Table 1.
Natural selection analysis of S2-MacroD and prediction of functional consequences.
| residue | variant(s)a,b | total no. variants | no. of sequencesc | occurrencec | conservation scored | classificatione | speciesb |
|---|---|---|---|---|---|---|---|
| pervasive positive | |||||||
| Asp218 | Glu(325), Asn(8), Tyr(2), Gly(1) | 336 | 75 640 | yes | 2 | N, SE | Glu: Bat-CoV-HKU9, HCoV-EMC, hMacroD1, hMacroD2 Asn: AfuMacroD, Bat-CoV-HKU4 Tyr: n.f. Gly: CelMacroD, EriCoV-1, FCoV, RuV, TGV |
| Pro340 | Ser(108), Leu(81) | 189 | 75 664 | yes | 1 | N | Ser: ChRCoV-HKU24, HCoV-OC43, MgrMacroD Leu: AfuMacroD, AfuYmdB, FCoV, HCoV-229E, TGV |
| Gly282 | Val(97), Cys(12), Ser(4) | 113 | 75 646 | no | 3 | N | — |
| His295 | Tyr(53), Arg(4) | 57 | 75 666 | yes | 1 | N, SE | Tyr: AfuYmdB, DppMacroD, hMacroD1, hMacroD2, ISKNV, LchMacroD, LisYmdB, SACIV, TRBIV Arg: AthMacroD, AtrMacroD, HCoV-229E, PpaMacroD |
| Thr350 | Ile | 55 | 75 659 | yes | 3 | N, SE | SARSr-CoV-HKU3-13 |
| Thr237 | Ile(19), Ala(6), Pro(4) | 29 | 75 667 | yes | 3 | N, SE | Ile: n.f. Ala: Bat-CoV-HKU9 Pro: Hp-betaCoV |
| Ala333 | Val | 17 | 75 665 | yes | 6 | N | Bat-CoV-512, FCoV, TGV |
| Leu292 | Phe | 13 | 75 667 | no | 0 | Df (Phe) | — |
| Asp309 | Asn(3), Gly(2) | 5 | 75 668 | yes | 0 | N | Asn: HEV Gly: EEEV, GETV, ONNV, RuV, RRV, SDV, TONV, VEEV, WEEV |
| Ala243 | Val | 4 | 75 669 | no | 11 | Ag | — |
| episodic | |||||||
| Glu206 | Lys(23), Ile(1) | 24 | 75 666 | yes | gap | N, SE | Lys: n.f. Ile: DppMacroD, RoBat-CoV GCCDC1 |
| Asn276 | Arg(6), Ile(1), Thr(1), Ser(1) | 9 | 75 664 | yes | gap | N, SE | Arg: AthMacroD, ChRCoV-HKU24 Ile: n.f. Thr: Bulbul-CoV Ser: FCoV, PDCoV, PpaMacroD |
| pervasive negative | |||||||
| Leu287h | n.a. | n.a. | 75 669 | ||||
| Leu326h | n.a. | n.a. | 75 669 | ||||
| Leu357h | n.a. | n.a. | 75 669 | ||||
| Val228h | n.a. | n.a. | 75 669 | ||||
| Ala254h | n.a. | n.a. | 75 669 | ||||
| Ser332h | n.a. | n.a. | 75 668 | ||||
| Glu229 | Gly(7), Asp(2), Ala(1) | 10 | 75 668 | yes | 2 | N, SE | Gly: GETV, RRV Asp: ChRCoV-HKU24, RuV Ala: Bat-CoV-512 |
| Ala242 | Val | 5 | 75 665 | no | 7 | Ai | — |
| Leu297 | Phe(3), Ile(1) | 4 | 75 663 | yes | 7 | Df (Phe) | Phe: AfuYmdB, HCoV-229E, HCoV-NL63 Ile: AfuMacroD, AthMacroD, AtrMacroD, CelMacroD, DppMacroD, EEEV, GETV, HEV, hMacroD1, hMacroD2, ISKNV, LisYmdB, MmaYmdB, MgrMacroD, ONNV, PpaMacroD, RRV, RuV, SACIV, SDV, TONV, TRBIV, VEEV, WEEV |
| Asn305 | Asp(1), Tyr(1), Ile(1), Ser(1) | 4 | 75 661 | yes | 2 | N, SE | Asp: AthMacroD, AtrMacroD Tyr: CelMacroD, hMacroD1 Ile: n.f. Ser: AfuYmdB, EEEV, GETV, ONNV, PDCoV, RRV, SACIV, TRBIV, WEEV |
| Pro302 | Gln | 3 | 75 668 | no | 10 | Df | — |
| Val355 | Ala | 3 | 75 668 | no | 7 | N | — |
| Asp366 | Asn(2), Glu(1) | 3 | 75 666 | yes | 1 | N, SE | Asn: Bat-CoV-HKU4, PpaMacroD, Glu: AfuYmdB, Bat-CoV-HKU9, BtRI-betaCoV, CelMacroD, EriCoV-1, LchMacroD, MgrMacroD, SACIV, SARS-CoV, TRBIV, Whale-CoV-SW1 |
| Leu257 | Ile(1), Phe(1) | 2 | 75 669 | yes | 9 | Df (Phe) | Ile: AfuMacroD, AfuYmdB, AthMacroD, AtrMacroD, Bat-CoV-HKU4, Bulbul-CoV, CelMacroD, ChRCoV-HKU24, DppMacroD, EcoYmdB, EriCoV-1, FCoV, HCoV-EMC, HCoV-HKU1, HCoV-NL63, HCoV-OC43, hMacroD1, hMacroD2, Hp-betaCoV, IBV, ISKNV, LchMacroD, LisYmdB, MHV-A59, MgrMacroD, MmaYmdB, PpaMacroD, PDCoV, RuV, SACIV, TGV, TRBIV Phe: HEV |
aNo. of occurrences given in parenthesis.
bn.a., not applicable; n.f., not found.
cNo. of sequences used for codon analysis (variability due to sequencing quality).
doccurrence and conservation score based on full alignment (electronic supplementary material, figure S2); conservation scores 11 and 10 are reported on the full alignment as * and +, respectively; gap: insertion/deletion in the sequence alignment.
eN, neutral; D, destabilizing (main destabilizing variant given in parenthesis); A, ADPr binding interface; SE, surface-exposed (defined using the findSurfaceResidues module in PyMOL with a cut-off value of 2.5 Å2, i.e. atoms with > cut-off Å2 exposed to solvent are considered exposed).
fThe protein variants L257I/F, L292F, L297I/F and P302Q will most likely destabilize the structure/folding of the protein due to clashing with neighbour residues, with only slight impact expected for the Leu-to-Ile variants due to the comparable physico-chemical properties.
gAla243 is part of the conserved NAAN motif and situated in the catalytic pocket. It is the α-face of the distal ribose. The increase in Van der Waals volume associated with the A243V variant may destabilize the ligand binding.
hSequences were picked up due to the occurrence of silent mutations.
iAla242 is part of the conserved NAAN motif situated in the catalytic pocket. It is facing the ADPr phosphates. The distance between the A242V variant and the phosphates is reduced from 3.5–4.3 Å to 2.1–2.9 Å, most likely destabilizing the ligand binding.