Protein-Ligand Binding Interactions

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

MASTER OF SCIENCE IN CHEMISTRY

Submitted by Manisheel Gautam Roll No: 09103009

Under the Supervision of Dr. Nand Kishore,

DEPARTMENT OF CHEMISTRY, INDIAN INSTITUTE OF TECHNOLOGY BOMBAY-400076

NOVEMBER, 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, Indian Institute of Technology, Bombay, India, under the supervision of Dr. Nand Kishore. This report Protein-Ligand Binding Interactions is submitted for the partial fulfilment of the degree of Master of Science (M.Sc.) in Chemistry at IIT Bombay, has not been submitted elsewhere for a degree. In keeping with the general practice of reporting scientific observations, due acknowledgements has been made wherever the described is based on finding of other investigation.

Manisheel Gautam 09103009 Department Of Chemistry

Declaration

I declare that this project titled Protein-Ligand Binding Interactions has been carried out by Manisheel Gautam, under my supervision.

------------------------------Signature of the Guide Dr. Nand Kishore Dept. of Chemistry, IIT Bombay

CONTENTS 1. Introduction 2. Objective and Approach PART 1 3. Docking

3.1 Literature review 3.2 Computational Details 3.3 Results and Observations 3.4 Interaction Profiles 3.5 Clustering of Conformations PART 2 4. Molecular Dynamics Simulation

4.1 Introduction 4.2 Computational Details 4.3 Setting up of simulation 4.4 Result 10. Future Work 11. References

1. INTRODUCTION The synergising effect of Glycine Betaine and urea is known to negate denaturing effect of urea on proteins .Glycine Betaine stabilizes proteins. 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. As there will be different solvation properties when both Glycine Betaine, salt are present in the solution. In the study we will analyse distribution functions, hydrogen bonds, and energetics to understand different interactions and their solvation properties. 2. OBJECTIVE AND APPROACH To study more about the synergising effect of urea and Glycine Betaine over denaturation of 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. We study how to find active site in a protein, see how inhibitors of a protein work and how they bind to proteins. We perform docking on several ligands with HIV protein and then examined them to find the best fit configuration. 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. In the simulation we solvate Glycine Betaine molecules in water with NaCl molecules. From the results we get we plot the radial distribution function, 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

PART 1

3. DOCKING

3.1 LITERATURE REVIEW First of all, we will have a brief look at the subject of discussion here i.e Protein-ligand binding interactions Protein can selectively bind to other molecules. This function is very important for: Enzyme function, Receptor actions, Self-organization cellular structures and multicomponent protein complexes. Both quantitatively and qualitatively it becomes very important to understand it. Protein-ligand binding is a spontaneous process in which forces at work are similar to forces in Protein folding. Function of proteins is defined through its interactions with other molecules A protein can often have sites where multiple ligands can come and bind. The second ligand which binds to the site is termed as an inhibitor if it is not biologically productive. A Substrate comes and fits into an enzyme with a complementary shape and form a product. But when a structurally similar compound comes and binds it does not necessarily lead to formation of a product. But when both the structurally similar compounds are present it might reduce ability of enzyme to form the product with the incoming substrate. Therefore the other molecule inhibits the reaction and is termed as an inhibitor. 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 .so the use of protease inhibitor combined with a reverse transcript inhibitor is growing to negate the effect of mutations in treating HIV. 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. Also drug resistant mutated viruses may develop. SO we work with a combination of such ligands to negate the effect of mutations on the treatment 3.2 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. Molecular docking uses various methods and

algorithms like molecular dynamics, Monte Carlo stimulation, 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. 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. 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. It was used to study the binding of different HIV inhibitors with HIV protease and check the final docking file for the conformations. We use VMD to visualise the different conformations 3.3 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. In AutoDock we take a flexible ligand where we restrict its torsions and bind it to a rigid molecule. We write flexible residues in a different pdbqt file giving it the name hsg1_rigid.pdbqt.[9] Preparing the Grid Parameter File. The grid parameter file has the information about the grid map. By checking the PDB file of protein we find out the active site of the protein and set up a grid box around it. We set up the grid maps around the site around the receptor site and also type of the maps .A different map is calculated for every atom type found in the ligand and also we have an electrostatics map.[9] Setting up of ligand A number of steps are involved in setting up a ligand: Ligand is loaded on AutoDock. ADT detects whether charges already exist on the ligand or not. Gasteiger charges are added if there are no charges. 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. The bonds which are rotatable are the single bonds. Normally amide bonds are not rotatable. After the ligand is prepared it is saved as a pdb file. [9]

3.4 RESULTS AND OBSERVATIONS Docking was done on the HSG1 protein and 9 different inhibitor ligands Saquinavir, Indinavir, Ritonavir, Nelfinavir, Amperanavir, Lopinavir, Atazanavir, Tipranavir and Darunavir with 50 conformers of each ligand. The search algorithm used was Genetic algorithm with 2500000 number of evaluations Number of generations 27000, and number of genetic algorithm run -150 .After docking all docking log files were checked and best conformers of each docking were tabulated with the docking energy. 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 -10.8 -7.8 -8.0 -8.2 -10.6 -8.7 -7.4 -4.4 -7.2

But maximum Binding Energy doesnt mean it is the most effective treatment for HIV . Sometimes combination of inhibitors are used for treatment

3.5 INTERACTION PROFILES

Fig 1: Interactions between Amperanavir ligand and protein residues The Interaction image shows the residues of the protein interacting with the ligand. In case of Amperanavir Lys, Gly, Val, 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. The dimer consists of two amino acid monomers. Each monomer has acid residue which are important for catalysis [7]

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

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. 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. After the comparing similar conformations are clubbed together into clusters this process is called clustering of conformations. [9] In the Clustering process the starting clustering rmsd is 0.5. AutoDock clusters at this rmsd if no changes are made to default settings. 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. When viewing clustering results, we can see the different rmsd between different cluster families

PART II

4. MOLECULAR DYNAMICS SIMULATION

4.1 INTRODUCTION GROMACS is a software used to perform molecular dynamics, i.e. to simulate the Newtonian equations of motion for systems with hundreds to millions of particles[8].

we use Gromacs to simulate biochemical molecules like proteins , nucleic acids etc. 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. 4.2 COMPUTATIONAL DETAILS Molecular dynamics simulation were performed for aqueous Glycine Betaine + NaCl system. The simulation were carried out using NVT-NPT ensemble at a temperature of 300 K. Periodic boundary conditions were applied in x, y and z directions. The integrator used for simulations was leap-frog integrator at a time step of 2 fs. Brendsen Coupling algorithm was used to maintain the temperature and pressure with a time constant of 0.1 ps. In all simulations OPLS force field was used for Glycine Betaine and NaCl 4.3 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. The box was then solvated with water molecules 75 molecules of salt (NaCl) molecules were put in the box Approx. 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, 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, the system may collapse. We need the system to attain a temperature here the simulation is going to take place and have the proper orientation

After the correct temperature is reached pressure is applied to have the correct density where final MD run will take place. We conduct the Equilibration in two phases. We must first stabilize the temperature of the system and then the pressure of the system. In the first phase we stabilize the temperature where we use NVT ensemble where number of particles, 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. Equilibration of pressure is conducted under an NPT ensemble, where the Number of particles, Pressure, and Temperature are all constant. Also known as the "isothermal-isobaric" ensemble, 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. After these steps have been finished we start the production MD for data collection 4.4 RESULTS After the MD run was finished the log file, 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- ions in the system shows that a maximum density of Cl- is found near the Nitrogen of Glycine Betaine due to the positive charge on Nitrogen. The Density then decreases as distance increases and reaches a stable value

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

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