PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands
Abstract
:1. Introduction
2. Materials and Methods
2.1. Rosetta Flexible Peptide and High-Resolution Docking
2.2. Protein Expression and Purification
2.3. Microscale Thermophoresis
2.4. PIRT and Calmodulin Pull-Down Assay
2.5. Nuclear Magnetic Resonance-Detected Binding Assay
3. Results
3.1. Bioinformatic Analysis of PIRT Predicts Calmodulin-Binding Motifs
3.2. Flex Pep Dock
3.3. PIRT Specifically Interacts with Calmodulin
3.4. NMR Titration Shows Residues with Ligand-Dependent Perturbation Including the Predicted Calmodulin Binding Site
3.5. Two Cholesterol-Binding Motifs Found on PIRT with Bioinformatic Analysis
3.6. PIRT and Cholesterol Ligands Screening with Microscale Thermophoresis
3.7. β-Estradiol Induced PIRT Chemical Shift Perturbation
3.8. Microscale Thermophoresis Shows PIRT Binds to Other Trp Channel Modulators
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Ligand | Kd (M) | RMSE (M) | Ligand Type |
---|---|---|---|
Calmodulin (apo, free) | 350 × 10−9 | 40 × 10−9 | CaM, Intracellular Protein |
Calmodulin (holo, bound) | 60 × 10−6 | 30 × 10−6 | Calcium Bound CaM |
Cholesteryl HS | 103 × 10−6 | 6 × 10−6 | Steroid Hormone Precursor |
Cortisol | 790 × 10−6 | 50 × 10−6 | Cortical Steroid Hormone |
β-Estradiol | 800 × 10−6 | 100 × 10−6 | Sex Steroid Hormone |
Cholecalciferol | 2.1 × 10−3 | 0.4 × 10−3 | Secosteroid Hormone |
Oxytocin | 7 × 10−6 | 1 × 10−6 | Peptide Hormone |
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Sisco, N.J.; Luu, D.D.; Kim, M.; Van Horn, W.D. PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands. Biomolecules 2020, 10, 478. https://doi.org/10.3390/biom10030478
Sisco NJ, Luu DD, Kim M, Van Horn WD. PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands. Biomolecules. 2020; 10(3):478. https://doi.org/10.3390/biom10030478
Chicago/Turabian StyleSisco, Nicholas J., Dustin D. Luu, Minjoo Kim, and Wade D. Van Horn. 2020. "PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands" Biomolecules 10, no. 3: 478. https://doi.org/10.3390/biom10030478
APA StyleSisco, N. J., Luu, D. D., Kim, M., & Van Horn, W. D. (2020). PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands. Biomolecules, 10(3), 478. https://doi.org/10.3390/biom10030478