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

2. 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

3. 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

4. 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: 2785-91. 2. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.

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

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

7. 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

8. 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, W5-9. + Edgar, et al. (2002) Nucleic Acids Res. 30, 207-210

9. 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

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

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

12. Type: ssh –X username@host DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

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

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

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

16. 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

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

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

19. 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

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

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

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

23.  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

24.  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

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

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

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

28.  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

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

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

31.  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

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

33.  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

34.  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

35.  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

36. 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

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

38. HM Validation: Ramachandran Plot Model assessment using RAMPAGE 1 webserver DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking 1. http://mordred.bioc.cam.ac.uk/~rapper/rampage.php

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

40. 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

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

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

43. 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

44. 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

45. 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.

46. 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

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

48. 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

49. Force Fields for Autodock Energy Function http://autodock.scripps.edu/resources/science/equations/ 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

50. Molecular Docking: Protocol http://Autodock.scripps.edu DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

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

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

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

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

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

56.  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

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

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

59.  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

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

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

62. 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

63. 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)

64. 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

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

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

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

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

69. 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

70. 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

71. 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

72. 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

73. 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

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

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

76. 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

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

78.  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

79.  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

80.  3D protein structure  Homology model of Col α1 (XI) NPP Protein Structure Viewed in PyMOL DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

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

82. Protein-Ligand Complex in PyMol Arixtra Lowest Ki = -6.51 kcal/mol DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

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

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

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

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