Vol. 11, No. 2 (2018) pp. 87− 92 https://doi.org/10.13160/ricns.2018.11.2.87
3D Structure Prediction of Human 5-Hydroxytryptamine Receptor 7 (5-HT
7R)
Thirumurthy Madhavan†
Abstract
5-Hydroxytryptamine receptor 7 (5-HT7R) is one of G-Protein coupled receptors, which is found to be involved in the pathophysiology of various neurological disorders including depression, sleep disorders, memory deficiency and neuropathic pain. After activation of 5-HT7R by serotonin, it activates the production of the intracellular signaling molecule cyclic AMP. The availability of 3D structure of the receptor would enhance the development of new drugs. Hence, in the present study, homology modelling of human 5-hydroxytryptamine receptor 7 (5-HT7R) was performed using comparative modelling (Easy Modeller) and threading (I-TASSER) approaches. The generated models were validated using Ramachandran plot and ERRAT plot and the best models were selected based on the validation results. The 3D model developed here could be useful for identifying crucial residues and further docking study.
Keywords: 5-HT7R, Serotonin, Homology Modelling, Threading
1. Introduction
5-Hydroxytryptamine 7 receptor (5-HT7R) belongs to the G-Protein coupled receptor superfamily of cell sur- face receptors[1]. The 5-HT7 receptor is encoded by the HTR7 gene and is activated by the serotonin, a neu- rotransmitter. After activation, 5-HT7 leads to a cascade of events starting with release of the stimulatory G pro- tein Gs from the GPCR complex which further activates the production of cyclic AMP, an intracellular signaling molecule[2]. Serotonin receptors have been divided into seven families, 5-HT1R to 5-HT7R. 5-HT7R is the recently cloned subtype of serotonin receptor family which is expressed in a variety of human tissues, includ- ing brain, the gastrointestinal tract, and in various blood vessels[3]. 5-HT7 receptor is involved in learning, mem- ory, thermoregulation, circadian rhythm, and sleep and also plays an important role in smooth muscle relax- ation within the vasculature and in the gastrointestinal tract[4]. 5-HT7 receptor has been reported in the patho-
physiology of various neurological disorders including depression, sleep disorders, memory deficiency and neuropathic pain. They are also found to down regulate after antidepressant administration[5]. These findings suggest that the 5-HT7 receptor antagonists can be used as antidepressant agents[6,7] and its activation produced anti-hyperalgesic effects in the animal models[8]. It is also speculated that this receptor may be involved in mood regulation suggesting that 5-HT7 receptor can be a useful target in the treatment of depression[9,10].
5-HT7 receptor (5-HT7R) is one of the most recently discovered serotonin receptors and could be a valuable target for the managing of cognitive decline during age- ing[11], in learning and memory[12,13] and in hippocam- pus-dependent cognitive processes[14]. The three dimensional structure of 5-hydroxytryptamine 5-HT7R is not reported to have been resolved. Hence, in this study we have generated the 3D models of 5-hydroxy- tryptamine receptor by comparative modelling and threading approaches. Homology modelling makes use of the fact that evolutionary related protein shares sim- ilar structures. Therefore the protein with unknown structure can be modeled using known structure (tem- plate) if both shares high sequence similarity. Therefore, comparative modelling was performed using Easy- Modelling software and threading approach was carried out using I-TASSER server.
Department of Genetic Engineering, School of Bioengineering, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Chen- nai 603203, India
†Corresponding author : thiru.murthyunom@gmail.com, thirumurthy.m@ktr.srmuniv.ac.in
(Received : April 17, 2018, Revised : June 15, 2018 Accepted : June 25, 2018)
2. Material and Methods
2.1. Template Selection
The amino acid sequence of human 5-hydroxytrypt- amine receptor 7 (5-HT7R) (accession No: P34969) was retrieved from the Uniprot database. The three-dimen- sional structure of 5-HT7R is not yet available; hence the present study was undertaken. Protein BLAST[15]
search was performed against PDB[16] with the default parameters to find suitable templates for homology modelling. Templates were selected based on sequence identity, query coverage and E-value. Multiple sequence alignment was done using CLUSTALW[17]
program to find conserved residues between the target and the template protein.
2.2. Comparative Modelling and Threading The three dimensional structures of 5-HT7R was
modeled using EasyModeller 4.0[18] which uses MOD- ELLER 9.12[19] and Python 2.7.1 in the backend and I- TASSER[20] server which is a protein structure model- ling approach based on the secondary-structure enhanced profile-profile threading alignment (PPA) and the iter- ative implementation of the Threading ASSEmbly Refinement (TASSER) program. In threading approach, the target sequence is first threaded through a PDB structure library to search for the possible folds by four simple variants of PPA methods employing the hidden Markov model, PSI-BLAST profiles, Needleman- Wunsch and Smith-Waterman alignment algorithms.
2.3. Validation of 3D models
The predicted models were validated using Ramach- andran plot[21] and ERRAT plot[22]. The Ramachandran plot gives us information about the percentage of resi- dues in allowed and disallowed regions. The ERRAT
Fig. 1. Sequence alignment between the target (5-HT7R) and template (4IAQ).
program is well suited for evaluating the progress of crystallographic model building and refinement and analyzes the statistics of non-bonded interactions between different atom types[23,24].
3. Results and Discussion
3.1. Template Selection
To select the templates for comparative modelling, sequence identity plays a major role. The crystal struc- ture of chimeric protein of 5HT-1B-BRIL in complex with dihydroergotamine (4IAQ) with sequence identity 36%, query coverage 66% and E-value 1e-61 was selected. The template belongs to GPCR family having seven transmembrane helices topology. Since the query coverage and identity of the template was little low, we have generated the models using threading which incor- porated different templates during modelling. The align- ment between the target and template sequence was shown in Fig. 1.
3.2. Model Generation
The three dimensional structure of 5-HT7R was pre- dicted using the comparative modelling program, Easy Modeller4.0 and online threading server I-TASSER.
Using Easy Modeller, 9 models using crystal structure of chimeric protein of 5HT-1B-BRIL (4IAQ) were gen- erated. Multiple template based approach was carried
out with the aim of identifying. The protein sequence of 5-HT7R was submitted to I-TASSER 3D structure prediction server, which produced five similar models.
I-TASSER uses multiple templates for the generation of 3D models. The use of multiple templates generally increases model accuracy as it combines the informa- tion from multiple templates and thereby improves the structure quality.
Table 1. Validation of the generated model using RC plot and ERRAT plot
Model No Template Ramachandran Plot
ERRAT Favored (%) Allowed (%) Disallowed (%)
1 4IAQ 91.2 5.9 2.9 54.92
2 4IAQ 90.6 6.3 3.1 46.60
3 4IAQ 91.0 6.9 2.1 52.86
4 4IAQ 91.2 5.9 2.9 61.24
5 4IAQ 92.2 5.0 2.7 57.54
6 4IAQ 91.4 6.3 2.3 59.35
7 4IAQ 91.8 5.9 2.3 57.26
8 4IAQ 89.9 7.1 2.9 52.58
9 4IAQ 91.6 5.7 2.7 51.18
10 I-TASSER server 66.2 21.0 12.8 75.37
11 I-TASSER server 71.5 17.8 10.7 79.61
12 I-TASSER server 75.1 15.9 9.0 86.99
13 I-TASSER server 69.4 16.6 14.0 74.25
14 I-TASSER server 67.7 18.0 14.3 74.94
Fig. 2. Selected models for 5-HT7R (a) Easy Modeller (Model 4) (b) I-TASSER (Model 12).
3.3. Model Validation
The predicted structures using different techniques were validated using Ramachandran (RC) and ERRAT plot. RC plot and ERRAT values were tabulated in Table 1. Based on validation results, the best models were selected and shown in Figure 2 and its RC plot and ERRAT plot is illustrated in Fig. 3 and 4 respectively.
The selected best models has 97.1% and 89.3% of res-
idues in favored and allowed region and ERRAT showed overall quality factor of 61.2%, and 86.9%
which validates the quality of the generated models.
4. Conclusion
3D-models for human 5-hydroxytryptamine receptor- 7 receptor were generated using comparative modelling Fig. 3. Ramachandran plot for the selected models. (a) Easy Modeller (Model 4) (b) I-TASSER (Model 12)
Fig. 4. ERRAT plot for the selected models (a) Easy Modeller (Model 4) (b) I-TASSER (Model 12)
and threading approaches. Our results demonstrate that modelling of 5-HT7R using both the approaches suggest that the model generated using I-TASSER server shows better quality score in ERRAT plot but the RC plot shows the model generated using Easy Modeller has more residues in the allowed and favored region. The generated structures will serve as cornerstone for further analysis with 5-HT7R receptor.
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