TABLE 2.
Affinity constants and variations in free energy of interaction of wild-type and modified MT7 toxins for wild-type and mutated hM1 receptors
Data were inferred from binding experiments with [3H]NMS and calculated with the ternary complex equation. The free energy of interaction (ΔΔGint) values were calculated from ΔΔGint(A-B) = ΔΔG(A*;B*) − ΔΔG(A*) − ΔΔG(B*). ΔΔGint values exceeding 0.7 kcal/mol are in bold and indicate proximity between the two mutated residues. Data are mean values ± S.E. of at least three experiments (see “Experimental Procedures”).
| hM1 receptor | hM1 region | MT7 toxin | MT7 loop | pKX | ΔΔGint |
|---|---|---|---|---|---|
| kcal/mol | |||||
| WT | WT | 10.47 ± 0.04 | |||
| H90A | E1 | WT | 10.66 ± 0.20 | ||
| W10A | 1 | 11.62 ± 0.28 | −2.08 ± 0.40 | ||
| Y30A | 2 | 9.55 ± 0.28 | +1.14 ± 0.37 | ||
| Y51A | 3 | 10.42 ± 0.13 | +0.20 ± 0.11 | ||
| R52A | 3 | 10.42 ± 0.44 | −0.04 ± 0.59 | ||
| W91A | E1 | WT | 9.30 ± 0.06 | ||
| W10A | 1 | 7.69 ± 0.05 | +1.09 ± 0.07 | ||
| Y30A | 2 | 7.81 ± 0.07 | +1.64 ± 0.09 | ||
| Y51A | 3 | 8.79 ± 0.17 | +0.44 ± 0.24 | ||
| R52A | 3 | 8.54 ± 0.13 | +0.66 ± 0.17 | ||
| W101A | TM3 | WT | 9.79 ± 0.13 | ||
| W10A | 1 | 8.57 ± 0.14 | +0.57 ± 0.19 | ||
| Y30A | 2 | 9.08 ± 0.09 | +0.60 ± 0.11 | ||
| R34A | 2 | 7.74 ± 0.10 | −0.37 ± 0.21 | ||
| R52A | 3 | 9.05 ± 0.19 | +0.62 ± 0.25 | ||
| E170A | E2 | WT | 10.20 ± 0.07 | ||
| W10A | 1 | 8.42 ± 0.20 | +1.33 ± 0.25 | ||
| Y30A | 2 | 8.84 ± 0.28 | +1.47 ± 0.38 | ||
| S32A | 2 | 8.63 ± 0.16 | +1.43 ± 0.22 | ||
| R34A | 2 | 7.50 ± 0.36 | +0.60 ± 0.49 | ||
| M35A | 2 | 8.63 ± 0.13 | +0.87 ± 0.17 | ||
| Y36A | 2 | 7.97 ± 0.13 | +1.24 ± 0.17 | ||
| Y51A | 3 | 9.71 ± 0.31 | +0.41 ± 0.42 | ||
| R52A | 3 | 8.57 ± 0.18 | +1.77 ± 0.27 | ||
| R171A | E2 | WT | 9.50 ± 0.29 | ||
| K5A | 1 | 9.25 ± 0.18 | −0.10 ± 0.24 | ||
| W10A | 1 | 7.76 ± 0.18 | +1.26 ± 0.24 | ||
| Y30A | 2 | 8.22 ± 0.14 | +1.49 ± 0.43 | ||
| S32A | 2 | 8.46 ± 0.08 | +0.72 ± 0.11 | ||
| M35A | 2 | 7.68 ± 0.46 | +1.20 ± 0.62 | ||
| Y36A | 2 | 7.03 ± 0.21 | +1.55 ± 0.28 | ||
| K48A | 2 | 9.48 ± 0.02 | −0.13 ± 0.02 | ||
| Y51A | 3 | 8.67 ± 0.12 | +0.89 ± 0.16 | ||
| R52A | 3 | 8.35 ± 0.17 | +1.17 ± 0.23 | ||
| L174A | E2 | WT | 10.51 ± 0.01 | ||
| W10A | 1 | 9.19 ± 0.20 | +0.71 ± 0.27 | ||
| Y30A | 2 | 9.45 ± 0.17 | +1.08 ± 0.23 | ||
| S32A | 2 | 9.66 ± 0.20 | +0.48 ± 0.27 | ||
| R34A | 2 | 7.52 ± 0.13 | +1.19 ± 0.42 | ||
| M35A | 2 | 8.72 ± 0.10 | +1.16 ± 0.13 | ||
| Y36A | 2 | 8.59 ± 0.23 | +0.83 ± 0.31 | ||
| Y51A | 3 | 10.06 ± 0.23 | +0.37 ± 0.31 | ||
| R52A | 3 | 9.23 ± 0.22 | +1.35 ± 0.29 | ||
| Y179F | E2 | WT | 9.25 ± 0.18 | ||
| K5A | 1 | 9.15 ± 0.15 | −0.28 ± 0.20 | ||
| W10A | 1 | 7.62 ± 0.19 | +1.13 ± 0.25 | ||
| R34A | 2 | <6 | > +1.35 | ||
| K48A | 2 | 9.21 ± 0.01 | −0.09 ± 0.01 | ||
| R52A | 3 | <6 | >+4.00 | ||
| K392A | E3 | WT | 10.57 ± 0.18 | ||
| W10A | 1 | 9.88 ± 0.22 | −0.13 ± 0.29 | ||
| F11A | 1 | 10.19 ± 0.20 | +0.27 ± 0.27 | ||
| D393A | E3 | WT | 10.50 ± 0.23 | ||
| S8A | 1 | 10.05 ± 0.06 | +0.21 ± 0.08 | ||
| W10A | 1 | 10.10 ± 0.02 | −0.53 ± 0.02 | ||
| F11A | 1 | 10.21 ± 0.24 | +0.35 ± 0.09 | ||
| Y51A | 3 | 10.00 ± 0.34 | +0.42 ± 0.46 | ||
| E397A | E3 | WT | 10.54 ± 0.20 | ||
| W10A | 1 | 8.64 ± 0.22 | +1.58 ± 0.31 | ||
| F11A | 1 | 9.89 ± 0.21 | +0.64 ± 0.28 | ||
| W400A | TM7 | WT | 9.43 ± 0.02 | ||
| K5A | 1 | 9.60 ± 0.05 | −0.66 ± 0.07 | ||
| W10A | 1 | 7.61 ± 0.32 | +1.38 ± 0.44 | ||
| F11A | 1 | 8.29 ± 0.25 | +1.29 ± 0.33 | ||
| Y30A | 2 | 8.49 ± 0.08 | +0.90 ± 0.10 | ||
| S32A | 2 | 8.64 ± 0.15 | +0.38 ± 0.20 | ||
| R34A | 2 | <6 | >+1.58 | ||
| M35A | 2 | 8.52 ± 0.13 | −0.11 ± 0.12 | ||
| Y36A | 2 | 7.37 ± 0.29 | +1.00 ± 0.38 | ||
| K48A | 3 | 9.41 ± 0.06 | −0.12 ± 0.07 | ||
| R52A | 3 | 7.73 ± 0.08 | +0.91 ± 0.17 | ||
| E401A | TM7 | WT | 10.20 ± 0.10 | ||
| R34A | 2 | 7.41 ± 0.37 | +0.61 ± 0.45 |