TABLE 2.
Characteristics of high-scoring residues of hMC4R
| Res | Status | Effect | SNAPMAT | Loc | Hyd | Chg | Size | Cons | SSt | SA | Bn | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| L31 | Predicted | Neu | −58 | Ex | F | U | L | Position | E | B | −0.6 | |
| N62 | Mut: S | Imp | 66 | Tr | L | U | M | Res: 98% | L | B | −0.4 | |
| N72 | Predicted | Imp | 50 | Cy | L | U | M | Position | L | I | 0.3 | |
| P78 | Predicted | Imp | 57 | Cy | U | U | S | Res: 63% | H | B | −0.3 | |
| D90 | Confirmed | Imp | 59 | Tr | L | N | M | Res: 85% | H | B | −0.7 | |
| G98 | Predicted | Neu | −54 | Tr | U | U | S | Position | L | B | 0.3 | |
| T110 | Predicted | Neu | −56 | Ex | U | U | M | None | L | I | 1.7 | |
| D146 | Confirmed | Imp | 58 | Cy | L | N | M | Res: 79% | L | B | −0.6 | |
| R147 | Confirmed | Imp | 64 | Cy | L | P | L | Res: 91% | H | B | −0.5 | |
| Y157 | Mut: S | Imp | 57 | Cy | U | U | L | Res: 61% | L | B | −0.5 | |
| H158 | Predicted | Imp | 54 | Cy | U | P | L | None | E | B | −0.4 | |
| V163 | Predicted | Neu | −56 | Cy | F | U | L | None | H | I | −0.1 | |
| R165 | Mut: A/Q/W | Imp | 63 | Cy | L | P | L | None | H | B | −0.3 | |
| W174 | Predicted | Imp | 55 | Tr | F | U | L | Res: 45% | H | B | −0.9 | |
| F201 | Conflict: A | Imp | 57 | Tr | F | U | L | Res: 56% | H | B | −0.7 | |
| Y212 | Predicted | Imp | 52 | Tr | U | U | L | Position | H | B | −0.6 | |
| H214 | Conflict: A | Imp | 53 | Tr | U | P | L | None | L | B | 0.1 | |
| M215 | Predicted | Imp | 52 | Tr | F | U | L | Position | L | B | −0.5 | |
| A219 | Mut: V | Imp | 54 | Cy | U | U | S | Position | H | B | −0.4 | |
| H222 | Predicted | Imp | 59 | Cy | U | P | L | None | H | B | −0.7 | |
| C257 | Predicted | Imp | 62 | Tr | F | U | L | Res: 62% | H | B | −0.6 | |
| W258 | Mut: A | Imp | 61 | Tr | F | U | L | None | H | B | −0.7 | |
| C271 | Mut: A/Y/R | Imp | 53 | Tr | F | U | L | None | E | B | −0.7 | |
| N294 | Predicted | Imp | 52 | Tr | L | U | M | Position | L | B | −0.4 | |
| P299 | Mut: H | Imp | 52 | Tr | U | U | S | Res: 96% | L | B | −0.4 | |
| Y302 | Predicted | Imp | 60 | Tr | U | U | L | Res: 94% | E | B | −1.1 | |
| G324 | Predicted | Neu | −55 | Cy | U | U | S | None | L | E | 2.2 |
Table shows various features of residues predicted with high reliability as either neutral or important to hMC4R function (SNAPMAT≤−50 or SNAPMAT≥50, respectively). Res column indicates the WT residue and its position in sequence. Status indicates experimental status of the residue: predicted, no experimental data; conflict: X, experimental functional annotation of effect of substitution of WT residue by X is in conflict with prediction (Supplemental Table 1); Mut: X, experimental functional annotation of effect of substitution of WT residue by X confirms prediction (Supplemental Table 1); confirmed, experimental data (including but not limited to mutagenesis) confirms prediction (Supplemental Table 9). Effect prediction is derived from the SNAPMAT score (Neu, neutral/negative; Imp, important to function/positive). Loc indicates cellular location of the residue (Ex, extracellular; Tr, transmembrane; Cy, cytoplasmic), taken from the Swiss-Prot (25) MC4R_HUMAN entry. Rest of the values are extracted as described for SNAP method (Supplemental Fig. 1 and ref. 23). Hyd inciates residue hydrophobicity (F, hydrophobic; U, unassigned; L, hydrophilic), Chg indicates charge (N, negative; U, uncharged; P, positive), and Size indicates surface area (S, small; M, medium; L, large). Cons indicates conservation (whether the residue is conserved in alignment). Values are Res: X% (WT residue conserved in X%≥45% of sequences; position (residue not conserved, but at most 3 highest scoring residues make up ≥55% of the distribution), none (position is not conserved). Predicted values are SSt, 3-state secondary structure (H, helix; L, loop; E, extended strand), SA, 3-state solvent accessibility [B, buried (0–9% solvent accessible), I, intermediate (9–36%), E, exposed (>36%)], and Bn, normalized B value reflecting residue flexibility [where values are −3 (rigid)≤Bn≤3 (flexible)].