Homology Modelling of Urotension-2 Receptor (UTS2R): Potential Target for Human Pharmacotherapy

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http://dx.doi.org/10.13160/ricns.2016.9.3.185

Homology Modelling of Urotension-2 Receptor (UTS2R): Potential Target for Human Pharmacotherapy

Sathya. B^†

Abstract

Urotensin-2 receptor (UTS2R) is the most potent vasoconstrictor and plays a major role in the pathophysiology of various cardiovascular diseases and becomes a potential target for human pharmacotherapy. The crystal structure of Urotension-2 receptor has not yet been resolved. Hence, in the current study homology modelling of UTS2R was done utilizing the crystal structure of human delta opioid receptor as the template. Since the template has low sequence identity, we have incorporated both comparative modelling and threading approach to generate the three dimensional structure.

10 models were generated and validated. The reported models can be used to characterize the critical amino acid residues in the binding site of UTS2R.

Keywords: UTS2R, Homology Modelling, Threading

1. Introduction

Human urotensin II (UTS2R) is a cyclic peptide generated by proteolytic cleavage from a precursor prohor- mone. UTS2R was first isolated from the fish spinal cord and has been recognized as a hormone in the neu- rosecretory system of teleost fish. UTS2R has been identified as an endogenous ligand of the G protein-coupled receptor (GPR)-14^[1-3]. UTS2R is a highly con- served undecapeptide which is well represented in the nervous system, heart and kidney and was initially seen as a vasoconstrictor/cardiodepressant compound and implicated in myocardial and renal dysfunction^[4-6]. UTS2R mediates vascular tone and increased contrac- tile force in human atrium and ventricle and the effects of UTS2R are mediated by binding to the urotension receptor^[7,8]. UTS2R provokes trophic and/or mitogenic actions in vascular smooth muscle cells, cardiac myocytes and cardiac fibroblasts^[9]. Urotension receptor expression is increased in cardiac myocytes, endothelial cells and fibroblasts in the rat heart after coronary artery ligation and plays a role in the physiology and patho-

physiology of the cardiovascular system. Urotension II treatment increased collagen mRNA and protein levels in cardiac fibroblasts and augmented cardiac hypertro- phy in cultured neonatal cardiomyocytes after transfec- tion with recombinant urotension II receptor. Mammalian UTS2R is the most potent endogenous cardiostimula- tory peptide identified and emerging evidence in exper- imental models and in humans indicates that urotensin II may play a cardioprotective role in coronary heart disease and in chronic renal failure^[10,11]. Evidence has been also provided that UTS2R and UT-R are expressed in the adrenal gland and adrenal tumors^[12,13]. Plasma levels of urotension II have been found to be elevated in patients with heart failure, systemic hypertension, diabetes mellitus, and renal failure^[14,15]. With iontopho- resis of urotension II into the skin, urotension II mediated a dose-dependent vasodilator response in normal subjects but a dose-dependent vasoconstrictor response in patients with heart failure, suggesting that urotension II may contribute to the increased peripheral vascular tone that occurs in heart failure. UTS2R is a neuropep- tide and may play a role in tumor development. The development of antagonists may provide novel treatment for cardiovascular diseases.

The three dimensional structure of UTS2R is an essential component in the perspective of understanding its underlying biological functions at molecular level. In

Department of Genetic Engineering, School of Bioengineering, SRM University, SRM Nagar, Kattankulathur, Chennai 603203, India

†Corresponding author : sathyainfo26@gmail.com (Received: July 21, 2016, Revised: September 18, 2016, Accepted: September 25, 2016)

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the current study, comparative modelling and threading based model generation was done for UTS2R receptor.

Ten models were successfully generated, and validated using ERRAT and Ramachandran plot scores. The validation results obtained were acceptable and used to identify and suggest the best model for performing the further docking studies for the identification of the important binding site residues.

2. Materials and Methods

2.1. Template Selection

The template used for modelling directly determines the main folding of the target structures and influences the quality of the model. Hence, template selection is the most critical start point in homology modelling. In our study we retrieved the amino acid sequence for the human UTS2R from the Uniprot database (Q9UKP6) and submitted to NCBI blast^[16] tool to perform a search against the PDB^[17] database. The best templates were chosen based upon the criteria’s like high percentage of sequence identity, maximum query coverage and low E value.

2.2. Model Generation

The ultimate goal of protein modelling is to predict the structure from its sequence. In the current study the homology model for human UTS2R protein was successfully built using EasyModeller tool and I-Tasser server. Easy Modeller 4.0^[18] is GUI based tool for homology modelling which uses Modeller 9.12^[19] and Python. I-TASSER^[20] is an online server which uses the secondary-structure enhanced profile-profile threading alignment and the iterative implementation of the Threading ASSEmbly Refinement (TASSER) program.

2.3. Model Validation

The quality of the generated models were validated based on Ramachandran plot generated using RAM- PAGE tool^[21] and ERRAT^[22]. The Ramachandran plot checks for the stereo-chemical quality of a protein and gives information about the percentage of residues in allowed region. The ERRAT program analyses the sta- tistics of non-bonded interactions between different atom types, with higher scores indicating higher quality.

3. Results and Discussion

3.1. Template Selection

BLAST search was performed against the protein data bank (PDB) in order to identify the adequate template for the target protein UTS2R. The blast search resulted in 22 templates among which the templates identity ranges between 20%-31% with varying query coverage. The crystal structure of human delta opioid receptor (4N6H) was selected as template because it sat- isfies the template selection criteria’s such as sequence identity >30%, maximum query coverage and template crystal structure with good resolution.The alignment between UTS2R and template 4N6H is shown in Fig. 1.

3.2. Model Generation and Validation

The three dimensional structure of UTS2R protein was modeled using EasyModeller and I-tasser. Five models were generated using EasyModeller. Although the identity was small the generated models retain the seven transmembrane regions which is the predicted topology of GPCR family. Similarly five models were obtained from I-Tasser server with seven transmembrane regions. The validation for 10 models was performed using RAMPAGE tool to calculate the stereo- chemical properties of the model. We found only 1% to 1.5% of the residues were found in disallowed region

Fig. 1. Sequence alignment between the UTS2R (target) and with templates (4N6H).

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for the models obtained using EasyModeller whereas in I-Tasser models 7.5% to 9% of residues were observed in disallowed region. ERRAT program was used to ana- lyze the overall quality factor of all the generated models. The quality of the models was in the range of 70%- 80% with higher quality for I-Tasser models compared to models generated from EasyModeller. Based on the validation result, two models were selected one from comparative modeling and one from threading approach.

The model obtained from easy modeler shows 98.2% of residues in favored and allowed region and its quality value in ERRAT plot was found to be 78% whereas the model from I-Tasser shows 92.5% of residues in favored and allowed region and 94% quality value in ERRAT plot. The three dimensional structure of the selected models is shown in Fig. 2. The validation result obtained from Rampage and ERRAT server for the selected models is shown in Fig. 3 and 4 respectively.

Fig. 3. Ramachandran plot for the selected model obtained from EasyModeller (a) and I-Tasser (b).

Fig. 2. 3D structure of the selected best model for human UTS2R protein

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4. Conclusion

In this study, homology modeling and threading based approaches have been carried out to develop the three dimensional model for human UTS2R to explore its biological functions. Reliable models were con- structed using EasyModeller and I-Tasser server and validated using Ramachandran plot and ERRAT plot.

We hope that our model could be useful for identifying critical residues and for designing new inhibitors.

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