Table 1.
List of primers for PCR amplification and Sanger sequencing of TSHR gene.
Fig 1.
A representative chromatogram showing a mutation in exon 10 of TSHR gene.
Table 2.
Mutations detected in the TSHR gene of hypothyroid patients and analysis of the effect of mutations using different bioinformatics tools.
Fig 2.
Comparison of TSHR protein model structures.
Superimposed snapshots of TSHR protein, (A) TSHR368-764 WT (purple), (B) TSHR368-764 MT1 (orange), and (C) TSHR368-764 MT2 (lemon) with PDB:1F88 (Pink). (D) Full length TSHR protein predicted by AlphaFold. Mutations are presented in yellow color. (E) Superimposed snapshot of TM-domain and cytoplasmic domain of TSHR protein predicted by AlphaFold (blue) and I-TASSER (deep Pink).
Fig 3.
The structure of TSHR protein (368–764) showing crucial amino acids responsible for binding with MS437 and MS438 molecules.
Table 3.
Summary of the corresponding model numbers, C–score, TM–score and the RMSD–score of the predicted 3D structures of TSHR368-764 WT, TSHR368-764 MT1, and TSHR368-764 MT2.
Fig 4.
Non-covalent interactions of MS437 with corresponding predicted structures of TSHR368-764.
(A) TSHR368-764 WT and MS437, (B) TSHR368-764 MT1 and MS437 and (C) TSHR368-764 MT2 and MS437.
Fig 5.
Non-covalent interactions of MS438 with corresponding predicted structures of TSHR368-764.
(A) TSHR368-764 WT and MS438 (B) TSHR368-764 MT1 and MS438 and (C) TSHR368-764 MT2 and MS438.
Table 4.
Binding Affinity (kcal/mol) of MS437 with TSHR368-764 WT, TSHR368-764 MT1 and TSHR368-764 MT2 proteins, and AlphaFold predicted full-length TSHR proteins after flexible docking.
Table 5.
Non-covalent interactions of MS437 with TSHR368-764 WT, TSHR368-764 MT1, and TSHR368-764 MT2 proteins after flexible docking.
Table 6.
Binding Affinity (kcal/mol) of MS438 with TSHR368-764 WT, TSHR368-764 MT1 and TSHR368-764 MT2 proteins, and AlphaFold predicted full-length TSHR proteins after flexible docking.
Table 7.
Non-covalent interactions of MS438 with TSHR368-764 WT, TSHR368-764 MT1 and TSHR368-764 MT2 proteins after flexible docking.
Fig 6.
Analysis of 50 ns MD simulation of TSHR368-764 in complex with MS437 ligand.
(A) Root mean square deviation values of C-α atom. The structural changes of TSHR368-764 proteins by means of (B) radius of gyration, (C) solvent accessible surface area, (D) molecular surface area, (E) root means square fluctuations, and (F) total number of hydrogen bonds formed during the simulation.
Fig 7.
Analysis of 50 ns MD simulation of TSHR368-764 in complex with MS438 ligand.
(A) Root mean square deviation values of C-α atom. The structural changes of TSHR368-764 proteins by means of (B) radius of gyration, (C) solvent accessible surface area, (D) molecular surface area, (E) root means square fluctuations, and (F) total number of hydrogen bonds formed during the simulation.
Fig 8.
The snapshots of the generated conformers for TSHR368-764 and ligands: MS437 (red), MS438 (blue) over the 50 ns MD simulation.
(A) TSHR368-764-WT (cyan), (B) TSHR368-764-MT1 (pink), and (C) TSHR368-764-MT2 (green) structures.
Fig 9.
PCA analysis on 50 ns MD simulation.
(A, C) The score plots represents three clusters for TSHR368-764 wild-type and mutant protein structures, where each dot specifies one time point. The clustering is attributable as: WT-MS437 (sky blue), MT1-MS437 (pink), MT2-MS437 (navy blue), and WT-MS438 (sky blue), MT1-MS438 (orange), MT2-MS438 (navy blue), (B, D) Loading plots display the energy and structural profile data from principal component analysis.