# *Clostridioides difficile* Toxins Unhinged: Allosterically Switchable Network Orients Beta-flap

## Description
Allosteric proteins regulate function by substrate binding far from the active site. 
*Clostridioides difficile* toxins use allosteric binding by an endogenous co-factor to orchestrate self-cleavage from within the target cell. 
This binding event induces a conformational shift, primarily effecting a lever-like "beta-flap" region, with two known orientations. 
We uncovered a mechanism for this allosteric transition using extensive atomistic MD simulations and computational and experimental mutagenesis.
The mechanism relies on a switchable interaction network. 
The most prominent interaction is K600–E743, with K600 interactions explaining ~70 % of the allosteric effect.
Rather than gradually morphing between two end states, the interaction network adopts two mutually exclusive configurations in the active and inactive state.
Similar switchable networks may explain allostery more broadly.
This mechanism in particular could aid in drug development targeting the *Clostridioides difficile* toxins autoproteolysis.

### Conformation naming
The names given to conformations changed during the development 
of this project. The conformation named "closed" corresponds to the
lowered conformation, while the name "open" corresponds to the 
"raised" conformation.

### Residue numbering
Throughout the paper, residues are numbered based on full-toxin numbering. Residue numbering in the scripts is indexed from the first modelled residue. To convert to full-toxin numbering, add 543 for TcdB and add 541 for TcdA.

## File tree

### Paths to notable files
- Grid points for umbrella sampling

- Project configuration: config/settings.py
- Alignment structure: ref-structures/ref_all_atoms.pdb
- Alignment residues: ref-structures/core_res.npy
- Reference TcdB CPD conformations: ref-structures/lowered_ref_state.pdb, ref-structures/raised_ref_state.pdb
- Modified Amber14sb force field: reproduce/amber14sb_ip6.ff

### Analysis scripts
Scripts related to the analysis of MD data and figure generation. 

AllosteryCPD/
- README.md
- AlphaFold/
    - AF_PCA.py
    - CombindedRMSD.py
    - ... (other scripts for AlphaFold structure generation/analysis)
- Biased/
    - FES2D.py
    - WindowAves.py
    - ... (other scripts for analysis of umbrella sampling data)
- Config/
    - settings.py
- Other/ (paper-plots)
    - AveIPL_charges.py
    - TablePlot_network.py
    - ... (other scripts, mostly figure generation)
- Tools/ 
    - connect.py
    - traj_funcs.py
    - utils.py
- BasicMD.py
- VectorCoordCombo.py
- ... (other basic analysis scripts)

### Unbiased simulations
Key files for reproduction of simulation data are within the reproduce/ directory. In general, this looks like:

AllosteryCPD/
- reproduce/
    - example-system/
        - example-system.pdb (initial structure)
        - set_up.sh (define box, solvate, add ions)
        - run_sim.sh (run simulation)
        - cat_traj.sh (post-process simulation)
    - MDP-apo/ (molecular dynamics parameters for apo simulations)
        - em_steep.mdp (energy minimization)
        - NVT.mdp (thermal equilibration)
        - NPT.mdp (pressure equilibration)
        - Production (md run)
    - MDP-holo/ (molecular dynamics parameters for holo simulations)
        - em_steep.mdp (energy minimization)
        - NVT.mdp (thermal equilibration, restraining protein and ligand)
        - NPT.mdp (pressure equilibration, restraining protein and ligand)
        - NVT2.mdp (thermal equilibration, restraining only ligand)
        - NPT2.mdp (pressure equilibration, restraining only ligand)
        - Production (md run)
    - ... (various systems, force fields)

### Biased simulations
Key files for reproduction of biased sampling data using umbrella sampling are within the reproduce/ directory. In general, this looks like: