Enzyme Engineering with BioLuminate

Tutorial Created with Software Release: 2025-4
Topics: Enzyme Engineering
Products Used: BioLuminate, Desmond, Glide

Tutorial files

1.08 GB
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

 

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Abstract:

 

In this tutorial, you will investigate the effect of mutations in an alkene reductase from the Old Yellow Enzyme family on enzyme stability and ligand binding. By following along with this case study, you will learn how to combine various tools into a workflow for enzyme engineering. It covers essential steps for structure preparation, ligand interaction analysis, molecular docking, residue scanning calculations, and molecular dynamics simulations.

Tutorial Content

  1. Introduction to Enzyme Engineering

  1. Creating Projects and Importing Structures

  1. Preparing the Structure

  1. Running and Analyzing the Enzyme-Substrate Docking Job

  1. Finding Mutation Hostposts with MM-GBSA Residue Scanning

  1. Investigating the Impact of a Mutation on the Ligand Binding Mode through MD Simulations

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to Enzyme Engineering

Enzymes are widely used for industrial applications as they are highly specific, selective, safe and efficient in enabling chemical reactions. However, many wild-type enzymes can not be directly used at an industrial scale, as parameters like long half-life, retention of activity under extreme conditions (pH and temperature), tolerance towards organic solvents, and substrate specificity need to be optimized.

Enzyme engineering overcomes these limitations by altering the amino acid sequence using recombinant DNA technology, site-directed mutagenesis, or computational predictions to improve an enzyme’s activity, stability, selectivity or substrate specificity. There are various strategies for enzyme engineering, such as rational design, directed evolution, and semi-rational design. In this tutorial, we are using the rational design approach guided by enzyme structure and function. In this strategy, we analyze the enzyme’s structure, compare it with homologous sequences, and use computational tools to identify key residues that can be targeted for mutation. To know more about enzyme engineering strategies, please refer to this publication.

This tutorial uses the 4GE8 structure – an engineered alkene reductase which belongs to the Old Yellow Enzyme (OYE) family. The 4GE8 crystal structure is complexed with (S)-Carvone, which is a model substrate to explore the enzyme’s activity and stereoselectivity in biocatalytic applications. This enzyme structure originates from a mutagenesis study which aimed to overcome the steric limitations of the wild-type enzyme by expanding the active site volume, particularly around residues near the β-position of the substrate. The objective was to identify OYE mutants capable of accommodating bulkier alkene substrates and producing enantiomerically pure alkanes. To achieve this, Padhi et al. performed a site-saturated mutagenesis at position 116, substituting TRP with smaller amino acids. While the design was rationally guided, the discovery of the OYE1 W116I mutant’s unique stereochemical behaviour was somewhat unexpected. The ILE 116 mutant did not only improve activity, but altered substrate binding orientation leading to the formation of products with opposite stereochemistry compared to the wild-type. For more information, see this publication.

Therefore, we will begin with the ILE 116 mutant to validate whether the docking and residue scanning calculations align with the findings reported in this publication. By comparing the engineered ILE 116 and the wild-type TRP 116 variants, we aim to understand why certain mutations lead to improved substrate accommodation while others prove deleterious. We will also illustrate how computational tools can be applied prospectively to guide enzyme engineering.

In this tutorial, you will learn how to

  • prepare the enzyme structure for modeling applications,
  • perform ligand interaction analysis for identifying the key molecular contacts,
  • perform molecular docking calculations for exploring binding modes of the substrate,
  • perform residue scanning calculations to evaluate how enzyme engineering has significantly improved the enzyme properties compared to the wild-type, and
  • perform MD simulations to understand how mutation influences substrate binding and stability in the active site.

You will evaluate the effects of back-mutations, focussing on TRP 116 – ILE back-mutation. This analysis helps assess how specific engineered changes influence the enzyme’s stability and binding affinity relative to the native form. Please note that although we are applying this approach to examine back-mutations, the same methods can also be used in the forward direction – to identify potential mutation hotspots for enzyme engineering based on evolutionary conservation and variability among homologous sequences.

2. Creating Projects and Importing Structures

At the start of the session, change the file path to your chosen Working Directorythe location where 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 saved, the project is automatically saved each time a change is made.

Structures can be built in Maestro or can be imported using File > Import Structures (or drag-and-dropped), and are added to the Entriesa 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 Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table is located to the left of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. 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. The Toggle Table is another way to interact with structures and can be accessed via Window > Toggle Table.

  1. Double-click the BioLuminate icon.

Figure 2-1. Change Working Directory option.

  1. Go to File > Change Working Directory.
  2. Find your directory, and click Choose.
  3. Pre-generated 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/enzyme_engineering.zip
  4. After downloading the zip file, unzip the contents in your Working Directorythe location where files are saved for ease of access throughout the tutorial.

Figure 2-2. Saving the Project.

  1. Go to File > Save Project As.
  2. Change the File name to enzyme_engineering.
  3. Click Save.
    • The project is now named enzyme_engineering.prj.

Figure 2-3. The Get PDB File dialog box.

 

  1. Go to File > Get PDB.
    • The Get PDB File dialog box opens.
  2. For PDB IDs, type 4GE8.
  3. Click Download.
    • A banner appears and the structure is loaded into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Note: Imported structures in Maestro are 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 and 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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion by default. Please refer to the Glossary of Terms for the difference between includedthe entry is represented in the Workspace, the circle in the In column is blue and 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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries.

3. Preparing the Structure

Structure files obtained from the PDB, vendors, and other sources often lack necessary information for performing modeling-related tasks mostly due to experimental limitations of structural biology techniques and insufficient or ambiguous data. Typically, these files are missing hydrogens, partial charges, side chains, and/or whole loop regions. To make these structures suitable for modeling tasks, you will use the Protein Preparation Workflow to find and resolve common structural issues.

The Protein Preparation Workflow in Maestro involves a series of structural and functional checks to prepare structures for accurate molecular modeling and simulations. First, the structure is assessed for missing atoms or residues which are then added or corrected. Hydrogen atoms are added to ensure proper protonation states, and the structure is optimized for correct bond lengths, angles, and torsions. The overall geometry is checked for any steric clashes or unusual bond geometries. Functional checks include evaluating the protonation states of ionizable residues at the relevant pH and ensuring proper orientation of active site residues. The final structure is minimized to relieve any unfavorable interactions and prepare it for further computational analysis, such as docking or molecular dynamics simulations.

In this section, you will prepare the enzyme structure using the Protein Preparation Workflow panel to make the structure suitable for modeling tasks performed later in this tutorial. For detailed information on how to prepare protein structures, please refer to the Introduction to Structure Preparation and Visualization tutorial, and Best Practices for Protein Preparation.

Figure 3-1. Opening the Protein Preparation Workflow panel.

  1. Click Prepare in the banner or alternatively click Protein Preparation in the Favorites toolbar.
    • The Protein Preparation Workflow panel opens in the Preparation Workflow tab.

Figure 3-2. The Valences tab in Diagnostics showing the valence errors in the structure.

Before preparing the structure, you should review potential issues.

 

  1. Go to the Diagnostics tab and click Check Workspace Entry.

 

The Valences tab shows valence errors present in the structure. These are caused by crystallographically invisible Hydrogen atoms and will be resolved during the Preprocess step of preparation.

Figure 3-3. The Missing tab showing the missing atoms for ASP 164 residue.

The Missing tab shows missing side chain atoms for the ASP 164 residue in chain A. This will be automatically fixed during the Preprocess step of preparation.

Figure 3-4. The Alternates tab showing the alternate positions of the residues.

The Alternates tab shows several residues that can crystallize in two distinct positions. The precise positioning of most of these residues will not affect the subsequent analysis in this tutorial, as they are not located in or near the binding pocket. So you will proceed with the default positions.

 

The ILE 116 residue is positioned close to the binding pocket; it does not directly interact with the ligand. You will keep the default position as it is the predominant one, exhibiting higher average occupancy in the crystal structure.

 

  1. Shift+Click to select all table rows.
  2. Click Commit.
    • Alternate residue positions are deleted, and only the default positions are retained.

Figure 3-5. The Substructures tab.

You can also review the contents of the structure. This is an easy way to remove crystallographic artifacts or some of the chains (and ligands or solvents associated with them) if you need to.

 

  1. Go to the Substructures tab and click Load Workspace Entry.
    • The tables are populated.

Figure 3-6. Reviewing the structure and deleting the crystallographic artifacts before preparation.

Notice that the 4GE8 structure contains ions such as Na+, Mg2+ and Cl-, and pentaethylene glycol  molecules (labeled as 1PE). These are crystallographic artifacts and do not contribute to the enzyme’s catalytic activity. You will delete them as they have no biological relevance.

 

  1. In the Ligands, Metals, Other table, Ctrl+Click (Cmd+Click) to select all the MG XXX Mg, CL XXX Cl, NA XXX Na, and 1PE XXX entries.
  2. Click Delete From Entry.
    • The structure with deletions is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and included in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Notice that the Ligands, Metals and Other table has FMN 411 and 0WU 414 ligands. The FMN 411 is a flavin mononucleotide cofactor, a prosthetic group that plays a crucial role in catalysis by mediating hydride transfer during the reduction. 0WU 414 serves as the representative substrate to explore enzyme activity and stereoselectivity in biocatalytic applications.

For workflows such as molecular dynamics simulations, ions will later be added during system setup to neutralize the system, so you can remove any non-functional metal ions present in the original structure during preparation. Beyond neutralization, additional salt concentrations can also be introduced to more accurately mimic the physiological conditions. Please see the Best Practices for Protein Preparation for more details.

Figure 3-7. Checking the simulation pH in the Global Settings.

After reviewing the structure, you can now set up the preparation workflow.

 

  1. Return to the Preparation Workflow tab.
  2. Click Global Settings to check the preparation pH.

Note that the enzyme structure is being prepared at a physiological pH of 7.4, which is suitable since OYEs exhibit optimal activity in neutral to slightly alkaline conditions. However, when working with your own system, it’s crucial to prepare it at the physiologically relevant pH. After the preparation, you should also confirm the protonation states of the catalytic residues, cofactor (if any), and the substrate, as enzymes can exhibit uncommon protonation patterns which can be challenging for protonation state assignment algorithms. Maintaining the correct pH is vital for the enzyme’s catalytic efficiency and structural stability.

Figure 3-8. Changing the Minimize and Delete Waters default settings and running the preparation job.

We recommend keeping all the crystallographic water molecules throughout the structure preparation. You may delete them later for workflows that require dry structures.

 

  1. Under Minimize and Delete waters, click Settings.
  2. For Delete Waters, uncheck Distant from ligands (hets).
  3. Change the Job name to proteinprep_4GE8.
  4. Click Run.
    • This job takes ~ 2 minutes.
    • Once the job is completed, a banner appears and a new group proteinprep_4GE8-out is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

Figure 3-9. Viewing the multiple states of the FMN cofactor in the Substructures tab after structure preparation.

 

Ligands/cofactors can exist in multiple protonation or tautomeric states. You can check these states in the Substructures tab after preparing the structure.

 

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue 4GE8 - prepared.
  2. In the Protein Preparation Workflow panel, go to the Substructures tab.
  3. Click Load Workspace Entry.
    • Notice that the FMN 411 cofactor has three states. The most likely state i.e. S1 is checked by default.
    • The state assignments for the ligand and cofactor here are correct, so no adjustment is needed.
  4. Close the Protein Preparation Workflow panel.

By default, Maestro assigns the most probable state at physiological pH, which works well in most cases. However, for ligands with acidic/basic groups, metal coordinating groups, or when binding occurs in unusual pH environments, you can explore other states. After switching to the alternate state, you can rerun the H-bond optimization step of the Protein Preparation Workflow in Interactive mode for the most accurate geometry. For more information, please see the Protein Preparation Workflow panel documentation.

Figure 3-10. Duplicating the prepared structure.

To use the prepared structure for docking calculations, you will duplicate it and remove the water molecules from the duplicated structure. Note that Glide considers water molecules as the immutable part of the receptor. Read this article for detailed information.

 

  1. Right-click 4GE8 – prepared entry and choose Duplicate > Entries Only (In Place).

Figure 3-11. Renaming and deleting all water molecules in the duplicated structure.

  1. Double-click the duplicated entry and rename it to 4GE8 – prepared_dry.
  2. In the Hierarchy, expand Solvents.
  3. Right click Waters and choose Delete Atoms.
    • All the water molecules are deleted.

4. Running and Analyzing the Enzyme-Substrate Docking Job

In this section, you will learn how to perform an enzyme-substrate docking calculation. Please note that although the 4GE8 crystal structure already contains the bound substrate (S)-Carvone, we will use this system to demonstrate the docking workflow. By re-docking the substrate into the enzyme binding site, you will compare the predicted docking poses with the experimentally observed crystal structure pose to validate that the structural model, preparation, and docking setup are sound before making any changes to the sequence. Furthermore, docking extends beyond finding the favorable binding poses for active ligands. If any known non-binders or inactive mutants are available, then docking can be used to compare their binding patterns or assess how mutations disrupt key interactions within active sites. Such comparisons can provide valuable insights into ligand recognition, specificity, or the structural basis of activity loss.

4.1 Prepare the ligand using LigPrep

The LigPrep panel is used to set up ligand preparation calculations. Ligands are often 1D or 2D structures with unstandardized chemistry – missing hydrogens, incorrect geometries, inappropriate protonation states, or undefined chiral centres, which can lead to erroneous docking results if not addressed. Additionally, Glide requires ligands in specific formats, e.g., 3D structures with filled valences and optimized geometries to evaluate binding interactions effectively. So, you will prepare the (S)-carvone substrate using the LigPrep panel to meet all the necessary requirements for Glide docking.

Figure 4-1. Splitting the prepared dry structure into substrate and cofactor.

First, you need to isolate the substrate from the enzyme into its own entry.

 

  1. Right-click 4GE8 – prepared_dry and choose Split > Into Ligands, Water, Other.
    • A new group with separate entries for FMN cofactor, S-carvone substrate, and the enzyme is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and included the entry is represented in the Workspace, the circle in the In column is bluein the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 4-2. Renaming the splitted entries for the cofactor and the substrate.

To improve clarity, you will now rename the separated ligand entries.

 

  1. Double-click 4GE8 – prepared_dry_ligand1 and rename it to FMN_cofactor.
  2. Double-click 4GE8 – prepared_dry_ligand2 and rename it to S-carvone_substrate.

 

Note: The FMN cofactor will be considered as a part of the receptor (rigid) while generating the receptor grid.

Figure 4-3. Opening the LigPrep panel from Tasks.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue only S-carvone_substrate in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Find and open LigPrep (3D conversion) from Tasks.
    • The LigPrep panel opens.

Figure 4-4. Modifying the LigPrep panel default settings and running the job to prepare the substrate for docking.

  1. For Use Structures from, choose Workspace (1 entry).
  2. Under Stereoisomers, choose Determine chiralities from 3D structure for Computation.
  3. Change the Job name to ligprep_S-carvone.
  4. Click Run.
    • This job takes a few seconds.
    • Once the job is compleA new group ligprep_S-carvone-out is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  5. Close the LigPrep panel.  

Note: To learn about the requirements a ligand structure must meet for Glide docking, see the Ligand Preparation for Glide Documentation. For a more detailed introduction to using LigPrep, see Introduction to Structure Preparation and Visualization and Structure-Based Virtual Screening Using Glide tutorials.

4.2 Set up a docking calculation

Figure 4-5. Opening the Ligand Docking (Beta) panel from Tasks.

For docking, you should use the dry structure unless your system has water molecules mediating interactions between the enzyme and the substrate i.e. it has exceedingly stable (“structural”) waters.

 

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue 4GE8 – prepared_dry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Find and open Ligand Docking (Beta) from Tasks.
    • The Ligand Docking (Beta) panel opens in the Docking Setup tab.

Figure 4-6. Choosing to create a Receptor grid in the Docking Setup tab.

  1. For Configure settings for, choose Small molecules.
  2. Under Inputs, for Receptor grid, choose Create New.
    • The Receptor Grid Generation panel opens in the Receptor tab.

Figure 4-7. Identify the substrate molecule to generate the receptor grid.

 

Next, you can identify the binding site by using the co-crystal ligand in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

  1. Under Define receptor, confirm that the boxes for Pick to identify the ligand (Molecule) and Show markers are checked.
    • A banner in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed prompts you to click on an atom in the ligand.
  2. Click on an atom in the S-Carvone substrate (0WU 414) in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • A purple box appears around the ligand with all the ligand atoms highlighted. This box defines the region that the docked molecule(s) can occupy.
    • The ligand will be excluded from the grid generation.

Our example system has a bound substrate, which implies we know the location of the binding site in our target enzyme. Consequently, we have demonstrated how to generate a receptor grid for docking calculation using the centroid of the workspace ligand.

 

If you don’t know where the binding site of your target enzyme is, you can run a SiteMap calculation in Identify mode to identify putative binding sites. The identified binding sites are returned and ranked by SiteScore. You can then use the top ranked binding site to define the receptor grid by following these steps:

  • In the Receptor tab of the Receptor Grid Generation panel, confirm that boxes for Pick to identify the ligand and Show markers are checked.
  • Switch from “Molecule” to “Entry” in the dropdown menu.
  • Click on any of the site points (white spheres) of the top ranked site in the Workspace. Your site will be now distinguished from the receptor.
  • Run the Receptor Grid Generation job.

 

For detailed instructions on how to use the SiteMap panel to identify binding sites in your target structure, please refer to the Target Analysis with SiteMap and WaterMap tutorial and SiteMap panel documentation.

 

If you don’t have a substrate bound structure of your target enzyme, but you do know the active site residues that interact with the substrate, you can alternatively define the receptor grid by following these steps:

  • Switch to the Site tab in the Receptor Grid Generation panel.
  • Under Center, choose Centroid of selected residues.
  • Click the Specify Residues button.
  • Use the Search Toggle in the Hierarchy to locate the active site residues and select them in the Workspace one at a time.
  • Once you have finished adding all the desired residues, click OK.
  • Run the Receptor Grid Generation job.

 

Note: You may also define the receptor grid using the Centroid of selected residues when you have a SiteMap binding site but the centroid of the site is not optimally located or the site points extend over a broad region.

Figure 4-8. Running the receptor grid generation job.

 

  1. Click the Fit-all button to see the entire structure with the purple box around the ligand.
  2. Change the Job name to glide-grid_4GE8.
  3. Click Run.
    • A dialog box pops up warning about the presence of an unidentified ligand-sized molecule other than the reference ligand.This is due to the presence of the FMN cofactor near the binding site. It will be considered as part of the rigid receptor.
  4. Click Continue in the Dialog box.
    • This Job takes a few seconds.
    • A receptor grid file is written to your Working Directorythe location where files are saved.

 

Note: We are using a prepared enzyme structure for generating the receptor grid. If you are following with a different structure, make sure that your structure is prepared using the Protein Preparation Workflow.

 

  1. Close the Receptor Grid Generation panel.

Figure 4-9. Loading the generated receptor grid for docking.

You can now use the grid you just generated to set up the docking calculation.

 

  1. In the Ligand Docking (Beta) panel, under Inputs, for Receptor grid, choose Browse File.
  2. Locate glide-grid_4GE8 in your Working Directorythe location where files are saved and choose glide-grid_4GE8.zip.
  3. Click Open.

Figure 4-10. Running the docking job.

 

Now, specify the ligands to dock. In this case, this is the co-crystal ligand we prepared in the previous subsection.

 

  1. In the ligprep_S-carvone-out group in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, 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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries S-carvone_substrate.
  2. In the Ligand Docking (Beta) panel, for Ligand source, choose Project Table (selected entries).
  3. Click Load.

 

Note: The default settings works well for this enzyme-substrate complex. To know about when to deviate from default settings – such as for large or highly flexible ligands, or when applying constraints, please refer to the Structure-Based Virtual Screening Using Glide tutorial.

 

  1. Change the Job name to Docking_S-carvone_4GE8.
  2. Click Run.
    • This job takes a few seconds.
    • A banner appears when the job is completed.
    • A new group Docking_S-carvone_4GE8_pv is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  3. Close the Ligand Docking (Beta) panel.

4.4 Analyzing enzyme-substrate interactions in the docked pose

Visualizing the interactions can give you more insights into the complementarity  between the enzyme and the substrate. By examining hydrogen bonds, salt bridges, hydrophobic contacts, etc., you can understand how the substrate is stabilized within the binding pocket.

Figure 4-11. Setting up the Workspace for viewing the docked pose.

  1. Click the workflow icon next to the Docking_S-carvone_4GE8_pv group and choose View Poses.
    • The Pose Viewer panel opens.
    • The 4GE8 – prepared_dry entry is fixed and the docked pose 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.

Figure 4-12. Comparing the docked pose with the co-crystal pose.

  1. Ctrl+Click (Cmd+Click) to include 4GE8 – prepared_dry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Go to Presets and choose Binding Mode Comparison.
    • The docked orientation of (S)-Carvone is consistent with its co-crystal orientation.
  3. Close the Pose Viewer panel.

Figure 4-13. Visualizing the enzyme-substrate interactions.

The Interactions Toggle in the Workspace Configuration Toolbar allows you to inspect non-bonded interactions in the 3D Workspace, represented as colored dashed lines.

 

  1. Turn on the Interactions Toggle in the bottom right.
  2. Observe the interaction patterns of the ligand in the binding pocket.
  3. Optional: Right-click the interactions toggle to customize the display.

Figure 4-14. Opening the Ligand Interaction Diagram from Tasks.

The 2D Ligand Interaction Diagram provides a more sequence-focused view of the ligand-receptor interactions.

 

  1. Right-click the In circle of 4GE8 – prepared_dry and choose Exclude to exclude it from the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Find and open the Ligand Interaction Diagram from Tasks.

Figure 4-15. The Ligand Interaction Diagram.

  1. In the Ligand option menu, choose A: 0WU 414.
  2. Click the Configure View icon (settings cog) and choose LID Legend.
  3. Take your time to visually analyze the different types of enzyme-substrate interactions.  

Note: You can click the Clean up button (green tick mark) to generate a less-cluttered orientation of the diagram.

The carbonyl oxygen atom of (S)-Carvone is positioned to form hydrogen bonds with HIE 191 and ASN 194 residues. Its isopropenyl group occupies a hydrophobic pocket directly above THR 37 and makes hydrophobic contacts with the side chain of ILE 116 residue.

Figure 4-16. The Save Image option in the Ligand Interaction Diagram.

Optional: Click the Export icon and choose Save Image to export the Ligand Interaction Diagram as an image. For visual clarity, we recommend adjusting the resolution depending on the intended purpose while saving the image.

 

  1. Close the Ligand Interaction Diagram.

5. Finding Mutation Hotspots with MM-GBSA Residue Scanning

This section explains how to identify mutation hotspots in a protein sequence based on homologous structures. We will demonstrate how to use the homology criterion within the MM-GBSA Residue Scanning panel to evaluate the effects of back-mutations, focussing on the TRP 116 – ILE back-mutation. This analysis helps assess how specific engineered changes influence the enzyme’s stability and binding affinity relative to the native form. Please note that although we are applying this approach to examine back-mutations, the same homology-guided residue scanning method can also be used in the forward direction – to identify potential mutation hotspots for enzyme engineering based on evolutionary conservation and variability among homologous sequences.

The homology criterion is based on multiple sequence alignment (MSA) of your target structure and its homologs. Since evolution often conserves key residues that are critical for an enzyme’s active site, using homology criterion allows for informed mutations to either conserved or variable residues. This approach is computationally more efficient than scanning every single residue in a large structure.

5.1 Set up an MM-GBSA residue scanning calculation

Figure 5-1. Setting up the Workspace and changing the selection scope to molecules.

 

In order to calculate ΔAffinity values for each mutation, you need to assign a different chain name to the substrate ((S)-Carvone). This is a purely technical step because the affinity is calculated between two sets of chains.

 

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue 4GE8 – prepared_dry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Type L on the keyboard.
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed zooms to the ligands.
  3. Change your selection scope to Molecules. Alternatively, you can type “M” on your keyboard to change the selection scope to Molecules.

Figure 5-2. Selecting the substrate to change atom properties.

  1. Click on any atom of S-carvone (A:0WU 414) to select the entire molecule in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Right-click the selection and choose Additional Edits > Change Atom properties.
    • The Change Atom Properties panel opens.

Figure 5-3. Assigning chain B to the substrate.

  1. For Property, choose Residue/Chain Name.
  2. Check the box for Chain Name and type “B” in the text box beside it.
  3. Click Apply.
    • The 0WU 414 is assigned to chain B.
    • You can confirm this by expanding Ligands in the Hierarchy.
  4. Close the Change Atom Properties panel.

Figure 5-4. Changing the default settings in the MM-GBSA Residue Scanning panel.

Now, you can set up the MM-GBSA residue scanning calculation.

 

  1. Find and open MM-GBSA Residue Scanning Calculations from Tasks.
    • The MM-GBSA Residue Scanning panel opens.
  2. For Import structure from, choose Workspace.
  3. Click Import.
    • The Residues table is populated.

 

You need to specify the Calculation type and the Binding partners (substrate binding to the enzyme i.e. chain B binding to chain A).

 

  1. For Calculation type, choose Stability and Affinity.
  2. For Binding partners, uncheck A in the Chains menu.
    • Chain B (substrate) is binding to chain A (enzyme).

You can mutate your target structure in several ways depending on whether you are performing a data-driven analysis, broader sequence-based screening, or a targeted mutation study. If you already have a predefined list of mutations (for example, from literature or experimental data), you can import them directly using a Mutations file. When the objective is to identify potential hotspots for improving selectivity, stability, or binding affinity, the Homology criteria option can be used – this automatically suggests mutations based on residue conservation across homologous sequences, pointing out positions that are naturally variable or conserved. Alternatively, mutations can be defined manually by directly selecting residues in the structure and choosing the desired amino acid replacements. This approach is ideal when testing specific hypotheses, such as exploring the role of an individual residue in catalysis or substrate binding.

Figure 5-5. Selecting the Homology criteria option to identify the mutation hotspots.

In this case, you will use the Homology criteria option to identify the mutation hotspots.

 

  1. Click Import mutations and choose Homology criteria.
    • The Homology Suggestions Dialog box opens.

Figure 5-6. Running a BLAST search to find homologs of 4GE8 enzyme structure.

You will now select residues for mutation using homology modeling, which can be applied to select residues based on variability or conservation. The homologs can be imported or located using a BLAST search. In this tutorial, you will run a BLAST search to find homologs of 4GE8 structure.

 

  1. In the Homology Suggestions Dialog box, click Run a Blast search.
    • The Blast Search Settings dialog box opens.
  2. For Search Tool, choose BLAST.
  3. For BLAST Server, choose Remote (NCBI).
  4. Click Start Job.
    • This job takes ~ 2 minutes.
    • Once the job is finished, the BLAST Search Results dialog box opens.
    • The top 10 homologs are selected by default.

Figure 5-7. Selecting the top 50 homologs of 4GE8 from the BLAST Search Results.

  1. Shift+Click to select the top 50 homologs.

 

Note: You can also type the number of top homologs in the text box at the bottom and click Select Top.

 

  1. Click Incorporate Selected Rows.
    • The selected homologs are added to the Sequence Viewer.

You can apply both homology-based and 3D structure-based criteria to control which residues are considered for mutation.

 

  • Homology-based criteria
    • This criterion is based on sequence conservation across homologs. It helps filter out positions that are highly conserved or unlikely to tolerate mutations, ensuring that analysis focuses on residues with variability. You can set thresholds for variability at specific positions or residue types, ignore conserved groups, or exclude the parent sequence when applying filters. Additionally, it is possible to define a cutoff for how different a residue must be compared to homologs.  
  • 3D structure-based criteria
    • The parent structure 3D criterion allows filtering based on structural context, such as solvent accessibility, the number of side-chains interacting within the protein/enzyme, or whether a residue interacts with cofactors, ligands, or ions. These options make it possible to exclude residues that are buried, structurally critical, or directly involved in essential interactions, while targeting residues whose mutations are structurally and functionally feasible.

 

Mutating highly conserved residues is likely to significantly alter affinity and stability values, while mutating highly variable residues can help predict tolerant mutations, useful for introducing changes without disrupting overall enzyme function.

Figure 5-8. Aligning the incorporated homologs and applying variability at position criterion to identify mutable residues.

 

In this case, you will use the variability at position criterion, which selects residues based on how much sequence conservation they show across the selected homologs.

 

  1. Click Align Homologs.
    • The incorporated homologs are now aligned.
  2. Check the box for Variability at position and set the value to > 30%.
    • This means that over 30% of the sequences in the selected homologs do not have the same residue at a specific position.
  3. Press the Enter/Return key on your keyboard to apply the variability percentage and select residues in the homologs.
    • 10 out of 399 residues are selected.
  4. Click Save.
    • The selected residues with plausible mutations are now shown in the Residues table in the MM-GBSA Residue Scanning panel.

Figure 5-9. Running the MM-GBSA Residue Scanning job.

  1. Change the Job name to mmgbsa_residue_scanning_4GE8.
  2. Click Run.
    • This job takes ~10 minutes.
    • Once the job is completed, a banner appears and a new group mmgbsa_residue_scanning_4GE8-out is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
    • Alternatively, you can find pre-generated results in the tutorial files.
  3. Close the MM-GBSA Residue Scanning panel.

For an in-depth introduction to using MM-GBSA Residue Scanning for improving protein stability or binding affinity and identifying binding hotspots, see the Peptide Modeling with BioLuminate tutorial. For more information on the panel settings, refer to the MM-GBSA Residue Scanning panel documentation.

5.2 Analyze the MM-GBSA residue scanning results

Figure 5-10. The MM-GBSA Residue Scanning Viewer panel with the results loaded.

  1. Find and open the MM-GBSA Residue Scanning Results from Tasks.
    • The MM-GBSA Residue Scanning Viewer panel opens.
  2. For Load results from, choose File.
  3. Next to File name, click Browse.
  4. Find and choose mmgbsa_residue_scanning_4GE8-out.maegz in your Working Directorythe location where files are saved.
  5. Click Open.
    • The Mutations table is populated.
    • ΔStability (solvated) vs ΔAffinity graph is shown below the Mutations table.

ΔStability (solvated) and ΔAffinity and values are most helpful in assessing the potential impact of a mutation.

 

Interpreting ΔStability

Stability is the change in the Gibbs free energy of folding (ΔGfolding) i.e. it is the difference between the native (folded) state and the denatured (unfolded) state of a protein. A more negative ΔGfolding means a more stable protein. ΔStability measures how a mutation changes the overall stability of a protein i.e. it measures the change in the folding free energy of the protein (enzyme) itself upon mutation – independent of ligand (substrate) binding.

 

     ΔStability = ΔGstability (mutant) – ΔGstability (wild-type)

 

Where,                                                 ΔGstability = Gfolded – Gunfolded

                                             

Interpreting ΔAffinity

Affinity is the change in the Gibbs free energy of binding (ΔGbinding) i.e. it is the energy difference between the bound and unbound states. A more negative ΔGbinding means a stronger, more favorable binding interaction. ΔAffinity measures how a mutation changes the binding strength between a protein (enzyme) and its binding partner.

 

ΔAffinity = ΔGaffinity (mutant) – ΔGaffinity (wild-type)

 

Where,                                               ΔGaffinity = Gbound – Gunbound

Figure 5-11. Investigating the outlier in the graph.

You will now investigate the outliers in the graph. Note that highly positive ΔStability (solvated) and the ΔAffinity values correspond to deleterious mutations.

 

In this tutorial, we will focus on ILE 116 –TRP mutation as the wild type OYE1 has TRP at position 116. This will give us an opportunity to compare the stability and affinity of (S)-carvone substrate with the wild-type and the engineered variant.

 

  1. On the graph, click and drag to select the outlier.
  2. Confirm that the Sync selection with mutations table box is checked.
    • The filtered result is now selected in the Mutations table.
    • The outlier corresponds to the ILE – TRP mutation.

The graph displays a correlation between ΔStability (solvated) and the ΔAffinity for the generated mutations. Each point corresponds to a specific mutation, with its position determined by how the mutation affects the system’s stability (X-axis) and binding affinity (Y-axis). Most mutations cluster near the origin, indicating minimal changes in both binding affinity and overall structural stability compared to the engineered ILE 116 variant. However, one distinct outlier – corresponding to the ILE-TRP back-mutation – shows a sharp spike in both ΔStability (solvated) and ΔAffinity values. This suggests that reintroducing TRP at this position significantly destabilizes the system and weakens binding, likely due to steric clashes or unfavorable orientation of bulky indole side chain.

Figure 5-12. Analyzing ΔAffinity values.

  1. Optional: Change the X-Axis Property to Mutant Row Number.
    • The graph updates to show ΔAffinity values for each mutation.
    • A sharp spike in ΔAffinity value is observed for the ILE – TRP mutation.

Figure 5-13. Analyzing ΔStability (solvated) values.

  1. Optional: Change the Y-Axis Property to ΔStability (solvated).
    • The graph updates to show ΔStability (solvated) values for each mutation.
    • Similar to ΔAffinity value, a sharp spike in ΔStability (solvated) value is observed for the ILE – TRP mutation.
    • Additionally, there is significant increase in ΔStability (solvated) value for LYS 393 – PRO mutation.
  2. Close the MM-GBSA Residue Scanning Viewer panel.

The sharp increase in the ΔAffinity and ΔStability (solvated) values observed in the graphs indicates that mutating isoleucine to tryptophan at position 116 is highly unfavorable. In the wild-type OYE1, (S)-Carvone binds in a normal orientation due to the bulky TRP restricting the active site. However, when TRP 116 is replaced by a smaller ILE, a much larger pocket is formed, causing (S)-Carvone to bind in a flipped orientation. Reverting to TRP reintroduces steric bulk into the pocket, creating clashes with the isopropenyl substituent of the flipped-bound substrate. These results are consistent with the findings reported in this publication, where replacing TRP 116 by ILE enlarges the active site to better accommodate the substrate.

 

It is important to note that during MM-GBSA calculations, minor steric clashes between the ligand and the protein are handled through a local minimization step that relaxes the structure to a nearby low-energy conformation. However, this minimization is limited in scope and does not involve any re-docking or large-scale conformational adjustments. Therefore, while small overlaps may be corrected during the MM-GBSA calculations, significant clashes – such as those arising from an incorrect binding mode or severe side-chain repulsion in back-mutated structures –  are not expected to be fully resolved.

 

Additionally, note that back mutations are useful for examining the effects of engineered changes, however, the same workflow can also be applied to identify mutation hotspots or introduce mutations in wild-type enzymes.

Figure 5-14. Visualizing the steric clashes between the flipped S-Carvone substrate and the TRP 116 residue.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue A:116 TRP mutant in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Using the Search Toggle in the Hierarchy, search for and select TRP 116 residue.
  3. Click Fit view to selected atoms button.
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed zooms to TRP 116 residue.
  4. Confirm that the Interactions Toggle in bottom right is turned on.

 

Take your time to visualize the flipped orientation of the (S)-Carvone substrate resulting in steric clashes with the TRP 116 residue in the wild -type variant.

In the wild-type OYE1, the bulky indole side chain of TRP 116 creates significant steric clashes with the bound (S)-carvone substrate. Additionally, the indole ring deviates from the ideal conformation (non-planar) to accommodate the substrate. 

6. Investigating the Impact of a Mutation on the Ligand Binding Mode through MD Simulations

In this section, you will perform molecular dynamics simulations on both wild-type and engineered W116I structures bound to the (S)-carvone substrate using Desmond. Our goal is to understand how the mutation influences substrate binding and stability in the active site. You will calculate missing torsion parameters for the substrate using the Force Field Builder – OPLS4/OPLS5 panel, set up systems consisting of enzyme, substrate, explicit solvent, and counter ions using the System Builder panel, and run the MD simulations and analyze the results by comparing the RMSD values of the substrate relative to its starting pose.

Note that while performing MD simulations, trajectories can sometimes get trapped in local minima of the potential energy surface. This means that, depending on the initial velocities assigned to atoms (which are determined by the random seed), the system might explore only a limited portion of the conformational space rather than the true equilibrium ensemble. To avoid this pitfall, we recommend running multiple independent simulations – each initiated with a different random seed. Comparing the results from these replicate runs helps ensure that the observed structural and energetic trends are reproducible and not artifacts of a single trajectory. This provides greater confidence in the statistical robustness and physical relevance of the conclusions drawn from the MD simulations analysis.

As an alternative to running multiple MD simulations, you could run a metadynamics MD simulation, which enhances the sampling of the underlying free energy space by biasing against previously-visited values of user-specified collective variables. For more details, refer to the Metadynamics panel documentation.

6.1 Generate missing torsion parameters for the substrate

Before performing molecular dynamics simulations, it is essential that all components of the system – including the protein (enzyme), the ligand (substrate), and the solvent – are fully described by the chosen force field. However, when a ligand or small molecule contains unusual chemical groups or torsional angles not covered by the default force field parameters (e.g., OPLS4), Maestro flags these as missing torsion parameters. These missing parameters must be generated using the Force Field Builder panel to ensure that the ligand’s conformational energetics are correctly represented during MD simulations. Without these parameters, the simulation may fail or produce unrealistic geometries and energetics. Therefore, generating missing torsion parameters is an important step to ensure that the ligand interacts accurately with the surrounding protein and solvent environment throughout the MD run.

Figure 6-1. Opening the Force Field Builder panel from Tasks.

  1. In the ligprep_S-carvone-out group, includethe entry is represented in the Workspace, the circle in the In column is blue S-carvone_substrate.
  2. Find and open Force Field Builder from Tasks.
    • The Force Field Builder - OPLS4/OPLS5 panel opens.

Figure 6-2. The Force Field Builder panel.

 

  1. For Start with project, (none - new analysis).
  2. Add structures from, choose Workspace (1 included).
  3. Click Preview New Missing Torsions to analyze the fragments with missing torsion parameters.
    • This takes ~1 minute.
    • A banner appears and the fragments with missing torsion parameters are shown.
  4. Click Location.
    • Set Custom Parameters Location dialog box opens.
  5. To specify the location of the OPLS custom parameters, click the dropdown and choose Browse.
    • The Custom Parameters Location dialog box opens.

Figure 6-3. Specifying the location of Custom Parameters and running the job.

  1. Set the location of Custom Parameters to your current Working Directorythe location where files are saved.
  2. Click OK.
    • A warning box confirming that the specified custom parameters location has been saved appears.
  3. Click OK.
  4. Change the Job name to ffb_S-carvone.
  5. Optional: Click Run.
    • This job is time and compute - intensive. To save time, you can find the output files in the tutorial archive.

Figure 6-4. Loading the custom parameter output.

  1. In the Force Field Builder panel, click the Start with project dropdown and choose Browse.
  2. Navigate to and choose ffb_S-carvone.zip in your Working Directorythe location where files are saved.
  3. Click Open.

 

Figure 6-5. Saving and merging the generated custom parameters to the local custom parameters in the Working Directory.

  1. Next to Update custom force field, click Save Parameters.
  2. Click Save in the Warning box.
    • The generated force field parameters are merged to the local custom parameters in your Working Directorythe location where files are saved.  

Note: See here for more information on how Maestro handles custom parameters within and between projects.

 

  1. Close the Force Field Builder - OPLS4/OPLS5 panel.

6.2 Prepare the systems for MD simulations

Figure 6-6. Choosing to duplicate the ILE 116 variant.

You will now prepare both the OYE1 (TRP 116 variant) and OYE1W116I (ILE 116 variant) structures for MD simulations. Setting up and running the MD will create additional entries, so you will make a group each for wild type and mutant to keep track of which steps have already been done for each of the systems.

 

  1. 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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries 4GE8 – prepared_dry.
  2. Right-click the selection and choose Duplicate > Into New Group.
    • The Duplicate into New group dialog box opens.

Figure 6-7. Duplicating the ILE 116 variant.

  1. Under New group title, type ILE116_mutant.
  2. Under Location of new group, choose At top level - End of table.
  3. Click Duplicate.
    • A new group ILE116_mutant is added at the end of the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table and 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.

Figure 6-8. Duplicating the TRP 116 wild-type variant.

  1. 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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the A: 116 TRP entry and duplicate it into a new group as shown in step 20.
  2. Under New group title, type TRP116_wild-type.
  3. Repeat steps 22 - 23.
    • A new group TRP116_wild-type is added at the end of the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.

Before running molecular dynamics (MD) simulations, the system must be properly set up to ensure  a realistic environment for the enzyme-substrate complex. The System Builder in Maestro automates this process by defining the simulation box, adding solvent molecules, neutralizing the system with counterions, and setting the appropriate salt concentration. It also assigns the force field parameters to all the components. This preparation ensures that the system is ready for equilibration and production MD runs.

 

Please see the Introduction to All-Atom Molecular Dynamics Simulations with Desmond for more in-depth instructions and insights into preparing your system and running MD Simulations.

Figure 6-9. Opening the System Builder panel from Tasks.

  1. From the newly created ILE116_mutant group, includethe entry is represented in the Workspace, the circle in the In column is blue the 4GE8 – prepared_dry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Find and open System Builder from Tasks.
    • The System Builder panel opens in the Solvation tab.

Figure 6-10. Modifying the settings in the Solvation tab of the System Builder panel.

 

  1. In the Solvent model section, choose Predefined : SPC water model.

 

Note: The SPC water model is the one with which the OPLS force field was originally parameterized.

 

  1. In the boundary conditions section, choose Orthorhombic as the Box shape.
  2. Optional: Check the box for Show boundary box option.
    • The box in which the enzyme-substrate complex will be placed is now shown in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Note: For oblong rather than globular proteins, we recommend using cubic boxes to minimize the risk of the protein interacting with its replica across the periodic boundary.

 

  1. Click on Minimize Volume.
    • The complex is centered in the box and the box volume is minimized by reorienting the water molecules.

 

We recommend using the OPLS4 force field (if available) due to its enhanced accuracy, broader chemical space coverage, and improved handling of small molecule torsions compared to its predecessor, OPLS_2005.

 

  1. For the Force field, choose OPLS4.
  2. Check the box for Use customized version.

Figure 6-11. Modifying the settings in the Ions tab of the System Builder panel and running the System Builder job for the OYE1W116I.

Ions are important to neutralize the system and mimic physiological salt concentrations, ensuring realistic electrostatic conditions during the simulation.

 

  1. Go to the Ions tab.
  2. Choose the option Neutralize by adding to add an exact amount of Sodium (Na+) ions to neutralize the system.
  3. Click the Recalculate button.
  4. Check the box for Add salt option and specify a concentration of 0.15 M in the Salt concentration text box.
  5. Change the Job name to desmond_setup_ILE116_mutant.
  6. Click Run.
    • This job takes ~ 2 minutes.
    • Once the job is completed, a banner appears and a new group MD: desmond_setup_ILE116_mutant is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

Figure 6-12. Running the System Builder job for OYE1 wild-type structure.

  1. From the newly created TRP116_wild-type group, includethe entry is represented in the Workspace, the circle in the In column is blue A: 116 TRP in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Repeat steps 29 - 38 to similarly prepare the TRP 116 wild-type variant for MD simulations.
  3. Change the Job name to desmond_setup_TRP116_wild-type.
  4. Click Run.
    • This job takes ~ 2 minutes.
    • Once the job is completed, a banner appears and a new group MD : desmond_setup_TRP116_wild-type is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  5. Close the System Builder panel.

6.3 Run the MD simulations

Figure 6-13. Opening the Molecular Dynamics panel from Tasks.

  1. From the MD: desmond_setup_ILE116_mutant group, includethe entry is represented in the Workspace, the circle in the In column is blue 4GE8 – prepared_dry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Find and open Molecular Dynamics from Tasks.
    • The Molecular Dynamics panel opens.

Figure 6-14. Modifying the settings and running the MD job for the ILE 116 variant.

  1. Under Model system, choose Load from Workspace and click Load.
    • The prepared ILE 116 variant is loaded.
  2. For Simulation time (ns), type 100 next to Total.
  3. Under Analysis, check the box for Run interaction analysis when simulation job completes.
    • This generates the event analysis file (.eaf file) as an automated step after the simulation completes.
  4. For Ligand, click the dropdown and choose ASL.
  5. Click Define.
    • Atom Selection dialog box opens.

Figure 6-15. Defining the ligand for running the interaction analysis.

  1. Go to the Residue tab and choose Residue Type.
  2. Choose 0WU from the list and click Add.
  3. Click OK.
    • This analyzes the interactions for the (S)-Carvone substrate once the simulation job completes.

Figure 6-16. Running the MD job for  OYE1W116I.

  1. Change the Job name to desmond_md_job_ILE116.
  2. Click the Job settings cog and choose the desired GPU host.
  3. Optional: Click Run.
    • This job is time intensive and requires a GPU host.
    • To save time, pre-generated results are included in the tutorial files.

Figure 6-17. Modifying the settings and running the MD job for OYE1.

  1. From the MD: desmond_setup_TRP116_wild-type group, includethe entry is represented in the Workspace, the circle in the In column is blue A:116 TRP in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Repeat steps 48-55.
  3. Change the Job name to desmond_md_job_TRP116.
  4. Click the Job settings cog and choose the desired GPU host.
  5. Optional: Click Run.
    • This job is time intensive and requires a GPU host.
    • To save time, pre-generated results are included in the tutorial files.

6.4 Analyze the results of MD simulations

Figure 6-18. Loading the event analysis file for OYE1W116I MD run.

  1. Find and open the Simulation Interactions Diagram from Tasks.
    • The Simulation Interactions Diagram panel opens.
  2. Click the Load button.
    • The Select Event Analysis or Trajectory file dialog box opens.
  3. Navigate to the desmond_md_job_ILE116 folder in your Working Directorythe location where files are saved and choose desmond_md_job_ILE116_run2.eaf.
  4. Click Open.
    • It takes a few seconds to load the Simulation Interactions Diagram.

Figure 6-19. The Simulation Interactions Diagram panel showing the PL-RMSD values for the OYE1W116I.

  1. Hover over the plot to show the frame number/simulation time and the corresponding RMSD values for a particular frame.

The PL-RMSD tab shows the Cα RMSD of the enzyme (teal curve) and substrate RMSD (magenta curve), both aligned on the enzyme backbone. The enzyme RMSD stabilizes around 1.2-1.4 Å, indicating that the overall enzyme conformation remains stable throughout the simulation. The ligand RMSD fluctuates between 0.5-1.5 Å, indicating that the substrate maintains a consistent binding mode within the active site with only minor conformational adjustments. These moderate fluctuations are expected as the ligand explores the binding pocket while maintaining key stabilizing interactions with HIE 191 and ASN 194 residues.

 

For an in-depth understanding of MD trajectory analysis, please refer to the Introduction to MD Trajectory Analysis with Desmond tutorial.

Figure 6-20. The Simulation Interactions Diagram panel showing the L-RMSF values for the (S)-Carvone substrate in OYE1W116I.

  1. Switch to the L-RMSF tab.
  2. Hover over the plot to see the atom name and type and the corresponding RMSF value.

L-RMSF data indicates that most atoms of the substrate exhibit low RMSF values – ranging from 0.5 to 0.8 Å – suggesting stable interactions with the enzyme.

Figure 6-21. Loading the MD trajectory for OYE1W116I.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue the newly added 4GE8 – prepared_dry entry in the MD: desmond_setup_ILE116_mutant group in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Click the “T” button and choose Load Trajectory.
    • This takes a few seconds.
  3. Click the Play button to play the trajectory.

Figure 6-22. Playing the trajectory for OYE1W116I.

Choose a visual representation that is most helpful to you while playing the trajectory. We have hidden all the water molecules, zoomed in to the (S)-Carvone substrate, and toggled on interactions.

 

Notice that the carbonyl oxygen of the (S)-Carvone consistently interacts with the ASN 194 and HIE 191 residues throughout the simulation.

Figure 6-23. The Simulation Interactions Diagram panel showing the PL-RMSD values for OYE1.

Now, you can load in the results for the TRP116 back-mutant, i.e. the wild type and compare.

 

  1. In the Simulations Interaction Diagram panel, click Load.
  2. Navigate to the desmond_md_job_TRP116  folder in your Working Directorythe location where files are saved and choose desmond_md_job_TRP116_run3.eaf.
  3. Click Open.

The substrate RMSD (magenta curve) initially shows a stable binding with an RMSD fluctuating between 0.4 Å and 1.0 Å up to ~ 85 ns. However, after 85 ns, the substrate RMSD shows a significant jump, rising sharply to ~ 4.0 Å and then further rising to ~ 6.4 Å. This spike strongly suggests a major reorientation/unbinding, implying that the initial binding pose (flipped orientation of (S)-Carvone in OYE1) is not stable over an extended timeframe.

Figure 6-24.The Simulation Interactions Diagram panel showing the L-RMSF values for the (S)-Carvone substrate in OYE1.

  1. Switch to the L-RMSF tab.
  2. Hover over the plot to see the atom name and type and the corresponding RMSF value.

Compared to the OYE1W116I with ILE at position 116, the L-RMSF values are higher in OYE1 with TRP at position 116. This clearly demonstrates that ILE mutation at position 116 greatly enhances the stability of the (S)-Carvone substrate binding, compared to the native TRP residue. This is expected, as the ILE residue has a smaller and less bulky side chain creating a larger active site that stabilizes the substrate effectively than the bulky, aromatic side chain of TRP 116 residue.

Figure 6-25. Playing the trajectory for OYE1.

 

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue the newly added A:116 TRP entry in the MD :desmond_setup_TRP116_wild-type group in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Click the “T” button and choose Load Trajectory.
    • This takes a few seconds.
  3. Click the Play button to play the trajectory.
    • Notice that the substrate starts drifting away from the binding pocket and loses contacts with HIE 191 and ASN 194 residues starting at ~ frame number 870. This results in a non-reactive binding orientation of the substrate in OYE1.
    • This observation is consistent with the sharp increase in the L-RMSD value after ~ 85 ns.

Comparing the MD results for wild-type OYE1 and the mutant OYE1W116I clearly suggests that ILE at position 116 provides a more favorable environment for binding of the (S)-Carvone substrate. These results demonstrate that MD simulations provide valuable insight into how mutations alter binding pocket dynamics, substrate orientation and overall complex stability. Such findings emphasize the importance of MD simulations as a powerful complement to static docking or MM-GBSA scoring, providing a deeper understanding of how residue substitutions shape substrate recognition and functional performance of enzymes.

 

Depending on the scientific question, more specialized MD-based methods can be more helpful to probe specific aspects of the system. For example, while the MD simulation showed that the binding pose for (S)-Carvone in the back-mutant was unstable, you did not obtain the "correct" stable binding mode for the ligand. To predict such induced-fit binding poses, we recommend using IFD-MD.

 

Other MD-based tools can help you understand how mutations affect hydration patterns in the binding pocket (WaterMap), or predict the effect of mutations on stability and affinity of the system with high-experimental accuracy (Protein FEP+). For more information, see the WaterMap and FEP Protein Mutation documentation pages.

7. Conclusion and References

In this tutorial, you learned how to prepare the enzyme-substrate structures for modeling applications like docking and MD simulations, identify key molecular contacts between enzyme and the substrate using 2D Ligand Interaction Diagram, and perform docking calculations to explore binding modes of the substrate. You also learned how to perform residue scanning calculations to identify mutation hotspots and evaluate how enzyme engineering improves enzyme properties. Further, you learned how to perform MD simulations to understand how mutation influences substrate binding and stability in the active site.

8. Glossary of Terms

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

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

Recent actions - This is a list of your recent actions, which you can use to reopen a panel, displayed below the Browse row. (Right-click to delete.)

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 Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries

Working Directory - the location where files are saved

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