Protein-Protein Interaction of Mutated Agouti Signaling Protein (ASIP) to Melanocortin Receptor 1 (MC1R) in Melanoma Skin Cancer: An Insilico Study

 

Keerthana Karunakaran¹, Jansi Rani Malaiyappan¹, Rajini Raja Muniyan²*

¹Department of Integrative Biology, School of Bioscience and Technology, Vellore Institute of Technology (VIT), Vellore-632014, TN, India

²Department of Biotechnology, School of Bioscience and Technology, Vellore Institute of Technology (VIT), Vellore-632014, TN, India

 

ABSTRACT:

Melanoma is a type of skin cancer which is rare and most serious. It is developed due to the defect in skin pigmentation pathway associated with the major receptor called melanocortin receptor 1(MC1R). Agouti signaling protein (ASIP) on binding to MC1R induced the melanoma by producing pheomelanin. In this study, our aim is to prevent the binding of ASIP to MC1R by mutating ASIP with various amino acids computationally.The MC1R protein sequence (Q01726) was modelled using MODELLER 9.18. Mutation study was performed with SPDBV software.  Protein-protein interaction was conducted with HADDOCK 2.2 server (High Ambiguity Driven DOCKing). Mutation was carried out in the active residue of ASIP-R117 with A, C, K, S based on their physiochemical property with wide variety. Docking result shows that, the parameters viz., haddock score, RMSD, electrostatic energy and z-score of MC1R-ASIP (R117A) were found to have -97.9±2.7, -9.5±0.2 Å, -61.0±25.4 kcal/mol and -2.0 respectively which was higher than that of the standard values given in the literature. Whereas the mutant R117K shows RMSD (0.8±0.5 Å), electrostatic energy (-193.0±25.8 kcal/mol) and z-score (-2.3) along with haddock score (-131.2±13.1)which was very low when compared with native form.   Hence, the mutant MC1R-ASIP (R117A) exhibited to have poor binding affinity. Moreover, MC1R-ASIP (R117A) complex does not show any hydrogen bond interaction. Therefore, the mutant ASIP (R117A) might be the probable choice to prevent the MC1R-ASIP complex formation. However, more detailed analysis need to be carried out to have an in-depth understanding on the in vivo significance of this bi-molecular interaction.

 

 

 

INTRODUCTION:

Melanoma -a type of skin cancerwhich is very serious and less common than that of the non-melanomas such as basal cell and squamous cell [1]. Melanomas are developed at any part of the skin due to the defect in skin pigmentation, however those are most proneto trunk in men and legs in women.

 

 

American cancer society (ACS) has reported that, there were 87110 new melanomas diagnosed and also reported that 9,730 people were estimated to have with melanoma death in the year 2017 [2]. Skin pigmentation is due to the accumulation of melanin granules which is synthesized in melanocytes and transferred tokeratinocytes [3]. Keratinocytes secrete cytokines and chemokines on UV exposure influencing the production of melanins [4].

 

Melanins are the product of two events: signal transduction and biosynthetic. Type of melanin to be produced depends on the type of ligand that binds to the receptor. Melanocortin 1 Receptor (MC1R) is mainly present in the membrane of melanocytes and synthesize two types of melanin such as eumelanin and pheomelanin [5,6]. MC1R is the class of GPCR (G-protein coupled receptor) family where an agonist alpha-MSH on binding to MC1R will activate the receptor and produce cyclic-AMP (cAMP). cAMP which is produced due to the receptor activation will further activate the downstream events and upregulate TYR, TYRP1, TYRP2 (Tyrosinase related protein) expression necessary for photo protective eumelanin production [7]. Eumelanin generated to protect our skin from UV induced DNA damage. The report shows that the competitive antagonist ASIP blocks the action of MC1R [8,9,10]. ASIP normally binds to the receptor and gets inactivated for the production of cAMP. Thus, further results in the down regulation of enzymes and photosensitive pheomelanin production. [11,12,13]. It is  reported that pheomelanin an aromatic pigment which has sulfur in its aromatic ring lowers the ionization potential of the pigment and makes it less stable. Pheomelaninutilizes sulfur compound known as glutathione for its formation. Glutathione act as a natural scavenger of Reactive Oxygen Species (ROS). As most of the gluatathione are utilized during pheomelanin production, ROS level get increased and result in the damage of DNA, thus causes melanoma [14,15].

 

Therefore, our aim of the study is to prevent the binding of ASIP to MC1R. Mutation of ASIP with various amino acids in the binding cavity region so as to reduce the interaction mechanism between MC1R-ASIP complexes and propose the amino acid which might repress bindings.

 

MATERIALS AND METHODS:

Data Collection:

The amino acid sequence of human MC1R was obtained from Uniprot (Q01726).In order to build the three dimensional structure of human MC1R, closely related homolog retrieved from the Protein Data Bank (PDB) ( ID: 2VT4_A). The complete structure of ASIP was obtained from PDB (ID: 2KZA) and used for further study.

 

Homology modeling of Human MC1R:

Using a template 2VT4_A, the three dimensional structure of  human MC1R was modelled using Modeller 9.18. The model was energy minimized using molecular mechanics force field UFF with Argus Lab 4.0 software and used for the docking study [16].

 

Mutations on ASIP:

Mutation of the amino acid was carried out using SPDBV software with the active residue of competitive antagonist ASIP-R117 with A(Alanine), C(Cysteine), K(Lysine) and S(Serine) based on their physiochemical property with wide variety. Stability of the mutated proteins were maintained using energy minimization.

 

Interaction of human MC1R and ASIP:

Protein-Protein interaction study was carried out using HADDOCK 2.2 server (High Ambiguity Driven DOCKing).  It is a rigid body docking generated 1000 models [17] among those models, best models were selected with the best haddock scores. The interaction study was examined with the help of Ligplot plus version 1.4.5and SPDBV software’s.

 

RESULTS AND DISCUSSION:

Molecular modeling of MC1R&mutation of ASIP:

The best hit has been selected based on the query coverage and the identity of aligned sequence were obtained to be 88% and 28% respectively between target (MC1R) and template (2VT4_A). In order to enhance the stability of modelled MC1R, energy minimization using molecular mechanics force field UFF was carried out. The overall quality factor was checked using Procheck and Whatcheck server as 93.38%, which shows that the modelled protein is more reliable (fig. 1A). ASIP is a competitive antagonist that induces the pheomelanin production. ASIP structure was retrieved from Protein Data Bank (ID: 2KZA) and depicted in fig.(1B).

 

 

Fig 1. A) 3D structure of Modelled MC1R protein and B) 3D structure of ASIP (ID: 2KZA)

 

RFF(117-119) is the most common motif in ASIP [18,19]. RFF motif was mutated with the active residues of competitive antagonist ASIP-R117 with A, C, K, S based on their wide range of physiochemical property.

 

Interaction of MC1Rwith native and mutant ASIP varieties:

Protein-protein interaction study was conducted to analyze the complexity between MC1R-ASIP native and MC1R-ASIP mutant variety. The active site residues of MC1R (Ser6, Asp117, Asp184,His260, Glu269, Thr272) and ASIP (Arg117, Phe118, Phe119)were previously reported [20,21]. Haddock generated the optimally refined clusters of MC1R and ASIP with the help of active site residues.  The best cluster was selected with less Haddock score and low Root mean square deviation (RMSD)value. Besides the HADDOCK score and other standard energies (Van der Waals, etc.), a z-score has been added. The z-score represents how many standard deviation the HADDOCK score of a given cluster is separated from the mean of all cluster. Four mutants of ASIP and native MC1R was docked. The clusters with low HADDOCK score, low Z-score and low RMSD were selected for each mutant ASIP-MC1R complex.

 

In order to analyze the interaction between MC1R and ASIP, we considered the parameters of HADDOCK such as HADDOCK score, Van der Waals energy, electrostatic energy, RMSD,buried surface area and z-score. HADDOCK score represents the average value calculated from the top four best-scoring structures within each cluster. Haddock results shows that the haddock scores forMC1R-ASIP (native form), MC1R-R117A, MC1R-R117C, MC1R-R117K, and MC1R-R117S were 104.3±25.8, -97.9±2.7, -110.2±8.8, -131.2±13.1 and -101.8±2.5 respectively between the protein-protein complexes as listed in table(1). Less negative the haddock score poorer the binding affinity [17]. However, our result shows that, MC1R-R117A had less negative (-97.9±2.7) when compared to the native complex (-104.3±25.8). Whereas, MC1R-R117S (-101.8±2.5) was less negative when compared to the remaining two complexes. Moreover, it was high negative (-131.2±13.1) with MC1R-R117K.  RMSDon the other hand, the acceptable RMSD valueof ≤8Å have possesses stronger affinity between two proteins [22]. Our results shows that, MC1R-R117A and MC1R-R117S were exhibited to have higher RMSD values of 9.5±0.2 and 9.5±0.6 respectively. Those werefar away with the RMSD (2.2±1.3) of MC1R-ASIP. However, MC1R-R117K shows low RMSD (0.8±0.5). We also considered the Van der Waals energy in which the two molecules are highly destabilized when they are much closer or further apart than the Van der Waals contact distance [23]. In our study, MC1R-R117A and MC1R-R117S were showsgreater Van der Waals energy of -36.5±1.2kcal/mol and -30.3±5.0 kcal/mol respectively when compared to the native form (-45.3±17.4 kcal/mol). Whereas, MC1R-R117C and MC1R-R117K have highly stabilized in their energy values. With respect to the electrostatic energy, MC1R-R117A and MC1R-R117K were higher with -61.0±25.4 kcal/mol, -52.1±8.0 kcal/mol respectively. Lowering the entropy of a complex by burying the amino acids is known as buried surface area (BSA) [23]. To have the highly disorded complex, BSA should be very less. By means of BSA, it was found that, MC1R-R117A(1037.6±30.1) and MC1R-R117S(1053.5±78.5) had least BSA value than the MC1R-ASIP complex(1093.7±313.5) and other mutant complexes. In addition to these, Z-Score is considered as the main parameter in our study as lower the z-score is better the complex [17]. The various parameters of Haddock 2.2 results, it was observed that,the mutant-ASIP (R117A) has poor binding affinity. Moreover, we have also found that ASIP-R117K has very strong affinity towards MC1R than that of native complex.

 

Table 1: Consolidate docking result of MC1R with ASIP native and mutant variety (R117A,R117C, R117K and R117S.

Mutated ASIP Docking parameters	ASIP  native form	ASIP-R117A	ASIP-R117C	ASIP-R117K	ASIP-R117S
Haddock score	-104.3±25.8	-97.9±2.7	-110.2±8.8	-131.2±13.1	-101.8±2.5
Cluster size	5	18	5	9	20
RMSD(Å)	2.2±1.3	9.5±0.2	10.8±0.2	0.8±0.5	9.5±0.6
Van der Waals energy(kcal/mol)	-45.3±17.4	-36.5±1.2	-53.0±4.5	-61.1±6.2	-30.3±5.0
Electrostatic energy(kcal/mol)	-80.6±18.0	-61.0±25.4	-138±21.7	-193.0±25.8	-52.1±8.0
Buried Surface Area	1093.7±313.5	1037.6±30.1	1402.4±64.1	1508±129.0	1053.5±78.5
Z score	-2.3	-2.0	-2.0	-2.3	-2.1
Number of Hydrogen bond interaction	2	0	2	2	1

 

Protein-protein 2D complex analysation:

In order to get insight on bond formation between protein complexes, we analyzedusing Ligplotplus 1.4.5 server. Proteins are destabilized by the absence of non-covalent interactions such as hydrogen bonds and they are formed between the main chain and side chain of proteins. Our view showed that there were two hydrogen bonds in the native complex, between the Asp184 residue of MC1R-ASIP (R117) of which implies the strong complex formation (fig. 2A). Among the four mutants obtained mutant R117C and R117K forms two hydrogen bond with MC1R in different residues. Though the equal number of hydrogen bonds in native complex and mutant R117K complex, the interacting residues vary in MC1R. Whereas in mutant R117C complex, there was no hydrogen bond formation between the active residue of MC1R-ASIP(R117C). However, active residue Glu269 of MC1R formed two hydrogen bond with the neighboring active residues of ASIP(Phe118, Phe119) (fig. 2C and 2D). In the case of mutant R117S complex, there was single hydrogen bond between Ala181 of MC1R which shows that affinity between the proteins was poor(Fig. 2E). With respect to MC1R-R117A complex, there was no any hydrogen bond formation between the active residues of and A117 (Fig. 2B).It gives us the conclusion that there was no affinity between the MC1R and mutant ASIP-R117A.

Figure 2: 2D interaction between MC1R (pink) and ASIP (brown), A) MC1R and ASIP R117A  B) MC1R and ASIP R117A C) MC1R and ASIP R117C D) MC1R and ASIP R117K and E) MC1R and ASIP R117S

 

In order to further conform the protein-protein interaction result, 3D interaction study was carried out for all the complexes using SPDBV software. However, it was observed that the similar result found in the interaction between proteinsespecially for MC1R-ASIP (R117A). Therefore our findings also proved that there was a poor binding affinity obtained in the mutant ASIP (R117A) (Figure 3A-E).

 

 

Figure 3: 3D interaction between MC1R (cyan), ASIP (pink) and mutated molecules (light green), A) MC1R and ASIP R117A  B) MC1R and ASIP R117A C) MC1R and ASIP R117C D) MC1R and ASIP R117K and E) MC1R and ASIP R117S

Overall, for the mutant ASIP (R117K) exhibited good binding affinity toward MC1R irrespective of the real binding site for the production of eumelanin. Whereas,ASIP (R117A) exhibited poor binding affinity with MC1R and blocks the production of pheomelanin which was more prone to photosensitive and leads to skin cancer.  Hence, the mutant ASIP (R117A) might be the probable choice to prevent the MC1R-ASIP complex formation.

 

CONCLUSION:

Agouti signaling protein (ASIP) on binding to MC1R induced the melanoma by producing pheomelanin leads to melanoma skin cancer. Among four mutant variety of ASIP, mutant ASIP (R117A) shows poor binding affinity with MC1R and might be the probable choice to prevent the MC1R-ASIP complex formation. However, more detailed analysis need to be carried out to have an in-depth understanding on the in vivo significance of this bi-molecular interaction.

 

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Accepted on 31.05.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 3913-3917.