DockoMatic: Automated Tool for Homology Modeling and Docking Studies DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking Tutorial

Learning Objectives  I. Create & validate a protein homology model  II. Use molecular docking to calculate the binding energy for a drug to the protein  III. Assessment of docking results  IV. Visualize the protein-drug complex DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Systems of Interest  Protein: Collagen α1 (XI) NPP domain  223 amino acid protein  2 disulfide bonds  A minor fibrillar collagen involved in thrombosis  Drug: Arixtra (a.k.a. Fondaparinux)  A heparin-based drug  An anticoagulant used to treat blood clots  Binds Col α1 (XI) NPP to prevent blood coagulation Arixtra structure DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

DockoMatic 2.0  User friendly graphical user interface (GUI) for automation  Homology model creation using the Timely Integrated Modeller (TIM) interface  Create, submit, manage AutoDock 1 molecular docking calculations  Track jobs & analyze results using PyMol 2 DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking 1. Morris, G. M. et al. J. Computational Chemistry 2009, 16: The PyMOL Molecular Graphics System, Version Schrödinger, LLC.

How many structures solved? The Protein Data Bank * * Bernstein, et al. (1977) J. Mol. Biol. 112, 535. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Part I: Homology Modeling DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Homology Modeling: Overview  Prediction of 3D-structure of a given protein sequence (target) based on alignment to known protein structures (templates)  A useful model requires 30% sequence identity between target and template  Models used to understand function, activity, ligand binding paradigms in drug design, etc. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Homology Model: Template Selection 1. Begin with the target amino acid sequence 2. Use the Basic Local Alignment Search Tool * (BLAST) to search for amino acid sequences of template proteins where the 3D structure is available 3. The principle repository for 3D protein structures is the National Center for Biotechnology Information + (NCBI) 4. Only amino acid sequences with greater than 30% identity between target and template should be considered. 5. If functionality is known for a class or family of proteins, look to protein templates with similar functionality as the target. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking *Johnson et al. (2008) Nucleic Acids Res. 36, W Edgar, et al. (2002) Nucleic Acids Res. 30,

Homology Modeling: Flow Chart Target Sequence Model Building for the Target Template Selection Alignment (Target- Template) Evaluate the Model Known (Template) Structures Homology Model YESNO DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Homology Model Creation: DockoMatic Procedure DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Open Terminal Window DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Type: ssh –X DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Enter password DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

This is the HOME directory DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Type: cd $SCRATCH DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Type: mkdir DOCKOMATIC-WORKSHOP Type: cd DOCKOMATIC-WORKSHOP Type: dockomatic DockoMatic will now open in a new window DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

DockoMatic 2.0 GUI DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

DockoMatic  Select: Use Modeller DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

DockoMatic: Accessing the TIM Wizard  Click on: Receptor  Timely Integrated Modeller (TIM) Wizard will open in a new window DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

DockoMatic – TIM Wizard Interface  TIM Wizard DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 1: Enter Template Sequence  Click on: Output Directory DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 1 (cont’d): Sequence Entry  Select: The desired output directory  Click: OK DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Output directory path is now established  Note this path for future reference TIM Step 1 (cont’d): Sequence Entry DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Click on: ‘Sequence’ Choose the target.pdb file OR  Copy/Paste Sequence: If you don’t have a.pdb, you can copy & paste the sequence here. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking TIM Step 1 (cont’d): Sequence Entry

 Name the target sequence  Click: Next TIM Step 1 (cont’d): Sequence Entry DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 2: Search for a Template  BLAST Template Search DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Template list  BLAST output DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking TIM Step 2 (cont’d): Template List

 Select a template  Templates listed from highest match to lowest  Click: Next TIM Step 2 (cont’d): Template Selection DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 3: Sequence Alignment  Alignment in progress DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Select sequence of template that has been aligned with the target TIM Step 3: Select Aligned Sequence DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Aligned template and target sequence display.  Alignment may be edited in this window.  Click: Next TIM Step 4: Edit Aligned Sequence DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 5: Model Generation  Model generation in progress DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 List of homology models for target protein created based on template structure DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking TIM Step 5: List of Models Created

 Select a homology model  Models listed from best to worst correlation  Click: Finish TIM Step 5: Select Homology Model DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Receptor field shows selected homology model.  Do not close this window; it will be required for molecular docking. End TIM Wizard: 3D Model for MD DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Open a terminal window. Enter output directory path (you noted this earlier). Type: ls The content of the working directory contains the.pdb files of the homology models for the target protein along with all other output files generated by DockoMatic. Location of Model Structure Files DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Homology Model Validation DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

HM Validation: Ramachandran Plot Model assessment using RAMPAGE 1 webserver DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking 1.

HM Validation: Sequence Alignment DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

HM Validation: Structure Overlay Homology model for Col α1 (XI) NPP PDB ID: 2UUR Template Root mean square deviation of Cα atoms = 1.12 Å DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Part II: Grid Parameter File Creation DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Open AutoDockTools (ADT) In the terminal type: adt & Click on the Icon created at installation DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Click on: Grid Then click ‘Macromolecule’ in the popup menu, then ‘Open…’ You can either load a.pdb or a.pdbqt created from an earlier ADT use. Change the file type to ‘.pdb’, unless loading the.pdbqt file type. Choose your file, then click open. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

The file will load then imidiately ask you to choose a location to save the converted.pdbqt Choose your location and filename, then click save

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking Click on: Grid Then click ‘Grid Box…’ in the popup menu This allows you to define the area of interest in the molecule. It is described by xyz coordinates. The molecule can be rotated by clicking and dragging.

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking Click on: Grid Then click ‘Output’ in the popup menu, then ‘Save GPF…’ Choose your location and filename, then click save

Part III: Molecular Docking DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Molecular Docking: Background  Docking : Molecular simulation of a ligand binding to a receptor  Output: Binding energies & scoring functions  Visualization: Binding poses for ligands  Docking tools  AutoDock  DOCK  Glide  MOE  GOLD DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Force Fields for Autodock Energy Function ΔG H-bond = W H-bond ∑ i, j E(t) * ( C ij / r ij 12 - D ij / r ij 10 + E hbond ) ΔG vdW = W vdW ∑ i, j ( A ij / r ij 12 - B ij / r ij 6 ) ΔG elec = W elec ∑ i, j ( q i * q j ) / ( ε(r ij ) * r ij ) ΔG desolv = W desolv ∑ i (C), j (S i * V j * exp ( -r ij 2 / (2 * σ 2 ) ) ) ΔG tor = W tor N tor ΔG = ΔG vdW + ΔG H-bond + ΔG elec + ΔG tor + ΔG desolv DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Molecular Docking: Protocol DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Click: Output Directory Molecular Docking in DockoMatic DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Select output directory  Click: OK MD: Getting Started DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Click: Ligand MD Step 1: Selecting a Ligand DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Select a ligand structure file (arixtra.pdb)  Click: OK MD Step 1 (cont’d): Ligand Selection DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Click: Receptor MD Step 2: Receptor Selection DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Select the homology model of Col α1 (XI) NPP from part 1 as the receptor  Click: OK MD Step 2 (cont’d): Receptor Selection DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Click: Box Coordinates MD Step 3: Receptor Binding Site Selection DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Select a box coordinate file  Click: OK DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking MD Step 3 (cont’d): Receptor Binding Site Selection

 Select number of AutoDock jobs to run: default=100; for this exercise select 10  Click: New Job MD Step 4: Job Setup DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Your job is not yet started  Job status displayed here MD: Job Status DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Left mouse click to select  Right click to access menu DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking MD: Starting the Docking Jobs

Start Delete Analyze View in PyMOl  Right click initiates pop- up window MD: Starting the Docking Jobs (cont’d) DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Start Delete Analyze View in PyMOl  Click: Start to submit jobs  While jobs are running, review input and output files. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking MD: Starting the Docking Jobs (cont’d)

Return to terminal window and type ‘ls’ to list files Make sure path is correct; if not, use ‘cd’ to change directory Starting a job creates dock directories where results are located Type: cd dock_1 DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking Review Files in Terminal Window

Part IV: Assessment of Docking Results DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Result output files Type: more arixtra.pdbqt DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking Viewing the Contents of a Structure File

arixtra.pdbqt contents Type: q Type: more dock.gpf DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking Protein Data Base File for Arixtra

dock.gpf contents Type: more dock.dpf DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking Viewing the Contents of a Grid Parameter File

View the contents of the dock.dpf file Type: more dock.dpf Viewing the Contents of a Docking Parameter File DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

DockoMatic: MD Required Files  DockoMatic automation requires a receptor, ligand, and grid parameter file as input  GPF creation: To run an autogrid job you would type: autogrid4 –p dock.gpf –l _gridLog.glg & *****You need not to run autogrid job*****  To run an autodock job you would have to type: autodock4 –p _.dpf –l _dockLog.dlg & *****You need not to run autodock job***** DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Check gridLog file Type: tail -100 _gridlog.glg _gridlog.glg end of file contents (last 100 lines) This means autogrid completed successfully DockoMatic: Grid Parameter Files DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Check dockLog Type: tail -100 _docklog.dlg _docklog.dlg end of file contents (last 100 lines) This means autodock completed successfully DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking DockoMatic: Job Status in Terminal

To check the Cluster Histogram data Type: less _docklog.dlg Scroll down a long way until you get to the Clustering Histogram section. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking MD Result Analysis: Cluster Histogram

Part VV Visualize the Protein-Ligand Complex DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Left mouse click to select  Right click to display the menu Viewing MD Results DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Start Delete Analyze View in PyMOl  Click: View in PyMOL  A 3D model viewer should open in a new window MD Result Analysis: Visualization DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Select the top ranked result  Click: OK MD Analysis: Selecting a Result DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Open PyMol  Click: File, then Open  This will open the arixtra_rank_1.pdb file.  This is the ligand binding pose for the lowest energy docked complex MD Analysis in PyMOL DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 Select a protein structure file  Click: Open  This will provide the structure of the protein receptor molecule PyMOL: View Protein Receptor DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

 3D protein structure  Homology model of Col α1 (XI) NPP Protein Structure Viewed in PyMOL DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Collagen α1 (XI) NPP domain DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking Ribbon Structure of Protein in PyMOL

Protein-Ligand Complex in PyMol Arixtra Lowest Ki = kcal/mol DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

PyMol View of Protein-Ligand Interactions DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Close up View of Interactions between Arixtra and Binding Site Residues DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Conclusions  DockoMatic  Homology model  Model Creation  Structure Validation  Molecular docking  Docking Studies  Result Analysis DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Questions! & Thank You DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking