Disulfide Bond Engineering

Tutorial Created with Software Release: 2024-2
Topics: Biologics Drug Discovery, Consumer Packaged Goods, Enzyme Engineering, Structure Prediction & Target Enablement
Products Used: BioLuminate, Prime

Tutorial files

51.9 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

 

Tip: You can hover over a glossary term to display its definition. You can click on an image to expand it in the page.
Abstract:

 

In this tutorial, you will explore how to use cysteine scanning to identify residues that could be mutated to cysteine to improve thermal stability and facilitate crystallization.

 

Tutorial Content

  1. Introduction to Disulfide Bond Engineering

  1. Creating Projects and Importing Structures

  1. Running the Cysteine Mutation Job

  1. Analyzing the Cysteine Mutation Results

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to Disulfide Bond Engineering

Disulfide bond engineering is the process of introducing cysteines in a protein to drive the formation of disulfide bonds. This is a commonly used technique in protein engineering to improve thermal stability, enhance chemical and enzymatic properties, and decrease the chance of misfolding.

In the Cysteine Mutation Panel, you have the ability to find residue pairs that can be mutated to cysteine to stabilize the protein by examining all residue pairs close enough to reasonably form a Cys-S-S-Cys disulfide bond, scoring all potential mutations with a combination of implicit solvent molecular mechanics (Prime) and empirical geometry terms based on disulfide bonds found in the Protein Data Bank.

Figure 1. Sample disulfide bond formation with the Cysteine Mutation panel.

In this tutorial we will focus on the LPA1 GPCR described in Chrencik et al. where the project team was trying to identify a series of ligands that would stabilize the receptor conformation to allow for structure determination. While three promising ligands were found from analytical size exclusion chromatography (aSEC), only two were able to facilitate crystallization. To facilitate the crystallization of the one uncrystallized complex experimented with inserting stabilizing disulfide bridge.

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 BioLuminate 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 Directorythe location that files are saved 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 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.

Maestro, BioLuminate, and MS-Maestro can all be used for this tutorial, though the instructions for how to access the Cysteine Mutation panel in Maestro and MS-Maestro each will differ compared to BioLuminate.

 

  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 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/disulfide_bond_engineering.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 Project.

  1. Go to File > Open Project
  2. Choose lpa1_disulfide.prjzip
  3. Click Open
    • 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
    • A structure 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 2-3. Save Project panel.

  1. Go to  File > Save Project As
  2. Change the File name to lpa1_disulfide, click Save
    • The project is now named lpa1_disulfide.prj

3. Running the Cysteine Mutation Job

Before loading anything into the Cysteine Mutation panel, the protein structure must already be prepared using the Protein Preparation Workflow. Please see the Introduction to Structure Preparation and Visualization tutorial for more details on using the Protein Preparation Workflow. Also see the Best Practices for Protein Preparation for more information. For more information on the Cysteine Mutation panel please click here. In this example we have already prepared the structure using the Protein Preparation Workflow, and modeled in the ONO-308573 ligand with the Ligand Docking panel.

Figure 3-1. Cysteine Mutation option in Biologics.

We have the pre-prepared 4Z35 structure with the ONO-308573 ligand modeled in.We will now use the Cysteine Mutation panel to predict which potential disulfide bonds could improve stability of the complex

 

  1. Go to Tasks > Biologics > Cysteine Mutation
    • The Cysteine Mutation Panel opens

The following are the options in the Cysteine Mutation panel that one might most commonly look to adjust:

 

Filter: By default, after you click Analyze Workspace all possible residue pairs meeting the selected criteria will be added to the table.

 

Solvent-accessible surface area: We would recommend keeping your Solvent-accessible surface area below 80 Å2 as more exposed cysteines are prone to oxidation.


Minimization shell: Salam et al. note in their validation that performance was best when they set the minimization shell to just the residues involved in the formed disulfide bond.

 

Refinement: Salam et al. note a moderate improvement in performance with the implicit solvent refinement.

 

Figure 3-2. Analyze Workspace.

  1. Click Analyze Workspace
    • The table is now filled with 108 residues pairs that meet the default filter criteria

 

 

If you are only interested in potentially introducing disulfide bonds in certain regions of your protein structure you can first select the eligible residues in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and select Analyze only selected Workspace residues. In this specific case the project team was only interested in mutating residues in the extracellular region so if we wanted to we could have pre-filtered the list by those selection criteria.

Figure 3-3. Adjust Filter.

As we are looking to introduce a disulfide bond with two engineered cysteines (as opposed to a mutating a residue to form a bond with an already existing cysteine) we want to filter the table accordingly

 

  1. Click Filter and deselect Cys-Cys and Cys-X
    • The table now includes 99 pairs

 

Figure 3-4. Adjust Disulfide creation eligibility criteria, minimization shell, and refinement.

As would like to avoid residues that are particularly solvent exposed (due to the potential liability for oxidation for solvent exposed cysteines) we will filter out solvent exposed residues.

 

  1. Check Solvent accessible surface area and set it to < 80 Å2 and type Enter
    • The table now include only 80 pairs
  2. For the Minimization shell, choose None (only residues involved in the bond are minimized
  3. For the Refinement, select Implicit solvent minimization

 

Figure 3-5. Select all 80 residue pairs.

  1. Use shift+click to select all of the residue pairs in the table
    • 80 residue pairs will be explored as part of the Cysteine Mutation job

Figure 3-6. Run the cysteine mutation job.

  1. For Job name, write cysteine-mutation-4Z35
  2. Click Run
    • The job should take 25-35 minutes to complete
    • Once the job is completed, 85 new entries will be added the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion
    • If you don’t want to wait for the job to complete you can load in the cysteine-mutation-4Z35-sample-out file

4. Analyzing the Cysteine Mutation Results

Figure 4-1. Load cysteine mutation results.

We will now use the outputs from the Cysteine Mutation panel to triage possible constructs.

 

  1. In the Cysteine Mutation panel, click the Results tab
  2. Click Load Results from Previous Run
  3. If you ran the job yourself, select the cysteine-mutation-4Z35-out.maegz file - otherwise select the pre-generated cysteine-mutation-4Z35-sample-out.maegz file and click Open
    • The Results tab is populated with the outputs from the Cysteine Mutation job sorted by Weighted Score  

The Weighted Score is the weighted sum of change in interaction energy, change in strain energy, pre-minimization score and post-minimization score. The score includes a potential penalty of 10,000 if there is a violation of geometric criteria.

 

The Weighted Score should be viewed as more of a qualitative/binary predictor than as a mechanism for rank-ordering the engineered constructs. Generally, promising constructs would have a Weighted score in the 200-500 range - having a Weighted Score of > 10,000 (thereby indicating that there is a violation of geometric criteria) would suggest that it should not be prioritized for experiment.

 

According to this guidance, only 18 of the 80 constructs would be recommended to be advanced to wet-lab experiments.

Figure 4-2. Zoom in to inspect one of the generated disulfide bonds.

  1. Select the A:204-A:282 row
    • The structure is now included in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and the disulfide bond is colored in dark red.

Chrencik et al. selected the D204C V282C double mutant for crystallization studies as it was found to have an enhanced expression profile and improved thermostability compared to the other double mutants in the extracellular part of the receptor that were identified by the Cysteine Mutation panel as promising.

5. Conclusion and References

In this tutorial we recreated the workflow highlighted in Chrencik et al. to identify pairs of residues in the LPA1 GPCR  that could be mutated to cysteines to form disulfide bonds. We filtered the residues to be explored based on certain criteria, and then analyzed the results by looking at the Weighted Score.

6. Glossary of Terms

Entry List - 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

incorporated - once a job is finished, output files from the Working Directory are added to the project and shown in the Entry List and Project Table

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