The variation in temperature with time is plotted for free calcitonin, CT-CGRP receptor complex and CT-AM1 receptor complex. The graphs are shown below:.
A COMPUTATIONAL APPROACH TO STUDY THE BINDING OF CALCITONIN WITH CALCITONIN PEPTIDE FAMILY RECEPTORS
Arindam Ghosh B.Sc. (H) Biotechnology Roll no.:21008212001 Registration no.:122102410045 of 2012-13 Supervised by: - Dr Sumita Mondal Institute of Genetic Engineering
Table of Contents Acknowledgement ................................................................................................. 3 Aim of Project ........................................................................................................ 4 Calcitonin ............................................................................................................... 5 Biosynthesis and regulation ................................................................................ 5 Effects ................................................................................................................. 5 Inhibition osteoclast activity in bones .............................................................. 6 Stimulation osteoblastic activity in bones ........................................................ 6 Mechanism of Action and Pharmacodynamic Effects ......................................... 6 Calcitonin peptide family receptors ....................................................................... 7 AMY1 receptor.................................................................................................... 7 AMY2 receptor.................................................................................................... 8 AMY3 receptor.................................................................................................... 8 CGRP Receptor.................................................................................................... 8 AM1 Receptor..................................................................................................... 9 AM2 Receptor..................................................................................................... 9 Experimental steps involved ................................................................................ 10 Docking ............................................................................................................. 10 Molecular Dynamic (MD) Simulation ................................................................ 10 Steps involved in MD simulation .................................................................... 11 Visualisation ..................................................................................................... 11 Analysis ............................................................................................................. 11 Experiment .......................................................................................................... 12 MD simulation of Calcitonin.............................................................................. 12 Molecular Docking of CT with CGRP receptor ................................................... 13 MD simulation of CT-CGRP receptor complex ................................................... 13 Molecular Docking of CT with AM1 receptor .................................................... 14 MD simulation of CT-AM1 receptor complex .................................................... 14 Analysis ................................................................................................................ 15 Structural changes in free calcitonin ................................................................. 15 Structural changes in CT while bound to CGRP receptor ................................... 16 Structural changes in CT while bound to AM1 receptor .................................... 17
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Comparison of Potential Energies ..................................................................... 18 Comparison of temperature ............................................................................. 19 Comparison of RMSD ........................................................................................ 20 Comparison of RMSF ........................................................................................ 21 Comparison of RDF ........................................................................................... 22 Comparison of H-bond formation ..................................................................... 23 Summary ............................................................................................................. 25 References ........................................................................................................... 26
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Acknowledgement I would like to extend my sincere thanks to Dr Sumita Mondal, my project supervisor, who introduced me into the world of molecular dynamic simulations. Without her guidance and help the project would have remained incomplete. I would also like to thank the entire Computer Department for allowing me to work in the computer laboratory. Finally I would also like to thank Dr Amit Chakravarty, Director, Institute of Genetic Engineering and Dr Sudipa Chakravarty, Deputy Director, Institute of Genetic Engineering for allowing us to undertake a project at an undergraduate level.
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Aim of Project Calcitonin, a polypeptide hormone, binds to its receptors and induces osteoclastic and osteoblastic activity by participating in signal transduction pathway. To trace the initial events occurring in this process within nanosecond time scale, Calcitonin is allowed to dock with the two available receptors separately and then dynamics is provided to the complex in a simulated environment. The purpose of this work is to compare between the two receptors of calcitonin involved in this pathway.
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Calcitonin Calcitonin (also known as thyrocalcitonin) is a 32-amino acid linear polypeptide hormone that is produced in humans primarily by the Para follicular cells (also known as C-cells) of the thyroid, and in many other animals in the ultimobranchial body. It acts to reduce blood calcium (Ca2+), opposing the effects of parathyroid hormone (PTH). Calcitonin has been found in fish, reptiles, birds, and mammals. It belongs to the calcitonin-like protein family. The calcitonin peptide family comprises calcitonin, amylin, calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and AM2, also known as intermedin. The peptides range from 32 to 52 amino acids in length in humans. The calcitonin peptide sequence is:
Figure 1: SOLUTION CONFORMATION OF A NONAMYLOIDOGENIC ANALOGUE OF HUMAN [PDB ID- 2JXZ]
Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-PheHis-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2
Biosynthesis and regulation Calcitonin is formed by the proteolytic cleavage of a larger prepropeptide, which is the product of the CALC1 gene (CALCA). The CALC1 gene belongs to a superfamily of related protein hormone precursors including islet amyloid precursor protein, calcitonin gene related peptide, and the precursor of adrenomedullin. Secretion of calcitonin is stimulated by: an increase in serum [Ca2+] Gastrin and pentagastrin.
Effects The hormone participates in calcium (Ca2+) and phosphorus metabolism. In many ways, calcitonin counteracts parathyroid hormone (PTH). More specifically, calcitonin lowers blood Ca2+ levels in four ways:
Inhibits Ca2+ absorption by the intestines. Inhibits osteoclast activity in bones. Stimulates osteoblastic activity in bones. Inhibits renal tubular cell reabsorption of Ca2+ allowing it to be excreted in the urine.
However, effects of calcitonin that mirror those of PTH include the following: Inhibits phosphate reabsorption by the kidney tubules. In its skeleton-preserving actions, calcitonin protects against calcium loss from skeleton during periods of calcium mobilization, such as pregnancy and, especially, lactation. Other effects are in preventing postprandial hypercalcemia resulting from absorption of Ca2+. Also, calcitonin inhibits food intake in rats and monkeys, and may have CNS action involving the regulation of feeding and appetite.
Inhibition osteoclast activity in bones Osteoclasts are the cells responsible for breaking down bone. When bone is broken down, the calcium contained in the bone is released into the bloodstream. Therefore, the inhibition of the osteoclasts by calcitonin directly reduces the amount of calcium released into the blood. However, this inhibition has been shown to be short-lived.
Stimulation osteoblastic activity in bones Osteoblasts are the cells that help in synthesis of bones. The cells utilize the calcium in blood to synthesize bones. Stimulation of osteoblastic activity directly reduces the amount of calcium in blood.
Mechanism of Action and Pharmacodynamic Effects Calcitonin binds to the calcitonin receptor (found primarily in osteoclasts) and enhances the production of vitamin D producing enzymes. This results in increased calcium retention and enhanced bone density. Binding of calcitonin to its receptor also activates the adenylyl cyclase and phosphatidyl-inositol-calcium signalling pathways.
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Calcitonin peptide family receptors CT receptors are class B GPCRs, a family which includes receptors such as the secretin receptor, parathyroid hormone receptor and calcitonin receptor-like receptor. These receptors share conserved cysteines in their extracellular Ntermini of ~100-160 amino acids. A general mechanism for peptide binding to these receptors is predicted, whereby the C-terminal peptide region is thought to bind to the extracellular N-terminus of the receptor whereas the N-terminus of the peptide interacts with the juxtamembrane region of the receptor. This is consistent with recent structural determinations of complexes between peptides and the Ntermini of their receptors. Whilst the receptor for calcitonin is a conventional class B GPCR, the receptors for CGRP, AM and amylin require additional proteins, called the receptor activity modifying proteins (RAMPs). There are three RAMPs in mammals; they interact with the CT receptor to convert it to receptors for amylin. For CGRP and AM, the related calcitonin receptor-like receptor interacts with RAMP1 to give a CGRP receptor and RAMP2 or 3 to give AM receptors. Calcitonin receptor-like receptor by itself will bind no known endogenous ligand. Structurally, each RAMP has a short intracellular C-terminus with a single transmembrane spanning domain and an Nterminus of ~100 amino acids. The interaction of the N-terminus of the RAMP with the N-terminus of the GPCR determines the specific pharmacology of each receptor complex.
AMY1 receptor Sub-units: RAMP1, CT receptor. Tissue Expression: CNS, Lungs, Stomach, Spleen, Liver, Kidney. Physiological Function: Reduction of food intake, Glucose regulation, Inhibition of gastric emptying and acid secretion, Inhibition of pancreatic enzymes, Calcium homeostasis, Memory retention.
Figure 2: CRYSTAL STRUCTURE OF THE EXTRACELLULAR DOMAIN OF HUMAN RAMP1 [PDB ID - 2YX8]
The complete structure of AMY1 receptor has not yet been determined. However the structure of one of its sub-units, RAMP1 is known.
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AMY2 receptor Sub-units: RAMP2, CT receptor. Tissue Expression: CNS, Lungs, Stomach, Spleen, Liver, Kidney. Physiological Function: Reduction of food intake, Glucose regulation, Inhibition of gastric emptying and acid secretion, Inhibition of pancreatic enzymes, Calcium homeostasis, Memory retention.
Figure 3: STRUCTURE OF THE EXTRACELLULAR DOMAIN OF HUMAN RAMP2 [PDB ID- 2XVT]
The complete structure of AMY2 receptor has not yet been determined. However the structure of one of its sub-units, RAMP2 is known.
AMY3 receptor Sub-units: RAMP3, CT receptor. Tissue Expression: CNS, Lungs, Stomach, Spleen, Liver, Kidney. Physiological Function: Reduction of food intake, Glucose regulation, Inhibition of gastric emptying and acid secretion, Inhibition of pancreatic enzymes, Calcium homeostasis, Memory retention. The structure of AMY3 receptor has not yet been determined.
CGRP Receptor Sub-units: CRLR, RAMP1. Tissue Expression: CNS, PNS, Cardio vascular tissues, Blood vessels, Spleen, Vas deferens, Lung, Adrenal gland. Physiological Function: Regulation of blood pressure, heart rate, Figure 4: CRYSTAL STRUCTURE OF THE Respiratory regulation, Calcium ECTODOMAIN COMPLEX OF THE CGRP homeostasis, Control of fluid balance, RECEPTOR [PDB ID- 3N7P] diuresis, natriuresis, Inhibition of gastric acid secretion, Inflammation, Nociception.
AM1 Receptor Sub-units: CRLR, RAMP2 Tissue Expression: CNS, PNS, Cardiovascular tissues, Spleen, Vas deferens, Lung, Liver, Skeletal muscle. Physiological Function: Regulation of blood pressure (vasorelaxation) and heart rate, Control of fluid balance, diuresis, natriuresis, angiogenesis, cell growth, Regulation of food intake, Nociception.
AM2 Receptor Sub-units: CRLR, RAMP3 Tissue Expression: Not Known Physiological Function: Not Known
Figure 5: CRYSTAL STRUCTURE OF THE HUMAN CRLR/RAMP2 EXTRACELLULAR COMPLEX [PDB ID- 3AQF]
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Experimental steps involved Docking In the field of molecular modelling, docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex.
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Figure 6: Schematic diagram illustrating the docking of a small molecule ligand (brown) to a protein receptor (green) to produce a complex.
Docking servers used:
Figure 7: Patch dock docking server logo
Figure 8: Hex docking server logo
Molecular Dynamic (MD) Simulation It is a computer simulation of physical movements of atoms and molecules. The atoms and molecules are allowed to interact for a period of time, giving a view of the motion of the atoms. In the most common version, the trajectories of atoms and molecules are determined by numerically solving the Newton’s equations of motion for a system of interacting particles, where forces between the particles and potential energy are defined by interatomic potentials or molecular mechanics force fields. Software used:
Figure 9: Gromacs logo
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Steps involved in MD simulation 1. Pdb2gmx Generates a topology for the input coordinate file. 2. Editconf
Edits the solvation box.
3. Genbox
Solvates a system in.
4. Grompp
Makes a run input file for energy minimization.
5. Mdrun
Performs an energy minimization.
6. Grompp
Makes a run input file for position restrained simulation.
7. Mdrun
Performs a position restrained simulation.
8. Grompp
Makes a run input file for final MD simulation.
9. Mdrun
Performs the actual MD simulation.
Visualisation The structures of the molecules and the trajectories obtained by the MD simulation can be graphically viewed for changes occurring in the molecule. Software used:
Figure 10: Rasmol logo
Figure 11: VMD logo
Analysis The stability of the systems are verified from potential energy, temperature and root mean square deviation calculation. Final analysis for stability of the complexes is done by comparing root mean square fluctuation, van der Waals interaction, hydrogen bonding if any and radial distribution function.
Experiment MD simulation of Calcitonin
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Calcitonin is solvated in a box of water and then MD simulation is carried out for 10 ns.
Figure 12: Calcitonin solvated in a box of water
Molecular Docking of CT with CGRP receptor
Figure 15: CGRP receptor
Figure 13: Calcitonin
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MD simulation of CT-CGRP receptor complex The complex is solvated in a box of water and then MD simulation is carried out for 6.5 ns.
Figure 16: CT-CGRP complex solvated in a box of water
Molecular Docking of CT with AM1 receptor
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Figure 17: AM1 receptor Figure 18: Calcitonin
Figure 19: CT-AM1 receptor complex
MD simulation of CT-AM1 receptor complex The complex is solvated in a box of water and then MD simulation is carried out for 2 ns.
Figure 20: CT-AM1 receptor complex solvated in a box of water
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Analysis Structural changes in free calcitonin
The changes that occur in the structure of calcitonin during the 10 ns free calcitonin MD run are shown below:
0 ns
1 ns
6 ns
10 ns
Figure 21: Structural changes in free calcitonin
The changes in the number of hydrogen bonds, number of helices and number of turns with respect to time are shown in the following graphs:
Figure 22: Change in number of H-bonds with time
Figure 23: Change in number of helix with time
Figure 24: Change in number of turns with time
Structural changes in CT while bound to CGRP receptor
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The changes that occur in the structure of calcitonin while bound to CGRP receptor during the 6.5 ns MD run are depicted below:
Figure 25: Structural changes in CT while bound to CGRP receptor
The changes in the number of hydrogen bonds, number of helices and number of turns with respect to time are shown in the following graphs:
Figure 27: Change in number of H-bond with time
Figure 26: Change in number of helix with time
Figure 28: Change in number of turns with time
Structural changes in CT while bound to AM1 receptor
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The changes that occur in the structure of calcitonin while bound to AM1 receptor during the 2 ns MD run are depicted below:
Figure 29: Structural changes in CT while bound to AM1 receptor
The changes in the number of hydrogen bonds, number of helices and number of turns with respect to time are shown in the following graphs:
Figure 30: Change in number of H-bonds with time
Figure 31: Change in number of helix with time
Figure 32: Change in number of turns with time
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Comparison of Potential Energies
The variation in potential energies with time is plotted for free calcitonin, CT-CGRP receptor complex and CT-AM1 receptor complex. The graphs are shown below:
Figure 35: Time-potential energy plot for free calcitonin
Figure 34: Time-potential energy plot for CT-CGRP receptor complex
Figure 33: Time-potential energy plot for CT-AM1 receptor complex
The average potential energies for the three samples are: Free calcitonin= -129355 kJ/Mol CT-CGRP complex= -303776 kJ/Mol CT-AM1 complex= -382011 kJ/Mol From the graphs it is observed that the change in potential energy with time for each sample is relatively constant. This indicates that each sample is stable when solvated in water. The decrease in average potential energy of calcitonin while being bound to its complexes indicates that calcitonin is much more stable in complexes than when free. On comparing the two complexes individually, it is observed that calcitonin is more stable when bound to AM1 receptor than CGRP receptor.
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Comparison of temperature
The variation in temperature with time is plotted for free calcitonin, CT-CGRP receptor complex and CT-AM1 receptor complex. The graphs are shown below:
Figure 38: Time-temperature plot for free calcitonin
Figure 36: Time-temperature plot for CT-CGRP receptor complex
Figure 37: Time-temperature plot CT-AM1 receptor complex
The average temperatures for the three samples are: Free calcitonin= 305.442 K CT-CGRP complex= 308.489 K CT-AM1 complex= 308.913 K Since the temperature change in free calcitonin and the complexes is minimal, the system can be considered to be relatively stable.
Comparison of RMSD
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The root-mean-square deviation (RMSD) is the measure of the average distance between the atoms (usually the backbone atoms) of superimposed proteins. The RMSD plots for free calcitonin, CT-CGRP receptor complex and CT-AM1 receptor complex are shown below:
Figure 41: RMSD for free calcitonin
Figure 40: RMSD for CT-CGRP receptor complex
Figure 39: RMSD for CT-AM1 receptor complex
Comparison of RMSF
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The root mean square fluctuation (RMSF) is a measure of the deviation between the positions of the particle from some reference position over a fixed interval of time. The RMSF plots for free calcitonin, CT-CGRP receptor complex and CT-AM1 receptor complex are shown below:
Figure 42: RMSF of free calcitonin
Figure 44: RMSF of CT-CGRP receptor complex
Figure 43: RMSF of CT-AM1 receptor complex
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Comparison of RDF
The radial distribution function (RDF) [g(r)] in a system of particles (atoms, molecules, colloids, etc.), describes how density varies as a function of a distance from a reference point. The RDF plots for CT-CGRP receptor complex and CT-AM1 receptor complex are shown below:
Figure 45: RDF of CT-CGRP receptor complex after 1ns
Figure 47: RDF of CT-AM1 receptor complex after 1ns
Figure 46: Points of Van der Waals interaction between CT and CGRP receptor
Figure 48: Points of Van der Waals interaction between CT and chain A of AM1 receptor
Figure 49: Points of Van der Waals interaction between CT and chain B of AM1 receptor
Comparison of H-bond formation
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The change in the number of hydrogen bonds formed between the hormone and the receptor is plotted against time. The graphs are shown below:
Change in number of H-bond formed between CT and CGRP receptor in the time interval 3ns and 4ns
Figure 50: Plot of number of H-bond against time (Time 1ps=10 frames)
From the above graph it is observed that the number of hydrogen bonds formed is 5.
Figure 51: H-bond (dotted) between CT (red) and CGRP receptor (blue)
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Change in number of H-bond formed between CT and AM1 receptor in the time interval 1.75 ns and 1.8 ns
Figure 52: Plot of number of H-bond formed between CT and chain A of AM1 receptor against time (Time 1ps=10 frames)
Figure 55: Plot of number of H-bond formed between CT and chain B of AM1 receptor against time (Time 1ps=10 frames)
Figure 53: H-bond (dotted) between CT (yellow) and chain A of AM1 receptor (red)
Figure 54: H-bond (dotted) between CT (yellow) and chain B of AM1 receptor (blue)
From the above two graphs it is observed that the number of hydrogen bonds formed between calcitonin and chain A of AM1 receptor is 2 while it forms 4 bonds with chain B i.e. a total of 6 hydrogen bonds are formed between calcitonin and AM1 receptor.
Since calcitonin forms more number of hydrogen bonds with AM1 receptor than CGRP receptor, the CT-AM1 receptor complex can be considered to be more stable than CT-CGRP receptor complex.
Summary After docking there is significant increase in the stability of the system.
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There are significant changes in the structure of free calcitonin but its flexibility decreases in the docked complexes due to the presence of hydrophobic interaction offered by the receptor. Significant fluctuation is observed for calcitonin in CT-CGRP receptor complex as compared to CT-AM1 receptor complex. Due to the occurrence of more hydrogen bonds in CT-AM1 complex than CT-CGRP complex the former is considered to have greater stability. The two complexes are highly stable and due to this high stability it participates in the signal transduction pathway and helps in maintaining calcium homeostasis.
References Research Articles:
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Shen et al. BMC Bioinformatics 2013, 14(Suppl 14):S6 J. W. Orry and B. A. Wallace, Biophysical Journal Volume 79 December 2000 3083–3094. Absalom et al., American Journal of Agricultural and Biological Sciences, 2013, 8 (1), 89-106. M. SAHIHI et al., Chem. Biochem. Eng. Q., 27 (4) 417–422 (2013). Fahrul Huyop et al., Int. J. Mol. Sci. 2012, 13, 15724-15754. Sandeep et al., Molecular docking & dynamics of PRKACA
Websites: http://www.gromacs.org/ http://rasmol.org/ www.rcsb.org/ http://www.sigmaaldrich.com/ www.guidetopharmacology.org http://www.biomedcentral.com/1471-2105/14/S14/S6
