Comparative Analysis of Docking of Regulator of G-protein Signaling 9 (RGS9)-[Homo sapiens] and its Mutated Structure

1. Rida Khalid
BS-BIOINFORMATICS
COMSATS INSTITUTE OF INFORMATION TECHNOLOGY, ISLAMABAD
1

2. Introduction of RGS9-protein
 The official name of RSG9 gene is “Regulator of g-protein signaling 9.”
 RGS9 gene encodes a member of the RGS family of GTPase(Guanosine Tri-
phosphatase) activating proteins that function in various signaling
pathways .
 This protein is anchored to photoreceptor membranes in retinal cells and
deactivates G proteins in the rod and cone photo-transduction cascades.
 Mutations in this gene result in bradyopsia, a rare condition that affects
vision.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 2

3. Cont…
 The RGS9 gene provides instructions for making two versions
(isoforms) of the RGS9 protein, known as RGS9-1 and RGS9-2. They
are found in different parts of the nervous system and have very
different functions.
 RGS9-1 is produced in the retina, which is the specialized tissue at the
back of the eye that detects light and color.
 Within the retina, RGS9-1 is associated with light-detecting cells called
photoreceptors. When light enters the eye, it stimulates specialized
pigments in these cells. This stimulation triggers a series of chemical
reactions that produce an electrical signal, which is interpreted by the
brain as vision. (This process is known as photo-transduction.)
 Once photoreceptors have been stimulated by light, they must return
to their resting state before they can be stimulated again. RGS9-1 is
involved in a chemical reaction that helps return photoreceptors to
their resting state quickly after light exposure.COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 3

4. Location of Gene
 RGS9 cytogenetic location is 17q24.
 More precisely, the RGS9 gene is located from base pair
65,137,338 to base pair 65,227,703 on chromosome 17 on the
long (q) arm at position 24 .
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 4

5. Mutation identification
 The Mutation occurred at position 299 where Threonine (T)
was replaced by Cysteine(C) which results in Bradyopsia, a
rare condition that affects vision.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 5

6. 3D structure prediction of RGS9 and
Mutated RGS9
 One of the best Models is to be selected on the basis of
RMSD value and its structure validation results, so Swiss-
Model server model is the best model which was generated
against same template “2pbi.2.A” for normal and mutated
Protein.
RSG9 CHIMERA view Mutated RSG9 CHIMERA view
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 6

7. Superimposition
 We use Chimera for the superimposition of Normal
and Mutated protein. RMSD value of this
superimposition is 0.660 as shown below.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 7

8. Domain Identification
 Domains are the functional and structural units in a
protein.
 They are responsible for a particular function or interaction
of a protein.
 In order to identify the domains of RGS9 we used following
servers.
SERVERS USED DOMAIN NAME DOMAIN RANGE
Superfamily (HMM library &
genome environment
server)
Regulator of G-protein Signaling
(RGS)
59-189
ScanProsite RGS domain 73-188
Interpro (Protein seq
analysis & classification)
RGS domain 59-188
Motif Scan Result RGS domain 73-187
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 8

9. ScanProsite Results
 Domain range 73-188 is selected.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 9

10. Binding Pocket Identification
 Binding Pockets are the area of protein known to be active
in forming of compounds.
Castp Results
 Binding sites and active sites of proteins and DNAs are
often associated with structural pockets and cavities as
shown below.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 10

11. Binding Pockets falling in Domain region
Pocket-
ID
Residues along with their respective positions VOLUME AREA
24 SER30, ASN31, ALA33, GLY37, CYS38, LEU39, PHE51, LEU54, ASN55, THR63, LYS64,
MET65, ARG66, ARG69, GLU193, PRO194
278.4 334
23 LYS57, ILE78, PRO81, ARG84, TRP103, GLU104, GLU107, ASP108, TYR111, GLY112 96.3 239.7
22 VAL16, GLY20, LYS23, TYR24, GLN27, PHE72, ASN73, PHE74, SER75, LYS171 113.5 246.3
21 MET8, ARG9, SER10, LYS142, ILE146, TYR157, VAL158, ASP160, ALA161, THR164 102.3 559.9
20 CYS106, LEU109, LYS110, ARG156, TYR157, LEU159, ASP160, GLN163 84.8 67.5
19 GLN88, GLY97, LEU100, GLY101, GLU104 31.4 32.1
18 SER75, ILE78, ARG79, TRP103, GLU107, TYR167 46.5 53.4
17 MET1, ARG9, HIS155, TYR157, ASP160 21.9 192.7
16 ILE78, LEU100, TRP103, TYR167, MET170 43.9 26.5
15 SER30, ASN31, MET65, GLU68, PHE72 41.6 32
14 ILE61, PRO62, MET187, LYS190, ALA191 34.7 18.7
13 ARG66, ARG69, TRP70, ASP80 18.1 14
12 LEU109, LYS110, GLY112, GLN114, VAL117, ARG156 29.7 19.3
11 TYR89, ILE183, ASP186 7.3 7.1
10 TRP70, SER77, GLY83, PHE87, TYR178 48.3 29
9 LEU54, VAL59, ILE61, THR63, ILE192 39.5 19.6
8 THR147, VAL158, LEU159, ALA161, ALA162 30.1 15.2
7 PHE74, SER77, PHE87, TYR175, TYR178 51.1 28.4
6 ILE146, THR147, VAL158, ALA161 31.4 15.9
5 PRO40, SER41, ASP48, PHE51 29.5 14.1
4 LEU109, ASP113, VAL117, LYS120 36.4 16.7
3 PRO40, GLN50, PRO194, GLN195 14.9 8.3
2 SER75, GLU76, ARG79 16.6 9.1
1 SER75, GLU76, ARG79 24.7 11.5
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 11

12. Retrieval of Binding Partners(Ligands)
 For this purpose String database is used which allows
searching of protein-protein interaction of RGS9
protein. Results are shown below.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 12

13. Selection of Ligand
 Selected ligands on the basis of least no. of amino
acids in it.
Ligand
#
Name Description No. of Amino
acids
Pdb ID
1 GNAT1 Guanine nucleotide-binding proteins
(G proteins) are involved as
modulators or transducers in various
transmembrane signaling systems.
350 aa 2ju4
2 PDE6H Participates in processes of
transmission and amplification of the
visual signal. These are the effector
molecules in G-protein-mediated
photo-transduction in vertebrate rods
and cones.
83 aa 1got
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 13

14. Molecular Docking
 When the ligand is docked onto the receptor and the
interactions are checked, the scoring function generates
scores depending on which the best fit ligand is selected.
For docking HEX server is used.
HEX server
 HEX is an interactive protein docking and molecular
superposition program. The first 10 solutions are saved as
both.pdb for each docking.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 14

15. Result of Hex
COMPLEX# Normal
receptor with
ligand#1 (2ju4)
Normal
receptor with
ligand#2 (1got)
Mutated
receptor with
ligand#1 (2ju4)
Mutated
receptor with
ligand#2 (1got)
1 -749.46 -513.68 -653.47 -745.57
2 -697.18 -510.31 -647.83 -680.30
3 -693.69 -490.68 -639.52 -654.98
4 -691.90 -475.57 -631.21 -629.29
5 -689.50 -471.50 -623.87 -606.62
6 -660.35 -468.26 -621.84 -603.53
7 -652.47 -464.42 -620.93 -588.93
8 -646.40 -457.91 -614.65 -587.69
9 -643.44 -457.56 -608.78 -586.71
10 -643.37 -450.76 -606.47 -583.95
The Energy values of 10 solutions each is shown below
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 15

16. Ligand#1 with Normal and Mutated Structure
Figure:
(A) Best solutions after docking of Ligand (2ju4) with Normal structure where E-value= -
749.46
(B) Best solutions after docking of Ligand (2ju4) with Mutated structure where E-value= -
653.47 COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD
16

17. Ligand#2 with Normal and Mutated Structure
Figure: (C) Best solutions after docking of Ligand (1got) with Normal structure where E-value=
-513.68
(D) Best solutions after docking of Ligand (1got) with Mutated structure where E-value= -
745.57
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 17

18. Ligplot+ Results
 The program automatically generates schematic
diagrams of protein-ligand interactions from the 3D
coordinates PDB file(Edited by PDB- Editer).
 In figures Maroon eyelashes represents hydrophobic
interactions with receptor while
 Pink eyelashes represents hydrophobic interactions with
Ligands.
 Green dotted lines show Hydrogen bonding between
receptor and ligand.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 18

19. Ligplot+ output of Ligand#1 (2ju4) with Normal
structure
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 19

20. Ligplot+ output of Ligand#1 (2ju4) with Mutated
structure
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 20

21. Ligplot+ output of Ligand#2 (1got) with Normal
structure
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 21

22. Ligplot+ output of Ligand#2(1got) with Mutated
structure
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 22

23. CONCLUSION
 The Protein-Ligand interaction plays a significant role in structural based drug
designing. By analyzing the HEX docking results it is concluded that after the
mutation occurs there is the desperate change between the E-values of same
protein normal protein and its mutated protein when respectively docked by
two ligands.
 The Ligplot+ results that the hydrogen bonded residues and hydrophobic
residues of normal receptor with ligands are different from those of mutated
receptor that are involved in bonding with respective ligands.
 Due to mutation in sequence at position 299 from T-C which resulted in the
change of binding position between the receptor and ligand as a result
Bradyopsia disease occurs.
COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 23

24. COMSATS INSTITUTE OF INFORMATION
TECHNOLOGY, ISLAMABAD 24