1. Protein molecular modeling of spike protein fragment
Gene Bank accession number for SARS-CoV-2 S is QHR63250.2, LOCUS QHR63250, Accession MN996527.1is used for protein molecular modeling of spike protein fragment.
Primary amino acid sequence of spike protein fragment (331 to 524) is as follows
NATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL HAPATV
2. Primary and Secondary structure analysis
Primary structure analysis shows that this spike protein fragment SARS-CoV-2 have 193 amino acid residues. Secondary structure analysis with PDBsum [31], shows that this protein fragment contains 3 sheets, 1 beta hairpin, 2 beta bulges, 9 strands, 6 helices, 1 helix-helix interaction, 14 beta turns, 4 gamma turns and 2 disulfide bonds.
Table 2 Templates for 3D structure of the spike protein fragment
Template
|
Seq Identity
|
Oligo- state
|
QSQE
|
Found by
|
Method
|
Resolution
|
Seq Similarity
|
Coverage
|
Description
|
6lzg.1.B
|
100.00
|
monomer
|
-
|
HHblits
|
X-ray
|
2.50Å
|
0.62
|
1.00
|
SARS-CoV-2
Spike receptor- binding domain
|
6m0j.1.B
|
100.00
|
monomer
|
-
|
HHblits
|
X-ray
|
2.45Å
|
0.62
|
1.00
|
SARS-CoV-2
receptor- binding domain
|
6w41.1.C
|
100.00
|
monomer
|
-
|
HHblits
|
X-ray
|
3.08Å
|
0.62
|
1.00
|
Spike glycoprotein receptor binding domain
|
6m17.1.C
|
100.00
|
monomer
|
-
|
HHblits
|
EM
|
NA
|
0.62
|
1.00
|
SARS-coV-2
Receptor Binding Domain
|
3. 3D structure modeling and validation
3D structure of the spike protein fragment has been modeled by using SWISSMODEL [32] server. Template 6lzg.1.B is selected for modeling protein with the sequence identity 100% and coverage 100% compared to the other two templates (Table 2) for modeling.
The SWISS-MODEL template library (SMTL version 2020-04-08, PDB release 2020-04-03) is searched with BLAST [19] and HHBlits [20] for evolutionary related structures matching the target sequence in Table 2. Overall, 101 templates are found.
Modelled structure obtained from SWISSMODEL server [32] has -2.87 QMEAN score, shown in Figure 4 (a). QMEAN value is intended as a linear combination of four statistical potential terms and transformed to a Z score relating it to high resolution X-ray structures of similar size. Higher Z score is related to more favorable model. Ramachandran plots are drawn for this model by using two web servers e.g. Molprobity [24] and PDBsum [31], are shown in Figure 4 (b) and 4 (c). For this model the overall average value of G -factors is -0.18 which is not unusual for dihedral angles and main-chain covalent forces. The value of G-factors provides a measure of how unusual or out-of -the ordinary, a property is. From MolProbity version 4.4 [33] it is calculated that, modelled structure has 94.44% residues in favored regions, 0.56% residues in outlier region and 3.18% in rotamer outlier region. Ramachandran plot statistics from PDBsum [34] for modelled structure of spike protein fragment, 136 (86.1%) residues in most favored regions [A, B, L], 21 (13.3%) residues in additional allowed regions [a, b, l, p], 1 (0.6%) residues in generously allowed regions [~a, ~b, ~l, ~p] and 0 (0.0%) residue in disallowed regions [X, X].
4. Molecular docking between spike protein fragment and human ACE2 receptor
Human ACE2 receptor (PDB ID 1R42) [35] is considered as receptor protein for molecular docking study of spike protein fragment with its receptor in human host.
By using ClusPro [25] web server, docking structure of A chain of human ACE2 receptor, binds with spike protein fragment, is obtained. SARS CoV2 spike protein binds with human ACE2 receptor protein with binding energy -779.8 Kcal/mole. A conformational change occurs in ACE2 receptor protein after binding with spike protein fragment (Figure 5).
Amino acids present in distorted site of ACE2 are ASP136, ASN 137, PRO 138, GLN139 and interacting amino acids of spike protein fragment are GLN 403, LYS 451 and ASP 416 (Figure 6).
Bound structure of SARS CoV2 spike protein fragment with ACE2 receptor protein is considered as therapeutic target for SARS-CoV2 treatment.
5. Molecular docking study of phytochemicals from Indian medical plants
5.1 Spike protein binding with ACE2 in presence of hesperidin
In Figure 7, spike protein fragment (331 to 524) is shown in red colour, hesperidin molecule in stick model and human ACE2 is shown in blue colour. Hesperidin binds with spike protein fragment and its receptor ACE2 with binding energy -8.99 Kcal/mole. This docked structure is stabilized by two H binding (shown in Figure with green lines) at PHE 457 of spike protein with O7 atom of hesperidin, with bond length 2.618Å and H atom of small molecule hesperidin with O atom of GLU 455 of spike protein fragment with a distance 2.067 Å. Hesperidin binds at ASN 63, ALA 71, LYS 74 and SER 44 amino acids of ACE2.
5.2 Spike protein binding with ACE2 in presence of emodin
The phytochemical emodin, obtained from Rheum emodi or Himalayan rhubarb [36], binds with spike protein fragment and its receptor human ACE2 protein [37], at the same cleft (Figure 8), same to that of hesperidin. But binding energy is less for emodin binding (-6.19 Kcal/mole) compared to that of that of hesperidin (-8.99 Kcal/mole).
5.3 Spike protein binding with ACE2 in presence of anthraquinone
Though anthraquinone can bind with bound structure of spike protein fragment and its receptor ACE2 molecule, with releasing binding energy -6.15 Kcal/mole, but the binding site of this phytochemical is totally different from that of hesperidin and emodin (Figure 9).
5.4 Rhein binding with bound spike protein and ACE2 receptor protein
The phytochemical rhein binds with docked structure of spike fragmented protein and human ACE2 receptor with Δ G value -8.73 Kcal/mole. But the binding site of this chemical totally different from earlier substances (Figure 10). Rhein can bound with only spike protein fragment. It has no interaction with human ACE2 receptor protein molecule.
5.5 Chrysin binding with bound spike protein and ACE2 receptor protein
Chrysin binds with the spike protein fragment and its ACE2 receptor with binding energy -6.87 Kcal/mole (Figure 11). This phytochemical binding site is almost similar with that of spike protein fragment molecule and its receptor. A conformational change occurs in ACE2 receptor molecule after spike protein fragment binding. Chrysin binding cleft is nearly located to that site as shown in Figure 12.
Energy parameters of bound structure of phytochemicals with spike protein fragment and ACE2 receptor are shown in Table 3.
Table 3 Energy parameters of bound structure of phytochemicals
Name of phytochemicals
|
Energy/ Simple fitness
|
FullFitness
|
ΔGvdw
|
ΔG (Kcal/mole)
|
Hesperidin
|
59.4535
|
-2147.5469
|
-52.5659
|
-8.99
|
Emodin
|
19.599
|
-2301.9927
|
-23.3637
|
-6.19
|
Anthraquinone
|
17.7976
|
-2234.7346
|
-21.5368
|
-6.15
|
Rhein
|
36.5174
|
-2310.458
|
-107.401
|
-8.73
|
Chrysin
|
15.8545
|
-2266.9272
|
-31.1973
|
-6.87
|
Considering the lowest binding energy, the phytochemical hesperidin is considered as most suitable ligand for target molecule, which is formed by binding with spike protein fragment and its human host ACE2 receptor.
In six docking structures interacting amino acids of ACE2 receptor and spike protein fragment are summarized in Table 4.
Table 4 Interacting amino acids in docking structures
Docking structure
|
Interacting amino acids of
ACE2 receptor
|
Interacting amino acids of
spike protein fragment
|
Spike protein fragment with
ACE2
|
ASP136, ASN 137, PRO 138,
GLN 139
|
GLN 403, LYS 451, ASP 416
|
Hesperidin binding with spike
protein and ACE2
|
ASN 63, ALA71, LYS 74,
SER 44
|
VAL 472, GLY 474, GLY
471, PHE 475, GLU 473
|
Emodin binding with spike
protein and ACE2
|
ALA 71, ASP 67, LYS 74
|
VAL 472, GLY 474, ALA
464, ASN 448
|
Anthraquinone binding with
spike protein and ACE2
|
SER 105, ASN 103, GLN
102, LEU 100, PHE 28
|
No interacting amino acids
|
Rhein binding with spike
protein and ACE2
|
No interacting amino acids
|
SER 388, VAL 401, THR
333, ASN 332, ASN 353
|
Chrysin binding with spike protein and ACE2
|
THR 129, ILE 126, THR 125
|
ARG 443, SER 448, ASN
449, TYR 410, PHE 486,
TYR 484, THR 487, ASN
488, LYS 406
|
Considering the docking structures and interacting amino acids of both ACE2 receptor and spike protein fragment, chrysin can act as most competent inhibitor for spike protein binding with ACE2 receptor.