705-708In ornamental fisheries, body ornamentation, including the body colour, is of great economic importance. Melanocortin-1 receptor (MC1R) gene is primarily responsible for pigmentation in the majority of the vertebrates, and its modulation will help to understand the pigmentation and external body coloration. The present study was aimed to predict the MC1R tertiary protein structure of Tetraodon nigroviridis through homology modelling and to identify putative modulators. The tertiary protein structure of the MC1R protein of T. nigroviridis was homology modelled and validated. BLAHtetrone with binding efficiency of -9.7 kcal/mol was identified as a putative modulator of the MC1R protein

Jahageerdar, Shrinivas

Pandey, Saumya

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Journal of Scientific & Industrial Research Vol. 79, August 2020, pp. 705-708 Computational Modelling of Tetraodon nigroviridis Melanocortin-1 Receptor (MC1R) Protein and Identification of Natural Compounds as Putative Modulators Saumya Pandey and Shrinivas Jahageerdar* Fish Genetics and Biotechnology Division, ICAR-Central Institute of Fisheries Education, Mumbai– 400 061, India Received:31 July 2018; revised 05 March 2020; accepted 23 May 2020 In ornamental fisheries, body ornamentation, including the body colour, is of great economic importance. Melanocortin-1 receptor (MC1R) gene is primarily responsible for pigmentation in the majority of the vertebrates, and its modulation will help to understand the pigmentation and external body coloration. The present study was aimed to predict the MC1R tertiary protein structure of Tetraodon nigroviridis through homology modelling and to identify putative modulators. The tertiary protein structure of the MC1R protein of T. nigroviridis was homology modelled and validated. BLAHtetrone with binding efficiency of -9.7 kcal/mol was identified as a putative modulator of the MC1R protein.  Keywords: BLAHtetrone, Docking, G-protein coupled receptor, Homology modeling, Virtual screening Introduction The price structure of ornamental fish is primarily determined by the body coloration, as the customer preference is for attractive coloration and ornamentation. Pigmentation in fishes is responsible for a broad spectrum of colors, which commands a better price for the fish.1 In the vertebrate species, the melanogenesis pathway is primarily responsible for pigmentation. A polymorphic Melanocortin-1-receptor (MC1R) gene initiates the melanogenesis pathway.2 MC1R protein is the smallest member of class A (rhodopsin-like) melanocortin (MC) receptor family of G-protein coupled receptors (GPCRs).3 Eumelanin (dark coloration) and pheomelanin (red or yellow color) are produced in the active and inactive form of MC1R in the presence of α-MSH.4 Tetraodon nigroviridis is a marine and brackish water aquarium fish with black spots on its green colored body. T. nigroviridis has been widely used as a model organism in genetic studies, including the color inheritance in fish species. The MC1R gene plays a vital role in the color formation; hence, the present study was envisaged to computationally characterize the MC1R protein of T. nigroviridis and identify natural compounds that can act as putative modulators of the MC1R protein. Materials and Methods Sequence Retrieval and Analysis UniProt was used to retrieve the FASTA format of the MC1R protein sequence of T. nigroviridis with H3CV70(5) as its UniProtKB ID. Conserved domain regions of the MC1R protein sequence were identified with the help of the BLAST CD-search. The CLUSTALW was used for multiple alignments of MC1R protein sequences. MC1R Protein Structure Analysis and Active Site Prediction The physicochemical properties of the MC1R protein of T. nigroviridis were determined by the ProtParam tool. The TMHMM v.2.0 server determined the transmembrane information. The subcellular localization of the MC1R protein was predicted with the Hum-mPLoc v3.0 server.6 For the secondary structure prediction, the SOPMA server7 was used. The tertiary structure for the MC1R protein was predicted by employing homology modeling with the help of the SWISS-MODEL.8 The Ramachandran plot generated by the PDBsum server was used to validate the predicted tertiary model. The COACH server9 was used for predicting the active site residue.  Virtual Screening of MC1R Protein with Natural Compounds For virtual screening, a total of 180 313 natural compounds library was prepared from the ZINC12 database. The AutoDock-Vina10 was used for docking of the MC1R protein to the ligands. With a threshold —————— *Author for CorrespondenceE-mail: jahageerdar@cife.edu.inJ SCI IND RES VOL 79 AUGUST 2020   706 value of -7 kcal/mol, the putative ligands were selected based upon their lowest binding efficiencies and ADME properties, which were tested by the ADMETsar server.  Results and Discussion  Sequence Analysis The nucleotide sequence length of the MC1R gene of T. nigroviridis was 978 bp, which encodes an amino acid sequence length of 325. Conserved domain analysis affirmed that the MC1R protein belonged to the GPCR family, with three domains lying between the amino acids 62 to 302. The first one was the serpentine type 7TM GPCR Srsx domain, which was found at the N-terminal and is a chemoreceptor. A seven-transmembrane receptor from the Class A-Rhodopsin family was identified as the second domain. The third domain was a G-protein coupled chemokine receptor, which was found at the C-terminal end. Chemoreceptors in the first domain suggest that the MC1R protein is capable of intaking chemical compounds. Moderate to high homology ranging from 52% to 84% was found between MC1R protein of T. nigroviridis with human, Guppy, mouse, pig, cattle, and dog MC1R, depicting that MC1R protein is conserved across the species. Earlier studies have also suggested that MC1R is conserved across the species.11  MC1R Protein Structure Analysis and Active Site Prediction The molecular weight of H3CV70 was predicted  to be 37043.99 da and the theoretical pI 6.61, indicating a negatively charged, acidic protein. The Hum-mPLoc v3.0 and TMHMM server showed that the MC1R protein is localized in the plasma membrane and comprises seven trans-membranes. The transmembrane sequence positions are provided in Table 1. The secondary structure analysis of the MC1R protein revealed that the protein is composed of 44.00% Alpha helix (Table 2). The presence of a high percentage of α-helix implies that the MC1R protein of T. nigroviridis is highly robust and stable to mutations due to a higher number of inter-residue contacts in helices.12 Beta1 adrenergic receptor, a GPCR protein13 of turkey with a sequence identity of 31.44%, was used as a modeling template for predicting the tertiary structure of MC1R protein of T. nigroviridis. The predicted tertiary structure is shown in Fig. 1. The global model quality estimate (GMQE) of 0.57 and  Table 1 — Expression of MC1R protein amino acid residues in different regions Transmembrane region information Query accession number Server Position Start amino acid End amino acid H3CV70 TMHMM2.0 Outside 1 50 H3CV70 TMHMM2.0 Tmhelix 51 70 H3CV70 TMHMM2.0 inside 71 82 H3CV70 TMHMM2.0 Outside 83 105 H3CV70 TMHMM2.0 Tmhelix 106 124 H3CV70 TMHMM2.0 inside 125 147 H3CV70 TMHMM2.0 Outside 148 167 H3CV70 TMHMM2.0 Tmhelix 168 190 H3CV70 TMHMM2.0 inside 191 199 H3CV70 TMHMM2.0 Outside 200 222 H3CV70 TMHMM2.0 Tmhelix 223 242 H3CV70 TMHMM2.0 inside 243 265 H3CV70 TMHMM2.0 Outside 266 279 H3CV70 TMHMM2.0 Tmhelix 280 302 H3CV70 TMHMM2.0 inside 303 325  Table 2 — Secondary structure prediction of T. nigroviridis  using SOPMA server Protein structure unit No. of amino acids Structural unit (%) Alpha helix 143 44.00 310 helix 0 0.00 Pi helix 0 0.00 Beta bridge 0 0.00 Extended strand 89 27.38 Beta-turn 23 7.08 Bend region 0 0.00 Random coil 70 21.54 Ambiguous state 0 0.00 Other states 0 0.00    Fig. 1 — Predicted tertiary model of MC1R protein of T. nigroviridis by using SWISS-MODEL, based on homology modelling, viewed using PyMol PANDEY & JAHAGEERDAR: COMPUTATIONAL MODELLING OF MC1R OF T. NIGROVIRIDIS   707 qualitative model energy (QMEAN) of 3.44 was present between the MC1R protein and the template sequence. The Ramachandran plot analysis revealed that of the 272 residues, 234 (91.1%) amino acid residues were present in the most favoured core region. The rest 23 (8.9%) residues were present in the additionally allowed regions. The presence of a high percentage of residues in the favoured region indicates that the predicted structure of the MC1R protein of T. nigroviridis is reliable and was used for further analysis. The confidence score (C-score) for the MC1R protein of T. nigroviridis was found to be 0.34 with the 2ycxA_BS01_P32 template cluster. The amino acid residues of TM-5 and TM-6, i.e., I-127, D-128,  C-132, L-199, T-206, F-209, W-258, F-262, L-265, L-288, I-292, were predicted as the active site residues using the COACH server. These are the part of the conserved regions of the MC1R protein of  T. nigroviridis and humans and are suitable sites  for ligand binding activity.14  Virtual Screening and Molecular Docking  From a list of 180 313 natural compounds, three compounds 1H-Indole-2-methanol (ZINC20763420), BLAHtetrone (ZINC05220992) and 2-naphthyl BLAHone (ZINC04237106), had the lowest binding efficiency and acceptable ADME property and  were selected for usage in fishes (Table 3). With  the binding efficiency of -9.70 kcal/mol, the  natural compound BLAHtetrone (ZINC05220992), extracted from Camu camu berry, was found to be a better modulator of the MC1R protein of  T. nigroviridis than others. Figs 2 and 3 depicted  the 2D and 3D docked sites for BLAHtetrone to  the protein. Since the identified compounds are  bound in the active sites of the MC1R, they may  have the ability to act as the MC1R modulators, which further needs to be confirmed with  lab validation and appropriate biochemical experiments.  Table 3 — Binding efficiencies of the natural compounds to the MC1R protein of T. nigroviridis along with the ADMET properties of  top 3 ZINC compounds S.No. Ligands Binding efficiency (kcal/mol) Hydrogen  bond donors Hydrogen bond acceptors Molecular mass (Dalton) Log P Fish toxicity Chemical 1 1H-Indole-2-methanol (ZINC20763420) -9.90 2 8 497.60 3.28 low  2 BLAHtetrone (ZINC05220992) -9.70 0 8 468.43 4.39 low  3 2-naphthylBLAHone (ZINC04237106) -8.30 2 3 317.41 3.20 low     Fig. 2 — 2D ligand (BLAHtetrone) – MC1R protein interaction map viewed using AutoDock    Fig. 3 — 3D ligand (BLAHtetrone) – MC1R protein interaction map viewed using AutoDock J SCI IND RES VOL 79 AUGUST 2020   708 Conclusions MC1R protein is the primary protein that governs the melanogenesis pathway. The study of T. nigroviridis MC1R protein provides a useful insight into its protein structure. The identified putative modulators may help to modify the body pigmentation of T. nigroviridis. There is a high similarity among the MC1R sequences of various species. It is suggested that the T. nigroviridis MC1R protein can be used as a model to understand the protein structure of the majority of the other fish species. Further, it can also be used to explore the possibilities of developing novel MC1R modulators for commercially important aquarium fishes.  Acknowledgment The authors are grateful to the Director, ICAR-Central Institute of Fisheries Education, Mumbai, India, for providing the facility to carry this work. The authors are thankful to Ms Archana N Panche, Assistant Professor, MGM's Institute of Biosciences & Technology Aurangabad, Maharashtra, for the technical guidance. The first author acknowledges the financial support provided by the ICAR, New Delhi, in the form of a junior research fellowship (JRF).  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Computational Modelling of Tetraodon nigroviridis Melanocortin-1 Receptor (MC1R) Protein and Identification of Natural Compounds as Putative Modulators

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Computational Modelling of Tetraodon nigroviridis Melanocortin-1 Receptor (MC1R) Protein and Identification of Natural Compounds as Putative Modulators

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