1. Molecular Mechanics and Dynamics Characterization of an In Silico Mutated Protein: A Stand-Alone Lab Module or Support Activity for In Vivo and In Vitro Analyses of Targeted Proteins BaMBEd

2. This week Learn about the software for modeling – Use the software to create representations of your protein highlighting features of interest – Make a mutant form of your protein – Solvate your mutant protein – Start your mutant protein “shaking” to find configuration that gives you the energy minimum Measure the ATP-binding pocket in wild type CK1

3. Your lab report In your growing lab report, you’ll – Make revisions – Report the modeling you set up this week (not in detail) – Report the size of the ATP-binding pocket in the wild type CK1 (probably in table form) If you don’t get these measurements finished this week, you can come in and use the computers next week to finish. Lab report due in two weeks.

4. Downloading the CK1 Structure Set up a folder to do your work in – C:/Users/Student/My Documents/CK1-modeling We are going to use a crystal structure for a CK1 from Schizosaccharomyces pombe Go to the Protein Data Bank (www.pdb.org)www.pdb.org Search for 1CSN – 1CSN is the pdb identifier for casein kinase - 1

5. Downloading the CK1 Structure Click “Download Files” and select “PDB File (Text)”

6. Dowloading CK1 structure The file is downloaded into the “Downloads” folder Pull up the Downloads window and drag the 1CSN.pdb file into the CK1-modeling folder you created.

7. Viewing the PDB File Text Right click on the 1CSN.pdb file and choose “Open With…” Click the radio button for “Select the program from a List” – Double click “Word” – You may want to adjust the margins to “narrow” under Page Layout to allow the file to be displayed well.

8. Anatomy of the PDB File The Header

9. Anatomy of the PDB File The amino acid sequence and secondary structure

10. Anatomy of the PDB File The atoms, their amino acid, and their coordinates

11. Anatomy of the PDB File Non-protein atoms like Mg, ATP, SO 4, H 2 O, etc.

12. Anatomy of the PDB File Connection table and end of file

13. Viewing Casein Kinase - 1 Open the program VMD

14. Viewing Casein Kinase - 1 You will see three windows

15. Viewing Casein Kinase - 1 In the VMD Main window select “File → New Molecule…”

16. Viewing Casein Kinase - 1 In the Molecule File Browser window that opens click “Browse” and find your pdb file (1CSN.pdb).

17. Viewing Casein Kinase - 1 The “Filename” region should now have the path to your pdb file, and “Determine file type:” should have PDB Click “Load”

18. Viewing Casein Kinase - 1 The enzyme will appear in the OpenGL Display window. Notice the various colors. What do they mean? Left click on the black region and move your mouse around.

19. Viewing Casein Kinase - 1 In the “VMD Main” window, click “Graphics → Representations”. This will open a new window

20. Viewing Casein Kinase - 1 Try changing the Drawing Method to see various representations like, CDW, CPK, Ribbons, new Ribbons, Cartoon, etc. Now, set it to Ribbons

21. Viewing Casein Kinase - 1 You’re now going to highlight some of the interesting features of CK1s Click on Create Rep In the Selected Atoms box, type “resname ATP” Under the Draw style tab, set the Drawing Method to “Lines” and select “Color ID” under “Coloring Method”. This should highlight the ATP in a different color than the whole protein.

22. Viewing Casein Kinase - 1 Highlight the ATP-binding pocket Create Rep Use the CPK drawing method and the Color ID coloring method to highlight residue 13 (this is Gly 83 in Yck2) Create Rep Use the CPK drawing method and the Color ID coloring method to highlight residue 15 (this is Gly 85 in Yck2). Keep the color the same for these amino acids because they’re both part of the ATP- binding pocket. Repeat these steps for residue 18, 35, 148, 149, and 150.

23. Viewing Casein Kinase - 1 Highlight the RD pocket Create Rep Use the CPK drawing method and the Color ID coloring method to highlight residue 124 (this is Arg194 in Yck2). Use a different color for the RD pocket than you used for the ATP-binding pocket. Create Rep Use the CPK drawing method and the Color ID coloring method to highlight residue 153 (this is Lys223 in Yck2). Keep the color the same for the amino acids that make up the RD pocket. Repeat these steps for residue 170.

24. Viewing Casein Kinase - 1 Highlight the activation loop Click Create Rep In the “Selected Atoms” box, type “residue 151 to 181” This is the activation loop Click “Create Rep” On the “Draw style” tab select “ColorID” under “Coloring Method”. Choose a different color than you used for the RD pocket or the ATP-binding pocket.

25. Creating Representation Here is a representation showing some important residues.

26. Measuring Distances You can select atoms by changing the mouse behavior in the Mouse menu to “Label → Bonds” Click on two atoms, and the distance between them will be shown

27. Measuring the ATP-binding pocket You’ll use this feature to measure the distance between atoms that make up the ATP-binding pocket.

28. Saving Protein for Simulation Open the Tk Console under the Extensions menu

29. Saving Protein for Simulation In TkConsole, change directory to your modeling folder It will depend on your computer folder hierarchy, but the command is something like cd../../../Users/Student/Documents/CK1-modeling If you close VMD for any reason, you will need to re-enter this command to put yourself back in the proper working directory. The default directory for VMD on Windows is C:/Program Files (x86)/University of Illinois/VMD if you mistakenly save work without changing directory first.

30. Saving Protein for Simulation In TkConsole, set a variable “csn” that stores only the protein set csn [atomselect top protein] Write a new pdb file storing only the protein (no ATP, Mg, SO 4, H 2 O will be saved) $csn writepdb csnp.pdb

31. Saving Protein for Simulation You can select anything and everything you want rather than just the protein just like when you created representations. Some other options all protein residue resname E.g., if you wanted to keep the ATP, you’d use the boolean operator OR set csnatp [atomselect top protein OR resname ATP]

32. Check Your New csnp.pdb Open with Word and make sure only amino acids 6-298 are in the file Beginning of file End of file

33. Open Your New pdb File First, delete 1CSN by selecting it in the VMD Main window. Next, select Delete Molecule from the Molecule menu. Then, File → New Molecule…

34. Generate a psf file The simulation software (namd ) requires a.psf psf stands for protein structure file Tells namd about Charges and ionization state of histidine The nature (stiffness) of bonds (C-C, C-N, C-O, etc.) Non-bonded interactions Angles (e.g., dihedral) NAMD stands for NAnoscale Molecular Dynamics It is pronounced NAM-DEE

35. Generate a psf file Open the automatic psf builder VMD Main Extensions → Modeling → Automatic PSF Builder

36. Generate a psf file 1. Change the Output basename to csn 2. Leave the default topology 4. Click Guess and split… 5. Click Create chains. A message may pop up. This is fine. Click OK. 3. Click Load input files Just do this once! Don’t be impatient—you won’t see anything happen unless you can see the console window.

37. 1. You should see some warnings. These are OK as long as you see the two Info sections saying the psf and pdb were written. Wait until you see “Updating structures” and you are returned to a blinking cursor. Check the TkConsole Output

38. Your modeling folder should have two files named csn. One is a.psf, and one is a.pdb. You can open the pdb in Word to make sure it is still amino acids 6-298 again. Check the Output Files

39. Open Your New Files in VMD First, delete csnp.pdb and csn.psf by selecting them in the VMD Main window. Next, select Delete Molecule from the Molecule menu. Then, File → New Molecule… Load csn.psf first, then load csn.pdb

40. Look at your New Files in VMD Display When the psf was generated, VMD added hydrogen to all of the amino acids (white tips) Hydrogens are not present in x-ray crystal structures because they are smaller (0.5 Å) than x-ray wavelengths (≥1 Å), and the single electron does not interact with x- rays

41. Make Your Mutation Open the Mutator VMD Main Extensions → Modeling → Mutate Residue

42. Make Your Mutation If you correctly loaded psf and pdb files, these will be correctly filled in for you. Change the output name to csn_mutation. Enter the mutation target residue number. Enter the mutation you want. Remember, the numbering is different for this file: Yck2S. pombe CK1Mutated aa code Arg194Arg130GLN Lys223Lys159GLN Lys240Lys176GLN Ser243Ser179ASP Run mutator

43. Check Your Mutation Open Tk Console and check that it completed successfully. The mutation should have created a pdb and psf file. Open the pdb file and make sure your mutation was successfully introduced. The file should be in your working directory (see slide 29).

44. Double mutations If you are doing a double mutation (e.g., R194Q/S243D), repeat the mutator steps to generate your second mutation, but use the mutated files with the single mutation as the starting point. Again, check your pdb file to make sure that both mutations you want are generated.

45. Solvating Your Protein NAMD is an explicit solvent molecular dynamics simulator. This means it has water molecules surrounding the protein. Some simulators use implicit solvent that mimics water dielectric without the molecules being present in the calculations. Implicit solvents are faster to simulate but typically cause proteins to adopt more closed configurations. Open the Solvator VMD Main Extensions → Modeling → Add Solvation Box

46. Solvating Your Protein If you correctly loaded psf and pdb files, these will be correctly filled in for you. Change Output: csn_mutation_wb Use Molecule Dimensions Click Solvate

47. Solvating Your Protein VMD creates psf and pdb files with the water box (_wb). Your new solvated protein should have appeared. Delete the unsolvated molecule in VMD and create a nice representation of your solvated one.

48. Coordinates of Your Protein We need to know where your protein in its water box sits in 3D space. Use TkConsole. set everyone [atomselect top all] measure minmax $everyone Returns {x min, y min, z min }{x max, y max, z max } Store these somewhere (in a lab notebook or in a file) See image below for what the output looks like

49. Equilibrating Your Protein Calculate the center of your water box. Calculate the size of your water box

50. Equilibrating Your Protein We need to get the protein to adjust itself because the mutation and water introduce new forces. In physics, forces have the potential to push things around. This is potential energy. NAMD will attempt to rearrange the protein to minimize the potential energy. This is the same as trying to find where all the forces exactly cancel one another. Delete all open molecules using VMD Main. Open your mutated.psf that has the water box, and without closing the open window, browse to and open your mutated.pdb that has the water box in the same window. You can make a nice representation if you wish.

51. Equilibrating Your Protein Open the NAMD Graphical Interface Extensions → Simulation → NAMD Graphical Interface

52. Equilibrating Your Protein If your directory is already set in TkConsole, Working dir will be correct You will want to add “_min” to Simulation basename The input files should be correct. They were guessed based on what you have open. The parameter files should be okay too. Make sure Minimization is checked, and the number of steps is 10,000 (not 1000)

53. Equilibrating Your Protein Next we need to tell NAMD the type of simulation we want. Open the Ensemble panel Edit → Ensemble

54. Equilibrating Your Protein Next we need to tell NAMD the type of simulation we want. Open the Ensemble panel Edit → Ensemble We will use NVT Constant number of objects Constant volume of the water box Constant temperature of 310K Select Periodic boundary conditions… You will get an error about not finding and xsc file. Click OK, and reopen the Ensemble panel.

55. Equilibrating Your Protein Click Edit to open a panel for entering the periodic boundary conditions. In this panel you enter the values calculated earlier The values should be separated by a space Close these two windows by clicking Ok. Dr. Brame says: If the NAMDgui gives you an error message about -1, close the NAMDgui and go to VMD Main. Set the box on the bottom lefthand corner to zero and press the arrow at the bottom right. Then repeat the NAMDgui work.

56. Equilibrating Your Protein Finally, select Particle Mesh Ewald This speeds up the calculation of electrostatic (charge) interactions. Close the window.

57. Equilibrating Your Protein Open the Other Simulation Parameters panel Edit → Other Simulation Parameters

58. Equilibrating Your Protein Open the Other Simulation Parameters panel Edit → Other Simulation Parameters Check that all of the values are as shown. Close the window

59. Equilibrating Your Protein Click Run NAMD

60. Equilibrating Your Protein The minimization will take 30 – 60 minutes. It will say Status: Running until it completes

61. Equilibrating Your Protein You should have a bunch of new files with “_min”.

62. Simulating Your Protein Now, we’re ready to run a simulation. Go back to the NAMDgui panel. Change the basename to have “MD” Use the.coor or restart.coor file for the pdb input Use the restart.xsc for the xsc input Number of steps = 1,000,000 = 1 ns Select Molecular dynamics Select Continue simulation

63. Simulating Your Protein The molecular dynamics simulation attempts to make the enzyme behave as it would in a cell. When it is finished you will be able to watch the enzyme “jiggle”. You will also be able to make measurements to see if the mutation had an effect on the average distance between the activation loop and the RD pocket 1,000,000 steps will take from one day to one week to complete, depending on the speed of the computer. It can be broken into multiple runs of fewer steps using the Continue simulation feature.

64. Simulating Your Protein Double-check that nothing has happened to your ensemble values. Edit → Ensemble

65. Simulating Your Protein Click “Run NAMD” and wait…

66. Measuring Distances After your simulation completes, you can load the.psf (same …wb.psf you have been using. csn_r130a_wb.psf in this tutorial) and the new.dcd file (csn_r130a_wb_MD.dcd). The psf file stores charge and other information for all of the atoms but nothing related to position. Therefore, nothing will appear in the VMD modeling window when a psf file is loaded. The dcd file is essentially a stack of pdb files that are snapshots of the atom positions through the simulation. As it loads in VMD, you will see it step through all of the snapshots. Select two atoms of interest (e.g., atoms within residues on different sides of the ATP- binding pocket).

67. Measuring Distances You can step through your simulation, and the distance between your selected atoms show you how the distance changes as the simulation ran.

68. Measuring Distances The root mean square distance can be calculated. This value tells you how far apart two things are. Will compare the distance between your mutation and We will use a script called distance.tcl to calculate the distance at each saved timestep. In the TkConsole enter the following two commands source distance.tcl distance “resid 243” “resid 130” 50 distance.csv histogram.csv Open the file histogram.csv in Excel

69. Measuring Distances You should have two columns of data corresponding to the distance between the center of mass of the two selections (residue 243 and 130) and the number of occurrences where the simulation has that distance Plot these as x = distance (Å) and y = occurences Distance Occurrences

70. Measuring Distances Plotting data from a wild type and a mutated (Yck2:S243D and 1CSN:S179D) simulation gives the following graph.