DOI: 10.5061/dryad.fttdz092h
title: Data from: Substrate interactions guide cyclase engineering and lasso peptide diversification

description: Lasso peptides are a diverse class of naturally occurring, highly stable
 molecules kinetically trapped in a distinctive rotaxane conformation. How
 the ATP-dependent lasso cyclase constrains a relatively unstructured
 substrate peptide into a low entropy product has remained a mystery owing
 to poor enzyme stability and activity in vitro. Here, we combine
 substrate tolerance data with structural predictions, bioinformatic
 analysis, molecular dynamics simulations, and mutational scanning to
 construct a model for the three-dimensional orientation of the substrate
 peptide in the lasso cyclase active site. Predicted peptide-cyclase
 molecular contacts are validated by rationally engineering multiple,
 phylogenetically diverse lasso cyclases to accept substrates rejected by
 the wild-type enzymes. Finally, we demonstrate the utility of lasso
 cyclase engineering by robustly producing previously inaccessible variants
 that tightly bind to integrin αvβ8, which is a primary activator of
 transforming growth factor β, and thus an important anticancer target.
description: MD simulations were performed using OpenMM 7.6.0 on distributed
 computing platform Folding@home by Song Yin [songyin2@illinois.edu] and Prof. Diwakar Shukla [diwakar@illinois.edu]. Resulting analysis code was created via python based scripts and softwares (Numpy 1.19.2, MDTraj 1.9.9, etc.). For more information about specific data and code, please refer to the paper and Github Repository: https://github.com/ShuklaGroup/lasso_rotaxane/.  This Dryad dataset has been made available in compliance with Data Availability guidelines by <em>Nature Chemical Biology</em>. For inquiries concerning further data availability, please contact Prof. Diwakar Shukla [diwakar@illinois.edu] and Prof. Douglas A. Mitchell [douglasm@illinois.edu].
description: # Data from: Substrate interactions guide cyclase engineering and lasso
 peptide diversification
 [https://doi.org/10.5061/dryad.fttdz092h](https://doi.org/10.5061/dryad.fttdz092h) ## Description of the data and file structure We conducted Molecular Dynamics (MD) simulations for five different FusA (Fusilassin, peptide) : FusC (Fusilassin cyclase, enzyme) complex systems. The system names and their explanations are as follows: * **FusA_FusC** represents FusA wildtype : FusC wildtype * **FusA_I11R_FusC** represents FusA I11R : FusC wildtype (I11R indicates that residue 11 I was mutated to R, same for below) * **FusA_I11R_FusC_R321G** represents FusA I11R : FusC R321G * **FusA_V13R_FusC** represents FusA V13R : FusC wildtype * **FusA_V13R_FusC_R321G** represents FusA V13R : FusC R321G These system names are used as prefixes for all the data files mentioned below. ### 1. MD Simulations Trajectories MD simulations were performed using OpenMM 7.6.0 on the distributed computing platform Folding@home. All the topology and trajectory files for each system are saved in `system name.tar.gz` files. **Files:** * **FusA_FusC.tar.gz (19.1 GB)**  (RUN0) * **FusA_I11R_FusC.tar.gz (19.1 GB)**  (RUN1) * **FusA_I11R_FusC_R321G.tar.gz (19.1 GB)**  (RUN2) * **FusA_V13R_FusC.tar.gz (19.1 GB)**  (RUN3) * **FusA_V13R_FusC_R321G.tar.gz (19.0 GB)**  (RUN4) Each `.tar.gz` file contains: - 1 topology file (`.parm7`). The topology file was generated using the CHARMM36m force field and shared among all the trajectories for each system. - 100 simulation trajectory (`.xtc`) files. Each trajectory file is named by `RUN[index]_CLONE[index]_traj.xtc`, where `RUN Index` specifies the corresponding simulation system as shown above (0-4) and `CLONE Index` is the index for trajectory (0-99). Each trajectory represents 500 ns simulations divided into 5000 frames, resulting in a total of 50 μs data per system. ### 2. Pairwise Residue Side Chain Heavy Distances Pairwise residue side chain heavy distances between the peptide and cyclase were calculated using MDTraj 1.9.9. We selected 112 FusC residues within 8 Å of FusA in the active site and all 18 FusA residues to study their interactions. These pairwise distances were calculated for each frame in every trajectory for each system and then saved in `system name_res_dist.pkl` files. **Files:** * **FusA_FusC_res_dist.tar.gz (17.7 GB)** - Including FusA_FusC_res_dist.pkl, FusA_I11R_FusC_res_dist.pkl, FusA_I11R_FusC_R321G_res_dist.pkl, FusA_V13R_FusC_res_dist.pkl, and FusA_V13R_FusC_R321G_res_dist.pkl. **Code:** [Pairwise_residue_distances.py](https://github.com/ShuklaGroup/lasso_rotaxane/blob/main/Pairwise_residue_distances.py) ### 3. Fraction of Native Contacts (Q) The fraction of native contacts (Q) of the peptide was computed using MDTraj 1.9.9 to demonstrate the formation of the pre-folded state of peptide. The Q values of FusA were calculated for each frame in every trajectory for each system and then saved in `system name_Q.pkl` files. **Files:** * **FusA_FusC_Q.tar.gz (8.4 MB)** - Including FusA_FusC_Q.pkl, FusA_I11R_FusC_Q.pkl, FusA_I11R_FusC_R321G_Q.pkl, FusA_V13R_FusC_Q.pkl, and FusA_V13R_FusC_R321G_Q.pkl. **Code:** [Native_Contacts.py](https://github.com/ShuklaGroup/lasso_rotaxane/blob/main/Native_Contacts.py) ### 4. Contact Probability Contact probability refers to the likelihood of exhibiting residue side chain heavy distances less than 5 Å within the subset of frames characterized by the peptide Q value exceeding 0.8. Contact probabilities calculated for each system were saved in `system name_contact_prob.npy` files and used to plot Fig. 2c, d, e, and Extended Data Fig. 5a, b, c, d, and e in the manuscript. **Files:** * **FusA_FusC_contact_prob.tar.gz (18.8 kB)** - Including FusA_FusC_contact_prob.npy (Fig. 2c, d, e, and Extended Data Fig. 5a), FusA_I11R_FusC_contact_prob.npy (Extended Data Fig. 5b), FusA_I11R_FusC_R321G_contact_prob.npy (Extended Data Fig. 5c), FusA_V13R_FusC_contact_prob.npy (Extended Data Fig. 5d), and FusA_V13R_FusC_R321G_contact_prob.npy (Extended Data Fig. 5e). **Code:** [Contact_probability_heatmap.py](https://github.com/ShuklaGroup/lasso_rotaxane/blob/main/Contact_probability_heatmap.py) ### 5. Fraction of Native Contacts (Q) Probability Density The fraction of native contacts (Q) probability density was calculated based on previous Q data and used to plot Fig. 3c and Extended Data Fig. 6d in the manuscript. The bins, mean, and standard deviation (SD) values of probability density for plotting were saved in `system name_Q_prob_density.pkl` files. **Files:** * **FusA_FusC_Q_prob_density.tar.gz (13.9 kB)** - Including FusA_FusC_Q_prob_density.pkl, FusA_I11R_FusC_Q_prob_density.pkl, FusA_I11R_FusC_R321G_Q_prob_density.pkl (Fig. 3c), FusA_V13R_FusC_Q_prob_density.pkl, and FusA_V13R_FusC_R321G_Q_prob_density.pkl (Extended Data Fig. 6d). **Code:** [Native_Contacts_prob_density.py](https://github.com/ShuklaGroup/lasso_rotaxane/blob/main/Native_Contacts_prob_density.py) ### 6. Peptide Residue Contributions to Contact Probability The contact probabilities between each peptide FusA residue and all selected FusC residues were summed. The proportion of each FusA residue's contact probability to the total was then calculated to evaluate the contribution. The calculated contribution values were saved in `system name_pep_res_contr.npy` files and used to plot Supplementary Fig. 10 in the manuscript. **Files:** * **FusA_FusC_pep_res_contributions.tar.gz (1.0 kB)** - Including FusA_FusC_pep_res_contr.npy, FusA_I11R_FusC_pep_res_contr.npy, FusA_I11R_FusC_R321G_pep_res_contr.npy, FusA_V13R_FusC_pep_res_contr.npy, and FusA_V13R_FusC_R321G_pep_res_contr.npy (Supplementary Fig. 10). **Code:** [Contact_probability_contribution.py](https://github.com/ShuklaGroup/lasso_rotaxane/blob/main/Contact_probability_contribution.py) ## Code/Software All the code used in this work is openly available at [https://github.com/ShuklaGroup/lasso_rotaxane/](https://github.com/ShuklaGroup/lasso_rotaxane/)