Protein-Ligand Binding Interactions

A Project Report Submitted as a Part of the Requirement for the Degree of


Submitted by Manisheel Gautam Roll No: 09103009

Under the Supervision of Dr. Nand Kishore,


Indian Institute of Technology. 2013 STAGE – 1 Statement I hereby declare that the matters presented in this project report are the results of investigation carried out by me in the department of Chemistry. Manisheel Gautam 09103009 Department Of Chemistry . This report “Protein-Ligand Binding Interactions” is submitted for the partial fulfilment of the degree of Master of Science (M. India. In keeping with the general practice of reporting scientific observations. has not been submitted elsewhere for a degree. Nand Kishore.NOVEMBER.Sc. Bombay.) in Chemistry at IIT Bombay. due acknowledgements has been made wherever the described is based on finding of other investigation. under the supervision of Dr.

under my supervision. Nand Kishore Dept. of Chemistry.Declaration I declare that this project titled “Protein-Ligand Binding Interactions” has been carried out by Manisheel Gautam. IIT Bombay . ------------------------------Signature of the Guide Dr.

Introduction 2.3 Setting up of simulation 4.4 Interaction Profiles 3. References .2 Computational Details 4.CONTENTS 1. Objective and Approach PART 1 3. Future Work 11. Molecular Dynamics Simulation 4.4 Result 10.3 Results and Observations 3. Docking 3.1 Literature review 3.1 Introduction 4.5 Clustering of Conformations PART 2 4.2 Computational Details 3.

and energetics to understand different interactions and their solvation properties. We study how to find active site in a protein. We use a two part approach wherein in first part we used docking to study binding interactions and in second part we use molecular dynamics to study solvation interactions between the molecules In first part we use Docking software. Interaction profiles of all the ligands to checked which residues of HIV protease were interacting with ligand residues and clustering of conformations was done to check the reliability of the results from the docking In the second part we use Molecular Dynamics Simulation to find out interactions of Glycine Betaine and NaCl salt in solution. 2. density distribution and Hydrogen bonding distribution are studied to understand the interactions between the molecules of the system. The concentration of Glycine Betaine is varied in the solution to check the change in interacting forces with the change in concentration of solute in the solution . hydrogen bonds. INTRODUCTION The synergising effect of Glycine Betaine and urea is known to negate denaturing effect of urea on proteins .1. In the simulation we solvate Glycine Betaine molecules in water with NaCl molecules. As there will be different solvation properties when both Glycine Betaine. It is essential to study effect of concentration of Glycine Betaine and NaCl on solvation of Glycine Betaine which is the important factor in the studying the overall effect of GB urea and salt. In the study we will analyse distribution functions. We perform docking on several ligands with HIV protein and then examined them to find the best fit configuration.Glycine Betaine stabilizes proteins. salt are present in the solution. From the results we get we plot the radial distribution function. see how inhibitors of a protein work and how they bind to proteins. OBJECTIVE AND APPROACH To study more about the synergising effect of urea and Glycine Betaine over denaturation of a protein.

PART 1 .

Therefore the other molecule inhibits the reaction and is termed as an inhibitor. DOCKING 3. Both quantitatively and qualitatively it becomes very important to understand it. The second ligand which binds to the site is termed as an inhibitor if it is not biologically productive. Protein-ligand binding is a spontaneous process in which forces at work are similar to forces in Protein the use of protease inhibitor combined with a reverse transcript inhibitor is growing to negate the effect of mutations in treating HIV. The inhibitor reduces the association affinity of ligand and the reduction will depend on the inhibitor concentration and also on the association constant of inhibitor for the protein We have inhibitors which inhibit the functioning of a protein this can be used in treatment of HIV where Inhibitors of HIV protein can be used to inhibit the protease function Mutation in HIV Protease can make treatment using many inhibitor drugs . But these inhibitor ligands bind to specific areas on the protein and interact with specific residues so even slight mutations in the protease can make the treatment unusable. Receptor actions. A Substrate comes and fits into an enzyme with a complementary shape and form a product. Function of proteins is defined through its interactions with other molecules A protein can often have sites where multiple ligands can come and bind. This function is very important for: Enzyme function.3. But when both the structurally similar compounds are present it might reduce ability of enzyme to form the product with the incoming substrate. we will have a brief look at the subject of discussion here i. Self-organization cellular structures and multicomponent protein complexes. SO we work with a combination of such ligands to negate the effect of mutations on the treatment 3.1 LITERATURE REVIEW First of all.e “Protein-ligand binding interactions” Protein can selectively bind to other molecules. Also drug resistant mutated viruses may develop. Molecular docking uses various methods and .2 COMPUTATIONAL DETAILS By using Molecular docking we try to predict the preferred orientation of a molecule bound to another molecule to form a stable molecule. But when a structurally similar compound comes and binds it does not necessarily lead to formation of a product.

3 SETTING UP OF DOCKING Setting up of Protein The protein molecule is opened in Autodock and extra molecules like water and iodine are removed from the file. The least energy conformation is the best fit configuration of the protein-ligand complex Different interaction between molecules are there as a consequence of forces between particles. Gasteiger charges are added if there are no charges. By checking the PDB file of protein we find out the active site of the protein and set up a grid box around it. and fragment based search methods In molecular docking of protein to a ligand we try to find the best fit configuration in which ligand is bound to the protein and we find it by checking the conformations found after docking.algorithms like molecular dynamics.[9] Setting up of ligand A number of steps are involved in setting up a ligand: Ligand is loaded on AutoDock. We set up the grid maps around the site around the receptor site and also type of the maps . The grid parameter file has the information about the grid map. These forces are divided into four categories: Electrostatic forces. steric forces. Solvent-related forces and other physical factors In our docking studies we use AutoDock molecular docking software. The bonds which are rotatable are the single bonds. After the ligand is prepared it is saved as a pdb file. Monte Carlo stimulation.pdbqt. In AutoDock we take a flexible ligand where we restrict its torsions and bind it to a rigid molecule. Normally amide bonds are not rotatable.[9] Preparing the Grid Parameter File. It was used to study the binding of different HIV inhibitors with HIV protease and check the final docking file for the conformations. ADT detects whether charges already exist on the ligand or not. Hydrogen are needed to be added to the ligand before we select the ligand and set number of Cyclic bonds are not rotatable also Bonds to leaf atoms cannot be meaningfully rotated. We write flexible residues in a different pdbqt file giving it the name hsg1_rigid.A different map is calculated for every atom type found in the ligand and also we have an electrostatics map. We use VMD to visualise the different conformations 3. [9] .

Nelfinavir. Amperanavir. and number of genetic algorithm run -150 . A total of 50 conformers were generated and best one was selected Interaction Profiles of all ligands were seen to check which residues of the HIV protease are interacting with the residues of the ligands The Clustering of conformations was done with different rmsd to check the reliability of the results and the final graphs were plotted Docking log files were checked to find the least the energy of best conformation for all the 9 Inhibitor ligands and the values were tabulated to compare the efficiencies of different ligands when bound to the HIV Protease Table 1: Docked energy of best conformation of each ligand LIGAND BINDING ENERGY (Kcal/mol) Saquinavir Indinavir Ritonavir Amperanavir Nelfinavir Lopinavir Atazanavir Tipranavir Darunavir -10.4 -4.0 -8. Ritonavir.6 -8. Lopinavir. Tipranavir and Darunavir with 50 conformers of each ligand. Indinavir.2 But maximum Binding Energy doesn’t mean it is the most effective treatment for HIV .After docking all docking log files were checked and best conformers of each docking were tabulated with the docking energy.4 -7. Sometimes combination of inhibitors are used for treatment .7 -7. Atazanavir.2 -10.8 -8.8 -7.3. The search algorithm used was Genetic algorithm with 2500000 number of evaluations Number of generations 27000.4 RESULTS AND OBSERVATIONS Docking was done on the HSG1 protein and 9 different inhibitor ligands Saquinavir.

In case of Amperanavir Lys.3. The dimer consists of two amino acid monomers.5 INTERACTION PROFILES Fig 1: Interactions between Amperanavir ligand and protein residues The Interaction image shows the residues of the protein interacting with the ligand. Each monomer has acid residue which are important for catalysis [7] . Asp and Leu residues are interacting with the ligand Fig 2: Interacting protein residues and Darunavir ligand The HIV protease is a dimer. It has C2 symmetry. Gly. Val.

Fig 3: Best conformation of protein after docking with Indinavir ligand The figures shows Indinavir inhibitor ligand docked to HIV-1 Protease in least Energy Configuration 3.6 CLUSTERING OF CONFORMATIONS Fig 4: Clustering in of Darunavir ligand Conformations rmsd: 5 .

AutoDock clusters at this rmsd if no changes are made to default settings. When viewing clustering results.5Å. AutoDock orders all conformations on the basis of their docked energy with lowest docked first and highest docked last. We start with the lowest energy conformation and then compare the remaining to it and according to the rmsd tolerance we have set it get clustered into different groups.Fig 5: Clustering in of Darunavir ligand Conformations rmsd: 3 To find out how reliable a docking experiment is we have to check similarity of final docked conformers. [9] In the Clustering process the starting clustering rmsd is 0. we can see the different rmsd between different cluster families . After the comparing similar conformations are clubbed together into clusters this process is called clustering of conformations. We take the rmsd of the best fit conformer which is the lowest energy conformer and also take rmsd of the other conformers we get from docking and then compare the rmsd.


we use Gromacs to simulate biochemical molecules like proteins .” to simulate the Newtonian equations of motion for systems with hundreds to millions of particles”[8]. The integrator used for simulations was leap-frog integrator at a time step of 2 fs. 4. concentration of both NaCl salt was 1 molar Processing of files before MD Run The steric clashes and inappropriate geometry of the system has to be removed before we start the md run. In all simulations OPLS force field was used for Glycine Betaine and NaCl 4.e.1 ps. the structure is relaxed through a process called energy minimization (EM). Equilibration of the solvent and ions around the protein should be done otherwise if unrestrained dynamics is attempted at this point.1 INTRODUCTION GROMACS is a software used to perform molecular dynamics.2 COMPUTATIONAL DETAILS Molecular dynamics simulation were performed for aqueous Glycine Betaine + NaCl system. The box was then solvated with water molecules 75 molecules of salt (NaCl) molecules were put in the box Approx. The simulation were carried out using NVT-NPT ensemble at a temperature of 300 K. i.4. nucleic acids etc. the system may collapse. MOLECULAR DYNAMICS SIMULATION 4. These molecules have lot of different interaction ranging from electrostatic to van der wal forces .but since GROMACS is extremely fast at calculating nonbonded interactions many groups are also using it for research on non-biological systems. Brendsen Coupling algorithm was used to maintain the temperature and pressure with a time constant of 0. We need the system to attain a temperature here the simulation is going to take place and have the proper orientation . Periodic boundary conditions were applied in x. y and z directions.3 SETTING UP OF SIMULATION Preparing topology and pdb file We create a PDB file for Glycine Betaine and use pdb2gmx command to create molecular topology file for it Solvation of Betaine Glycine A cubic box of side 5 nanometres and total volume 125 nm^3 was generated and molecules of Betaine Glycine were put in the cubic box.

We must first stabilize the temperature of the system and then the pressure of the system. topology files were studied for the final results Fig 5: radial distribution function of Nitrogen of Glycine Betaine v/s ClFrom the Plot of radial distribution function of Nitrogen of Glycine Betaine v/s Cl. volume and temperature remains constant also known as isothermal-isochoric. In the second phase after stabilizing the temperature we stabilize the pressure and thus also the density of the system. and Temperature are all constant. The Density then decreases as distance increases and reaches a stable value .After the correct temperature is reached pressure is applied to have the correct density where final MD run will take found near the Nitrogen of Glycine Betaine due to the positive charge on Nitrogen. After these steps have been finished we start the production MD for data collection 4. and most closely resembles normal conditions where most biological functions occur MD Run Before we start the MD run we need to ensure that energy minimization and equilibration have been finished and the system has been brought to stable temperature and pressure level. where the Number of particles. Pressure. Also known as the "isothermal-isobaric" ensemble.ions in the system shows that a maximum density of Cl. Equilibration of pressure is conducted under an NPT ensemble.4 RESULTS After the MD run was finished the log file. We conduct the Equilibration in two phases. In the first phase we stabilize the temperature where we use NVT ensemble where number of particles.

Fig 6: Radial distribution function of Oxygen of Glycine Betaine v/s Hydrogen of water Same can be said about Hydrogen around Oxygen of Glycine Betaine as solvent forms a layer around Glycine Betaine with hydrogen oriented towards oxygen of GB. Fig 7: Plot of Hydrogen bonds between GB and water Hydrogen Bonds form between oxygen of GB and Hydrogen of water molecules .

Brunton. J. Jat R. Freire E.. REFERENCES 1. Arch Biochem Biophys 2001. Direct measurement of protein binding energetics by isothermal titration calorimetry date. FUTURE WORK: Glycine Betaine is known to stabilize proteins and also counteract the denaturing effect of urea on it. The binding energetics of first and second generation HIV-1 protease inhibitors: implications for drug design. Using Autodock with AutoDockTools. Ernesto 9. Ruth Huey. Velazquez-Campoy A. Parjapati Gunjan ..S.gromacs... 7. www. Kishore N: J Chem Phys. K.. Drug Res. Arthur J Olson.L. Exact analysis of competition ligand binding by displacement isothermal titration calorimetry.. Sigurskjold BW. Anal Biochem 2000. Manju.Using AutoDock for ligandreceptor docking.. 11:560– 566 2. Lazo..139(11):115104 6.. Article.The Scripps research institute . Stephanie Leavitt... 2013. 277:260-266 4. 8.L. (2006) Goodman and Gilmans´s the Pharmacological Basis of Therapeutics (11th edition). 3. K. J.2 189197 5. 2012.. Kumar N. L.5. United States of America: McGraw-Hill. 390:169-175. Kiso Y. Parker. Current Opinion in Structural Biology 2001. Garrett M Morris. Tech. It is essential to study effect of concentration of Glycine Betaine and NaCl on solvation of Glycine Betaine which is the important factor in the study [5] Future work will be focussed on studying effects of changing concentration of both salt and Glycine Betaine on its solvation 6. Kharb...Gupta Anju: Int.

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