Effect of charged residues on SARS-CoV-2 sequence
Here D, E, H, R, K took as a charged residues and C, S, T, N, Q, Y, W took as uncharged polar residues. Amino acid compositions were calculated from the non-block format whereas block format was used to calculate disorder forming residues (Dis), order forming residues (Ofr), bulkiness, aliphatic index (AI) and polarity. GRAVY (grand average of hydropathy) is calculated by adding the hydropathy value46 for each residue and dividing by the length of the protein sequence. Is there a preference for amino acids in SARS-CoV-2 relative to SARS? To find that answer, we calculate all those physicochemical properties.
Spike proteins showed higher abundance (Fig. 1) of charged residues (except D) in SARS-CoV-2. Polar residues showed higher quantity (except T, W) in SARS-CoV-2. In nucleoproteins of SARS-CoV-2 D, K and R shows higher abundance and E, H shows lower abundance as charged amino acids. Polar residues in nucleoproteins also showed higher abundance (except T, N) in SARS-CoV-2. Surprisingly C is absent in both groups of sequence in nucleoproteins. Other proteins i.e. membrane proteins and ORF proteins showed almost similar abundance with those previous results. The number of disorder forming residues has higher abundance in SARS-CoV-2 than SARS. The number of order forming residues has lower abundance in SARS-CoV-2 than SARS. The higher number of disorder forming residues in SARS-CoV-2 indicates that it can easily create toxicity or disease in humans. The lower value of GRAVY (except nucleoproteins) indicates the hydrophilic nature of SARS-CoV-2. So, it can be easily mixed with aqueous or liquid medium. The aliphatic index is high in every SARS-CoV-2 proteins. High value of the aliphatic index in SARS-CoV-2 proved that SARS-CoV-2 is more thermally stable than SARS47.
When we check the polarity of those proteins, it showed slightly high values in SARS-CoV-2 proteins than SARS (Fig. 2). Due to the latter, bulkiness is also high in SARS-CoV-2 than SARS. The high value of bulkiness in SARS-CoV-2 indicates that they need longer heating periods in hydrolysis48. They can tolerate heat better than SARS. The Kyte-Dollitle hydrophobicity scale indicates that the SARS-CoV-2 is hydrophilic in nature (Fig. 3). The hydrophilic nature of SARS-CoV-2 gives a clue that it can easily interact with water or aqueous medium and spread easily than SARS49-50. The intrinsic disorder regions are very much high in SARS-CoV-2 than SARS. High abundance of intrinsic disorder regions of SARS-CoV-2 indicates that it will more interact with other proteins than SARS51-52.
Abundance of charged residues in helix of SARS-CoV-2
The building blocks of proteins i.e. amino acids are found in four positions of secondary structure; coil, helix, sheet, and turn.
Table 2. Amino acid abundance in protein secondary structures (turn, helix, coil and sheet) of SARS-CoV-2 (5R80) and SARS (2H2Z)
|
SARS-CoV-2 (5R80)
|
SARS (2H2Z)
|
|
Charged
|
Polar
|
Hydrophobic
|
Charged
|
Polar
|
Hydrophobic
|
Turn
|
1.63
|
2.45
|
2.86
|
0.90
|
2.28
|
3.59
|
Helix
|
11.47
|
5.32
|
14.75
|
8.16
|
3.92
|
14.70
|
Coil
|
2.04
|
8.60
|
11.47
|
1.63
|
5.88
|
8.16
|
Sheet
|
9.01
|
19.67
|
10.65
|
6.53
|
16.01
|
28.10
|
Charged residues showed higher abundance in every position (turn, helix, coil and sheet) of SARS-CoV-2 (Table 2) than SARS. Charged residues show higher abundance within the helix of both proteins. Introduction of high number charged residues in the helix results in proteins more resistant to the acidic environment or temperature denaturation and helps in increasing the stability53-54. Hydrophobic residues have higher abundance in SARS (except coil) than SARS-CoV-2. Polar residues also show higher abundance in SARS-CoV-2 than SARS.
Intra-protein interactions effect on stability of SARS-CoV-2
Salt bridges have a significant effect on protein stability55-58. Charged residues are participating in the formation of salt bridges. Normally two types of salt bridges are found in proteins, i.e. isolated salt bridge and network salt bridge. The increasing number of charged residues of SARS-CoV-2 indicates that charged residues might affect salt bridge formation to gain more stability. Other intra-protein interactions like, metal ion binding site59, aromatic-aromatic interactions60-61 are also helps in protein stabilization.
SARS-CoV-2 has large pocket area than SARS (Fig. 4), which gives it more protein-protein or protein-ligand interactions possibilities (Table 3). Volume of the protein is also high in SARS-CoV-2 than SARS. Protease from SARS-CoV-2 possess 7 isolated salt bridges and 1 network salt bridges, whereas SARS protease has 5 isolated and 1 network salt bridges. Result indicates that SARS-CoV-2 is highly stabilized by those salt bridges. Number of metal ion binding site is also high in SARS-CoV-2 than SARS. Free solvation energy is a thermodynamic factor that determines protein salvation or nature of denaturation62. By this property we can determine how fast proteins easily denature. Solvation free energy is also high in SARS-CoV-2 than SARS which indicates the SARS-CoV-2 protein not easily denature in contact with solvent.
Table 3. Volume, pocket area, isolated salt bridges (ISB), network salt bridges (NSB), metal binding site (MBS) and solvation free energy (ΔGsolv) of SARS-CoV-2 (5R80) and SARS (2H2Z)
Protein
|
Volume
|
Area
|
ISB
|
NSB
|
MBS
|
Solvation Free Energy (ΔGsolv)
|
5R80
|
669.47
|
1013.45
|
7
|
1
|
3
|
4786.55 Kcal/mol
|
2H2Z
|
228.27
|
835.26
|
5
|
1
|
2
|
3266.89 Kcal/mol
|
Aromatic-aromatic interactions show high number in SARS-CoV-2 than SARS (Table 4). Not only number, those residue (Phe8, Tyr37, Phe103, Tyr101, Phe150, Phe159) which participate in aromatic-aromatic interactions are forming a very long network, which is never been reported in any proteomics research. SARS-CoV-2 has 9 isolated and 1 network aromatic-aromatic interactions where as SARS has only 9 isolated aromatic-aromatic interactions.
Table 4. Aromatic-aromatic interactions of of SARS-CoV-2 (5R80) and SARS (2H2Z)
Protein
|
Position
|
Residue
|
Position
|
Residue
|
D(centroid-centroid)
|
Dihedral Angle
|
SARS-CoV-2 (5R80)
|
3
|
PHE
|
291
|
PHE
|
4.96
|
130.9
|
8
|
PHE
|
150
|
PHE
|
6.77
|
45.38
|
37
|
TYR
|
103
|
PHE
|
5.27
|
88.61
|
101
|
TYR
|
103
|
PHE
|
5.7
|
133.64
|
101
|
TYR
|
159
|
PHE
|
6.78
|
118.46
|
103
|
PHE
|
159
|
PHE
|
6.44
|
74.2
|
112
|
PHE
|
161
|
TYR
|
5.41
|
142.21
|
126
|
TYR
|
140
|
PHE
|
6.38
|
64.71
|
134
|
PHE
|
182
|
TYR
|
6.33
|
164.01
|
150
|
PHE
|
159
|
PHE
|
6.43
|
56.09
|
161
|
TYR
|
182
|
TYR
|
6.47
|
150.59
|
218
|
TRP
|
219
|
PHE
|
5.96
|
104.81
|
SARS (2H2Z)
|
3
|
PHE
|
300
|
CYS
|
4.72
|
116
|
54
|
TYR
|
44
|
CYS
|
4.29
|
52.78
|
66
|
PHE
|
22
|
CYS
|
4.61
|
29.56
|
112
|
PHE
|
160
|
CYS
|
4.18
|
149.49
|
126
|
TYR
|
128
|
CYS
|
4.58
|
86.27
|
181
|
PHE
|
85
|
CYS
|
4.87
|
85.18
|
182
|
TYR
|
130
|
MET
|
4.84
|
149.68
|
209
|
TYR
|
264
|
MET
|
4.98
|
8.08
|
230
|
PHE
|
265
|
CYS
|
4.58
|
166.08
|
The number of phosphorylation site (Fig. 5) in SARS-CoV-2 is 54, whereas the number of phosphorylation site in SARS is 45. That means SARS-CoV-2 has higher number of phosphorylation sites than SARS. The high number of phosphorylation site in SARS-CoV-2 increase the strength of protein-protein interactions and also helps in stability63.
SARS-CoV-2 has cyclic salt bridge
Generally proteins have two types of salt bridges, isolated and network salt bridges. Both proteins have only one network salt bridge. But SARS-CoV-2 has special engineered salt bridge (Fig. 6) which forms a cyclic salt bridge (R131-E290,K137-E290,R131-D197,K137-D197,R131-D289), whereas SARS has normal network salt bridge. Novel cyclic salt bridge might have a great role in its stability.
Favorable point mutations of SARS-CoV-2
Result of MSA (Figure 7) of both structure shows some point mutations occur in SARS-CoV-2. So, we have analyzed their effect on SARS-CoV-2 protein stability.
Total 11 mutations have been identified between which 8 are favorable and 3 are unfavorable for SARS-CoV-2 protein stability (Table 5). Residue number 35 which was threonine of SARS substitute by valine in SARS-CoV-2 after mutation, contribute high energy i.e. -2.24 Kcal/mol in protein stability. By those specific point mutations SARS-CoV-2 ultimately got -7.46 kcal/mol energies which make them more stable than SARS.
Table 5. Effect of amino acid mutations in SARS-CoV-2 with their contributing energies
Residue in SASS-CoV-2 (5R80)
|
Residue number
|
Residue in SARS (2H2Z)
|
Contributing energy in stability of SARS-CoV-2 (Kcal/mol)
|
Result of mutations in stability of SARS-CoV-2
|
V
|
35
|
T
|
-2.24
|
Stabilized
|
S
|
46
|
A
|
0.08
|
Destabilized
|
N
|
63
|
S
|
-1.17
|
Stabilized
|
V
|
86
|
L
|
-1.08
|
Stabilized
|
K
|
88
|
R
|
-0.59
|
Stabilized
|
A
|
94
|
S
|
-1.14
|
Stabilized
|
F
|
134
|
H
|
-0.98
|
Stabilized
|
N
|
180
|
K
|
0.08
|
Destabilized
|
V
|
202
|
L
|
-0.33
|
Stabilized
|
S
|
267
|
A
|
-0.13
|
Stabilized
|
L
|
286
|
I
|
0.04
|
Destabilized
|