (3.49) | 1.24 [3] (8.91±0.13) |
0.12 [3] (9.92±0.00) |
ND | ND |
| C140L (3.52) | 0.21
[3] (9.68±0.05) |
0.03
[3] (10.50±0.02) |
ND | ND |
| W264F (6.48) | 0.88
[3] (9.06±0.14) |
0.06
[3] (10.25±0.26) |
ND | ND |
| A305P (7.50) | 3.94
[3] (8.40±0.19) |
0.47
[3] (9.32±0.06) |
ND | ND |
| N312D (7.57) | 2.85
[3] (8.54±0.07) |
0.36
[3] (9.44±0.04) |
ND | ND |
| P95A (2.59) | No specific binding up to 7 nM [3H]-melatonin | |||
| M120A(3.32) | 0.44
[3] (9.42±0.16) |
0.028
[3] (10.7±0.3) |
0.055
[3] (10.28±0.09) |
0.052
[3] (10.35±0.18) |
| N175A(4.60) | 0.86 [3] (9.2±0.3) |
0.09
[3] (10.06±0.06) |
0.08
[3] (10.12±0.11) |
0.25
[3] (9.60±0.04) |
| F192A (ECL2) | Low expression, no specific binding up to 7 nM [3H]-melatonin | |||
| F192I (ECL2) | No specific binding up to 7 nM [3H]-melatonin | |||
| Q194A (ECL2) | 0.62 [3] (9.4±0.3) |
0.043
[3] (10.38±0.07) |
0.051
[3] (10.4±0.2) |
0.12
[3] (9.94±0.09) |
| Y200A (5.38) | 0.63 [3] (9.3±0.3) |
0.14
[3] (9.86±0.01) |
0.19
[3] (9.73±0.03) |
0.67
[3] (9.18±0.02) |
| A203F(5.41) | 0.82
[5] (9.09±0.01) |
0.12
[5] (9.94±0.06) |
0.19
[5] (9.47±0.19) |
0.42
[5] (9.37±0.19) |
| H208A (5.46) | 1.24
[3] (8.94±0.13) |
0.17
[3] (9.79±0.09) |
0.18
[3] (9.77±0.11) |
0.22
[3] (9.68±0.11) |
| N268A (6.52) | 0.96 [3] (9.3±0.4) |
0.09
[3] (10.08±0.09) |
0.12
[3] (9.92±0.05) |
0.20
[3] (9.69±0.03) |
| Y294A (7.39) | 1.07
[3] (8.99±0.09) |
0.042
[3] (10.38±0.04) |
0.049
[3] (10.33±0.09) |
0.10
[3] (10.04±0.10) |
| Y308S (7.53) | No specific binding up to 7 nM [3H]-melatonin | |||