Using IFD-MD on a Membrane-bound protein

Tutorial Created with Software Release: 2023-4
Topics: Free Energy Perturbation (FEP), Hit Discovery, Hit-to-Lead & Lead Optimization, Small Molecule Drug Discovery, Structure Prediction & Target Enablement
Products Used: IFD-MD

Tutorial files

16 MB
This tutorial is written for use with a 3-button mouse with a scroll wheel.
Words found in the Glossary of Terms are shown like this: Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.the 3D display area in the center of the main window, where molecular structures are displayed
Abstract:

 

In this tutorial, we will prepare and set up a membrane-bound protein for use with IFD-MD. Our protein of interest is beta-2 adrenergic receptor, ADRB2,which resides within a lipid bilayer environment within the cell membrane. Structures of numerous membrane proteins orientated as they would be within a membrane environment can be obtained from the Orientations of Proteins in Membranes (OPM) database. We will use this information to prepare an ADRB2 structure with a membrane for use in an IFD-MD. Finally, we will view results of a pre-run IFD-MD calculation to observe how induced-fit effects in the binding site are taken into account with docking a different ligand from another ADRB2 structure into the initial complex.

 

Tutorial Content

  1. Introduction to IFD-MD

  1. Creating Projects and Importing Structures

  1. Preparing a membrane-bound protein for IFD-MD

  1. Setting up and submitting IFD-MD calculations with a membrane-bound protein

  1. Visualization and analysis of IFD-MD results

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to IFD-MD

Many structure-based drug design methods, including free energy perturbation (FEP+), require accurate, atomic-level detail structures of the target protein complexed with a member of the ligand series being explored to perform optimally; consequently, the domain of applicability of structure-based drug design (SBDD) is limited by the availability of high-resolution crystal structures. Even when the exact protein-ligand complex structure needed for design work is not available, accurate atomic-level detail structures are often available for similar systems. There is often a significant lag between the identification of a hit and the structural-enablement of drug discovery work around that hit. A new ligand subseries sometimes results in a changed binding mode. IFD-MD enables structure-based drug discovery before the relevant co-crystal structure is available.

Often the only differences in protein structure between the available structure and the one that is needed for SBDD are the movements of a couple of side chains or a small loop motion. However, These small changes can have a large impact on the accessible ligand binding modes. IFD-MD uses a combination of docking algorithms, water thermodynamics, empirical scoring functions, implicit solvent force field energies, and explicit solvent metadynamics trajectories to explore the motions of the target protein and simultaneously determine their relative energetics. This technology makes it possible to create accurate “first looks” at the protein-ligand interactions for novel active compounds before a crystal structure is solved, and even allows for accurate structures of protein-ligand binding when starting from homology models. IFD-MD integrates multiple Schrödinger applications into a single solution for predicting binding poses, operating as a series of coupled CPU and GPU jobs:

 

Figure 1-1. IFD-MD Subjobs. Each dashed vertical line is a checkpoint. The job can be restarted from any checkpoint.

Initial Pose Generation (Phase, Prime, Glide)

Initial pose generation is done by first pharmacophore docking your ligand of interest (target ligand) onto the ligand of an existing holostructure ligand (template ligand). This ligand-based docking initially ignores clashes to the receptor. Pharmacophore docking generates thousands of conformations of your target ligand. For each conformation, clashes with the receptor are resolved using Prime to produce potentially a unique receptor conformation for each ligand conformation. The new receptor conformations are then re-docked using Glide to create an initial induced fit ligand receptor complex. For more details, please consult the references mentioned at the end of the tutorial.

Molecular Dynamic Stages

Concerted ligand-receptor changes are accomplished through short time scale (500 ps) MD simulations. The short MD time-scale also includes the placement of explicit water molecules, necessary for inclusion in scoring with Glide WS. The stability of a pose is evaluated using multiple metadynamics (MtD) calculations. Poses with more consistent conformations score more favorably. Poses with greater conformational variability score less favorably. IFD-MD incorporates this MtD stability as a component of its scoring function. A final 100 ns MD simulation is performed on the top two IFD-MD structures if the structure appears unconverged after 500 ps.

Note: Drastic backbone changes, i.e. DFG-in to DFG-out, are beyond the scope of IFD-MD predictions.

2. Creating Projects and Importing Structures

At the start of the session, change the file path to your chosen Working Directorythe location that files are saved. in Maestro to make file navigation easier. Each session in Maestro begins with a default Scratch Projecta temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project., which is not saved. A Maestro project stores all your data and has a .prj extension. A project may contain numerous entries corresponding to imported structures, as well as the output of modeling-related tasks. Once a project is created, the project is automatically saved each time a change is made.

Structures can be imported from the PDB directly, or from your Working Directory using File > Import Structures, and are added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion. and Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data.. The Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion. is located to the left of the Workspace. The Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data. can be accessed by Ctrl+T (Cmd+T) or Window > Project Table if you would like to see an expanded view of your project data.

  1. Double click the Maestro icon to start Maestro

Figure 2-1. Change Working Directory option.

  1. Go to File > Change Working Directory
  2. Find your directory, and click Choose
  3. Pre-generated input and results files are included for running jobs or examining output. Download the zip file here: https://www.schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/membrane_ifd-md.zip
  4. After downloading the zip file, unzip the contents in your Working Directorythe location that files are saved. for ease of access throughout the tutorial

Figure 2-2. Open the project.

  1. Go to File > Open Project > membrane_IFD-MDl.prjzip
  2. In Save scratch project, click OK
  3. Go to File > Save Project As
  4. Change the File name to membrane_IFD-MD-adrb2
  5. Click Save
    • The project is named membrane_IFD-MD-adrb2.prj
    • Structures are shown in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

3. Preparing a membrane-bound protein for IFD-MD

Before we perform our calculations, we must prepare our structures for use with IFD-MD. We will also remove parts of the structure (T4 lysozyme, ions, and waters) that are not needed for this job. Additionally, we will also download a template structure of the protein that has been aligned in a lipid membrane from the OPM database. In this tutorial, the complex containing our ligand of interest (6PS5) has already been prepared and aligned to our protein of interest (4LDE). While the 6PS5 protein and the 6PS5 ligand, propranolol, have each been prepared and aligned in advance, it is important to remember these steps when performing calculations.

Structures obtained from the PDB, vendors, and other sources often lack necessary information for immediately performing modeling-related tasks. Typically, these files are missing hydrogens, partial charges, side chains, and/or whole loop regions. In order to make these structures suitable for modeling tasks, such as IFD-MD, we will use the Protein Preparation Workflow to resolve these issues. Please see the Introduction to Structure Preparation and Visualization tutorial for further information on using the Protein Preparation Workflow. Here, we will use the Protein Preparation Workflow default settings.

Similarly, ligand files can be sourced from numerous places, such as vendors or databases, often in the form of 1D or 2D structures with unstandardized chemistry. LigPrep can convert ligand files to 3D structures, with the chemistry properly standardized and extrapolated, ready for use in modeling tasks. Even though we will be using a ligand from a crystal structure for this IFD-MD calculation, LigPrep has been used on this ligand as part of our recommended best practices.

3.1 Preparing proteins and ligands for use in IFD-MD

Figure 3-1. Get PDBs from the Protein Preparation Workflow panel.

  1. In the Favorites Toolbar, open the Protein Preparation Workflow
  2. Click Get PDB

Figure 3-1. Download the PDB structures.

  1. Next to PDB IDs, type 4LDE
  2. Next to Chain name (optional), type A
  3. Click Download
    • The structure is added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
    • 4LDE is includedthe entry is represented in the Workspace, the circle in the In column is blue. in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Optional: Type z to zoom your Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. to fit the structure.

 

Note: We will use the default settings for the Protein Preparation Workflow here. See the Protein Preparation Workflow user manual for more information on the setting options.

Figure 3-2. Select the proteins and click run.

  1. In the Protein Preparation Workflow panel, next to Use structures from, choose Workspace (included entry)
  2. Check the Preprocess, Optimize H-bond Assignments, and Minimize and Delete Waters options
    • We will use the default settings
  3. Next to Job name, type autoprep_adrb2 and click Run
    • This job will take a few minutes to complete
    • The prepared protein complex is added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  4. In the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion., includethe entry is represented in the Workspace, the circle in the In column is blue. the 4LDE - prepared structure

Figure 3-3. Apply white ribbons to the 4LDE - prepared structure.

  1. In the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion., includethe entry is represented in the Workspace, the circle in the In column is blue. 4LDE - prepared
  2. Click Style and Ribbons
  3. Choose Constant coloring and white
    • White ribbons are applied to the protein structure

Figure 3-4. Render the ligand in white ball and stick.

  1. Type L on the keyboard
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. is zoomed into the ligand
  2. Double-click an atom in the ligand to select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries. the ligand
  3. For Color Atoms, choose Element + Custom Ligand and white
    • The ligand carbons are rendered in white
  4. Finally, click Ball and Stick
    • The ligand is rendered in ball and stick

Figure 3-5. Delete the crystallization artifact from the structure.

  1. In the Structure Hierarchy, expand the Ligands dropdown
  2. Right-click the 1WV ligand and choose Delete Atoms
    • The crystallization artifact is removed

Figure 3-6. Delete the waters from the structure.

  1. Expand the Solvents dropdown
  2. Right-click the Waters and choose Delete Atoms
    • The crystal waters are deleted  

Note: It is advised that waters are removed from a structure unless coordinating to a metal. IFD-MD uses a combination of implicit and explicit solvent to sample and place waters. Pre-existing water molecules can interfere with this workflow and are not considered necessary. Please see the IFD-MD panel documentation and IFD-MD Best Practices for more information.

Figure 3-7. Delete the ion from the structure.

  1. Expand the Other and Metals/Ions dropdowns
  2. Right-click the NA 1402 and choose Delete Atoms
    • The ion is deleted

Figure 3-6. Delete the T4 lysozyme.

  1. In the Sequence Viewer, click-and-drag to select D858 to D1029
  2. Right-click the selection and choose Delete
    • The T4 lysozyme, which is not needed for this calculation, is deleted
    • The protein is ready for use in IFD-MD calculations

3.2 Downloading template proteins from the OPM

Figure 3-7. Search for the protein in the OPM database.

  1. Open a web browser, such as Chrome or Firefox
  2. Navigate to https://opm.phar.umich.edu/
  3. Search for 4LDE

Figure 3-8. Download the template protein to use for alignment.

  1. Under the protein image, click Download OPM File: 4lde.pdb
    • The file is downloaded to your computer
  2. Locate this file and rename it to 4lde_OPM.pdb
  3. Move 4lde_OPM.pdb to your Working Directorythe location that files are saved.

 

Note: Renaming and moving the file is not essential, but helpful to avoid confusion with the original PDB file and to keep all files related to this work in one location.

4. Setting up and submitting IFD-MD calculations with a membrane-bound protein

With our prepared ligand and protein, plus the downloaded template protein with the correct orientation for the lipid membrane, we are now ready to set up and submit the IFD-MD calculation. For compute intensive jobs such as IFD-MD, you may prefer to write out the calculation input files and transfer them to a remote cluster for running. Here, we show how to submit the calculation from your local computer to a remote cluster using Job Server.

Figure 4-1. Include and select the input structures.

  1. In the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion., includethe entry is represented in the Workspace, the circle in the In column is blue. 4LDE - prepared
  2. In the ligprep group, select the 6PS5 - prepared_ligand1  

Figure 4-2. Load the input structures into the panel.

  1. Navigate to Tasks > Receptor-Based Virtual Screening > IFD-MD
    • The IFD-MD panel opens
  2. In the IFD-MD panel, under Docking, choose Noncovalent
  3. Next to Target Ligand, choose Project Table (1 selected) and click Load
    • The 6PS5 - prepared_ligand1 structure is loaded into the panel
  4. Next to Template complex from Workspace, click load
    • The 4LDE - prepared structure is loaded into the panel
    • The ligand is automatically detected

Figure 4-3. Add the membrane protein template.

  1. Check Add membrane to protein to template and click Browse
  2. Choose 4lde_OPM.pdb and click Open
    • The 4lde_OPM structure is loaded into the panel
    • The 4LDE - prepared protein will be aligned to the 4lde_OPM protein to have a standard orientation in the lipid membrane
  3. Next to Job name, type IFD-MD_4LDE
  4. Click the Job Settings cog
    • The IFD-MD Job Setting panel opens

Figure 4-4. Choose the CPU and GPU hosts.

  1. Choose the CPU subhost and number of processors
  2. Choose the GPU subhost and number of processors
  3. As this is a lengthy job, do not click Run
    • Clicking Run will submit this job from your local computer
    • We will use pre-generated results

Figure 4-5. Optional: Write out the input files.

Note: If preferred, you can write out the input files for transfer to a remote cluster to run this job. Be sure that the appropriate hosts are chosen in the Job Settings.

5. Visualization and analysis of IFD-MD results

Simply rigid-receptor cross docking propranolol, the ligand from the 6PS5 ADBR2 structure, into the 4LDE ADBR2 structure results in a poor binding pose whereas the pose from IFD-MD has good agreement with the 6PS5 complex. Here, we will compare the pose of propranolol in 4LDE from pre-generated Glide docking results to the pose of the same structures with IFD-MD. There are small induced-fit motions at SER1203, and SER1207 which need to occur to accommodate propranolol in the correct binding pose into the 4LDE binding pocket. Rigid receptor docking with Glide does not take these motions into account.

Figure 5-1. Compare the 6PS5 structure (dark green) with the Glide docking results of the 4DLE receptor and 6PS5 ligand (gray).

  1. Shift-click to includethe entry is represented in the Workspace, the circle in the In column is blue. the 6PS5_prepared and both glide-dock_SP_4LDE-6PS5ligand1_pv1 entries
    • The Glide pose of the 6PS5 ligand (gray) is flipped from the 6PS5 complex pose (green)
    • SER1203 and SER1207 from 4LDE (gray sticks) clash with the 6PS5 ligand pose

Figure 5-2. Compare the 6PS5 structure (dark green) with the IFD-MD results of the 4DLE receptor and 6PS5 ligand (white).

  1. Ctrl-click (Cmd-click) to includethe entry is represented in the Workspace, the circle in the In column is blue. the 6PS5_prepared and the first IFD-MD_4LDE-out entry
    • The IFD-MD pose of the 6PS5 ligand (white) agrees with the 6PS5 complex pose (green)
    • SER1203 and SER1207 from 4LDE (white sticks) do not clash with the 6PS5 ligand pose

Figure 5-3. Compare Glide docking results of the 4DLE receptor and 6PS5 ligand (gray) with the IFD-MD results of the 4DLE receptor and 6PS5 ligand (white).

Optional: Use ctrl-click (Cmd-click) to includethe entry is represented in the Workspace, the circle in the In column is blue. both glide-dock_SP_4LDE-6PS5ligand1_pv1 entries and the first IFD-MD_4LDE-out entry. Note how the induced fit movements of SER1203 and SER1207 open the binding pocket to accommodate the correct propranolol pose.

Optional: Use the Binding Mode Comparison Preset representation to easily visualize results.

6. Conclusion and References

In this tutorial we learned how to prepare membrane-bound protein structures for use with IFD-MD, set up an IFD-MD calculation with a membrane-bound protein, and compared the results of the IFD-MD output against Glide docking. The pose produced by IFD-MD using the 4LDE protein showed much better agreement with the known pose of the ligand of interest based on a crystal structure (6PS5). Rigid receptor docking with Glide was not able to capture induced-fit motions of residues in the binding pocket that are necessary for producing the correct pose. If the 6PS5 crystal structure were not available, the IFD-MD output of the 6PS5 ligand into the 4LDE protein would be able to be used in further modeling tasks, such as FEP+.

For further reading:

7. Glossary of Terms

Entry List - a simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

Favorites Toolbar - buttons for tasks designated as favorites in the Task Tool. You can add panels to your Favorites Toolbar by checking the star icon beside the panel name in Tasks.

included - the entry is represented in the Workspace, the circle in the In column is blue.

Project Table - displays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data.

Scratch project - a temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project.

selected - (1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries.

Working Directory - the location that files are saved.

Workspace - the 3D display area in the center of the main window, where molecular structures are displayed.