TABLE 1.
Site-directed mutagenesis data
The figures for binding and displacement experiments are written in boldface type if significant changes were observed upon mutation of the receptor.
| CCR5 mutantsa | 2D7 geometric MFI (% WT-CCR5) | CTC5 geometric MFI (% WT-CCR5) | [125I]CCL3 binding (% WT -CCR5) | [35S]gp120 binding (% WT-CCR5) | MVC |
||
|---|---|---|---|---|---|---|---|
| [3H]MVC binding KDb | Displacement of [125I]CCL3 IC50b | ||||||
| TM1 | Y37A | 115 ± 13 | 106 ± 6.9 | 14 ± 1.1 (13)c | 41 ± 7.5 (37)c | 0.80 | 2.2 ± 1.3 |
| TM1 | Y37F | 86 ± 28 | 103 ± 11 | 78 ± 10 (88) | 53 ± 1.5 (59) | 1.10 | 0.9 ± 0.2 |
| TM2 | W86A | 117 ± 0.4 | 112 ± 1.6 | 40 ± 2.1 (35) | <10 | 42.9 ± 13 | |
| ECL1 | W94A | 140 ± 15 | 121 ± 7.3 | <10 | <10 | 0.79 ± 0.3 | |
| ECL1 | W94M | 103 ± 7.5 | 108 ± 3.4 | <10 | <10 | 1.18 ± 0.1 | |
| ECL1 | M100A | 114 ± 14 | 122 ± 17 | 95 ± 1.0 (82) | 127 ± 21 (107) | 1.0 ± 0.1 | |
| ECL1 | M100T | 79 ± 9.3 | 86 ± 10 | 42 ± 4.1 (52) | 53 ± 1.5 (65) | 0.63 | 1.3 ± 0.3 |
| TM3 | L104A | 131 ± 18 | 113 ± 1.7 | 81 ± 6.3 (67) | 127 ± 19 (97) | 1.07 | 0.9 ± 0.2 |
| TM3 | L104F | 128 ± 17 | 107 ± 8.2 | 43 ± 3.8 (37) | 53 ± 21 (46) | 2.05 | 1.2 ± 0.2 |
| TM3 | Y108A | 65 ± 21 | 92 ± 8.0 | 44 ± 13 (49) | <10 | 75.5 ± 6.4 | |
| TM3 | F109A | 91 ± 7.3 | 82 ± 6.8 | 159 ± 1.0 (186) | 23 ± 11 (27) | 1.43 | 1.4 ± 0.2 |
| TM3 | F109H | 106 ± 6.1 | 105 ± 18 | 179 ± 23 (174) | 24 ± 0.7 (23) | 1.5 ± 0.1 | |
| TM3 | F112A | 21 ± 1.5 | 28 ± 2.3 | 23 ± 7.6 (101) | 31 ± 7.6 (121) | 0.4 ± 0.2 | |
| TM3 | F112Y | 125 ± 25 | 112 ± 31 | 104 ± 19 (89) | 169 ± 3.6 (115) | 1.1 ± 0.3 | |
| TM4 | G145A | 137 ± 2.7 | 130 ± 6.1 | 103 ± 11 (78) | 150 ± 17 (113) | 0.60 | 1.3 ± 0.3 |
| ECL2 | R168A | 77 ± 8.7 | 67 ± 11 | 47 ± 8.5 (64) | 45 ± 11 (72) | 1.3 ± 0.5 | |
| ECL2 | T177A | 92 ± 2.2 | 118 ± 4.3 | 13 ± 4.4 (13) | 8.1 ± 6.1 (7.6) | 1.70 | 1.4 ± 0.5 |
| ECL2 | T177Q | 82 ± 13 | 98 ± 9.2 | 13 ± 1.6 (15) | 18 ± 6.7 (22) | 0.79 | 1.3 ± 0.2 |
| ECL2 | C178A | 0 ± 0 | 74 ± 37 | <10 | <10 | ||
| ECL2 | S180A | 124 ± 1.1 | 120 ± 13 | 85 ± 14 (69) | 139 ± 19 (115) | 1.10 | 0.9 ± 0.0 |
| ECL2 | S180P | 85 ± 1.7 | 94 ± 2.0 | 58 ± 8.9 (65) | <10 | 0.56 | 0.9 ± 0.1 |
| ECL2-TM5 | Y184A | 160 ± 37 | 134 ± 23 | 33 ± 7.7 (23) | 133 ± 27 (122) | 2.1 ± 0.7 | |
| ECL2-TM5 | Y187A | 88 ± 10 | 112 ± 8.5 | <10 | 144 ± 8.0 (161) | 1.19 ± 0.5 | |
| ECL2-TM5 | F189A | 57 ± 3.9 | 73 ± 4.4 | 71 ± 2.6 (110) | <10 | 1.5 ± 0.0 | |
| ECL2-TM5 | W190A | 22 ± 13 | 84 ± 46 | 16 ± 0.4 (19) | <10 | 1.6 ± 0.8 | |
| ECL2-TM5 | Q194A | 105 ± 4.5 | 110 ± 2.0 | 84 ± 5.8 (78) | 162 ± 40 (150) | 0.43 | 0.6 ± 0.2 |
| ECL2-TM5 | Q194H | 104 ± 1.4 | 113 ± 4.7 | 85 ± 6.4 (78) | 184 ± 40 (169) | 2.40 | 5.6 ± 0.9 |
| TM5 | I198A | 124 ± 14 | 118 ± 12 | 130 ± 4.7 (109) | 49 ± 0.8 (41) | 17.40 | 131 ± 29 |
| TM6 | W248A | 3.6 ± 2.3 | 14 ± 0.4 | <10 | <10 | ||
| TM6 | Y251A | 10 ± 2.4 | 31 ± 0.1 | 13 ± 4.8 (64) | <10 | 7.7 ± 2.3 | |
| TM6 | Y251F | 68 ± 0.8 | 80 ± 3.6 | 88 ± 7.8 (120) | 38 ± 9.3 (52) | 3.3 ± 1.2 | |
| TM6 | Y251I | 4.8 ± 2.1 | 20 ± 1.8 | 14 ± 2.2 (119) | <10 | 19.9 ± 2.1 | |
| TM6 | N252A | 101 ± 2.5 | 86 ± 3.9 | 46 ± 5.2 (49) | 92 ± 29 (98) | 0.80 | 1.1 ± 0.4 |
| TM6 | N252I | 141 ± 14 | 137 ± 8.4 | 16 ± 0.8 (12) | 147 ± 6.2 (106) | 0.44 | 1.2 ± 0.3 |
| TM6 | L255A | 39 ± 2.5 | 41 ± 0.5 | 49 ± 0.1 (123) | 30 ± 2.5 (76) | 2.3 | 0.9 ± 0.5 |
| TM6 | N258A | 91 ± 5.5 | 86 ± 8.6 | 68 ± 4.5 (77) | 123 ± 5.9 (140) | 0.48 | 0.4 ± 0.3 |
| TM6-ECL3 | N258M | 85 ± 6.0 | 87 ± 0.3 | 71 ± 0.4 (82) | 85 ± 3.3 (98) | 1.27 | 0.7 ± 0.2 |
| TM6-ECL3 | N258Q | 138 ± 2.9 | 141 ± 7.2 | 81 ± 2.4 (58) | 149 ± 28 (107) | 1.10 | 1.2 ± 0.2 |
| TM6-ECL3 | F260A | 40 ± 5.1 | 60 ± 15 | 46 ± 3.8 (97) | 13 ± 13 (32) | 0.8 ± 0.0 | |
| TM7 | Q280A | 106 ± 6.2 | 110 ± 2.0 | 84 ± 6.6 (78) | 128 ± 21 (119) | 0.74 | 1.1 ± 0.1 |
| TM7 | E283A | 70 ± 4.3 | 75 ± 8.1 | 21 ± 3.7 (30) | <10 | undd | >10,000 |
| TM7 | E283Q | 92 ± 3.2 | 99 ± 6.6 | 33 ± 11 (35) | 26 ± 12 (28) | undd | >10,000 |
a The seven transmembrane helices of CCR5 are defined as follows: TM1 from Ile-28 to Asn-57; TM2 from Met-64 to Gln-93; TM3 from Asn-98 to Val-130; TM4 from Thr-143 to Ile-165; TM5 from Lys-191 to Gly-216; TM6 from Arg-230 to Leu-255; and TM7 from Gln-277 to Tyr-297. The first, second, and third extracellular loops are defined using TM boundaries (ECL1 between TM2 and TM3, ECL2 between TM4 and TM5, and ECL3 between TM6 and TM7). ECL2-TM5 and TM6-ECL3 are ECL2 and ECL3 and are expected to adopt a helical structure according to CXCR4 x-ray structure.
b Fold change is compared with WT-CCR5 (KD = 0.69 ± 0.26 nm, lC50 = 1.05 ± 0.61 nm).
c Numbers in parentheses are corrected with the cell-surface expression level of CCR5 mutants compared with that of WT-CCR5 (for the CCR5 mutants Y108A and W190A, binding of radioligands was corrected with their receptor expression level determined using the CTC5 mAb only).
d Data were undetectable.