Autodock

Objective to dock Purpurogalline to zinc dependent beta-lactamase along with the refernce compound .

Installation.

The installation of the Autodock vina can be followed from the given website.

It is recommended to create a conda environment for installation of the autodock vina.

$ conda create -n vina python=3
$ conda activate vina
$ conda config --env --add channels conda-forge
# install the dependency
conda install -c conda-forge numpy swig boost-cpp sphinx sphinx_rtd_theme
# install using source code.
git clone https://github.com/ccsb-scripps/AutoDock-Vina

cd AutoDock-Vina/build/python

The autodock also needs other dependency such as ADFR and Meeko

installation of ADFR

Since macos does not allows non standard apps to get install we have to give a temporary access to the software.

sudo spctl --master-disable

Then install the ADFR

For that we have run

./install.sh
# export the path in ~/.bashrc or  ~/.bash_profile for macos
export PATH=/Users/sriram/Documents/thanet/docking/ADFRsuite_x86_64Darwin_1.0/bin:$PATH

# enabling the security
sudo spctl --master-enable

installation of Meeko

Meeko can be installed in the same conda enviroment as of the autodock vina

conda install -c conda-forge numpy openbabel scipy rdkit
pip install meeko

installation of the ADT tools for grid visualization.

Install mgltools for particular version using the following site

update in bash profile

~/.bashrc - export PATH="/Users/Shared/MGLTools/1.5.7/bin:"$PATH

adt GUI can be opened by adt command if path is set.

autogrid installation

Binary of the autodock4 can be downloaded from this website Only path has to be specified in basrc for two binary files such as autogrid4 and autodock4.

Tutorial to dock zinc related enzyme.

Beta-lactamase is an enzyme that are responsible for bacterial resistance to beta-lactam antibiotics, such as penicillins and cephalosporins. This enzymes hydrolyze the beta-lactam ring of the antibiotic, rendering it ineffective and allowing the bacteria to survive. The beta-lactamase enzymes are classified into different classes (Class A, Class B, Class C, and Class D), based on their molecular structure and mechanism of action. Zn-Dependent Metallo-Beta-Lactamase in Bacillus cereus corresponds to Class B beta-lactamases, also known as metallo-beta-lactamases (MBLs), are Zn-dependent enzymes. Unlike serine beta-lactamases, they have a metal ion at the active site that facilitates the hydrolysis of the beta-lactam ring. Bacillus cereus is one of the bacterial species that produce Zn-dependent metallo-beta-lactamase. Catalysis

In the Zn-dependent MBLs, one or two zinc ions are crucial for catalysis. In the structure take 4NQ6 there are two zinc complexed with bound inhibitor. Generally the zinc ions are located at the active site of the enzyme and coordinate the water molecules, which are subsequently involved in the hydrolysis of the beta-lactam ring. The catalytic mechanism often involves the formation of a transient enzyme-substrate complex, followed by hydrolysis, leading to the inactivation of the antibiotic. Catalytic Site

The catalytic site in Zn-dependent MBLs usually comprises specific amino acid residues that coordinate the zinc ions. These may include histidine, aspartate, and sometimes cysteine residues. In the case of the 4NQ6 it is HIS 81,83,144,205 ASP 85 and cysteine 163.

The zinc ions play a critical role in the catalytic activity of MBLs. They stabilize the transition state during hydrolysis, lowering the activation energy required for the reaction. Moreover, they help in orienting the water molecules and the substrate at the active site. In the current beta-lactamse , the presence of two zinc ions can dramatically increase the hydrolytic efficiency.

The study of Bacillus cereus Zn-dependent metallo-beta-lactamase and its catalytic site is critical for developing new antibiotics or inhibitors that can effectively target this enzyme. So previously obtained experimental compounds was taken for docking with zinc bound structure.

The 4NQ6 have negative density at zinc ions in the given structure, while the position in the zinc can be verified in other structures. . The same structure was taken as the it has a ligand bond structure. Negative density suggest absence of the electron density at the site.

Tutorial

Use chimera to prepare protein and ligand using dockprep and save as pdb separately.

prepare_receptor -r 4nq6.pdb -o protein.pdbqt -U nphs_lps_waters
pythonsh /Users/sriram/Documents/thanet/autodockZN_files/zinc_pseudo.py -r hydrogen_added_4nq6.pdbqt -o protein_tz.pdbqt

use the ligand generated using the chimera and load in the ADT tool and save as .pdbqt file.

to generate the affinity map

inbound ligand

pythonsh autodockZN_files/prepare_gpf4zn.py -l autodock_protein/inbound_ligand/4nq6_ligand.pdbqt -r autodock_protein/protein_tz.pdbqt \
-o autodock_protein/inbound_ligand/protein_tz.gpf -p npts=40,40,40 -p gridcenter=9.97,9.359,26.102 \
–p parameter_file=autodockZN_files/AD4Zn.dat

./autogrid4 -p autodock_protein/inbound_ligand/protein_tz.gpf -l autodock_protein/inbound_ligand/protein_tz.glg

vina --ligand autodock_protein/inbound_ligand/4nq6_ligand.pdbqt --maps autodock_protein/protein_tz --scoring ad4 \
--exhaustiveness 32 --out autodock_protein/inbound_ligand/4nq6_ligand_out.pdbqt | tee autodock_protein/inbound_ligand/4nq6_ligand.log

purpurogalin

pythonsh autodockZN_files/prepare_gpf4zn.py -l autodock_protein/ref_potassium_clavulanate/clavulanate.pdbqt -r autodock_protein/protein_tz.pdbqt \
-o autodock_protein/ref_potassium_clavulanate/protein_tz_clavulanate.gpf -p npts=40,40,40 -p gridcenter=9.97,9.359,26.102 \
–p parameter_file=autodockZN_files/AD4Zn.dat

./autogrid4 -p autodock_protein/pur/protein_tz_pur.gpf -l autodock_protein/pur/protein_tz_pur.glg

vina --ligand autodock_protein/pur/pur.pdbqt --maps autodock_protein/protein_tz --scoring ad4 \
 --exhaustiveness 32 --out autodock_protein/pur/pur_out.pdbqt

clavulanate

pythonsh autodockZN_files/prepare_gpf4zn.py -l autodock_protein/ref_potassium_clavulanate/clavulanate.pdbqt -r autodock_protein/protein_tz.pdbqt \
-o autodock_protein/ref_potassium_clavulanate/protein_tz_clavulanate.gpf -p npts=40,40,40 -p gridcenter=9.97,9.359,26.102 \
–p parameter_file=autodockZN_files/AD4Zn.dat

./autogrid4 -p autodock_protein/ref_potassium_clavulanate/protein_tz_clavulanate.gpf -l autodock_protein/ref_potassium_clavulanate/protein_tz_clavulanate.glg

vina --ligand autodock_protein/ref_potassium_clavulanate/clavulanate.pdbqt --maps autodock_protein/protein_tz --scoring ad4 \
 --exhaustiveness 32 --out autodock_protein/ref_potassium_clavulanate/clavulanate_out.pdbqt | tee autodock_protein/ref_potassium_clavulanate/clavulanate.log

without zinc inbound ligand

pythonsh autodockZN_files/prepare_gpf.py -l autodock_protein/inbound_ligand_without_zn/4nq6_ligand.pdbqt -r autodock_protein/inbound_ligand_without_zn/hydrogen_added_4nq6_no_zn.pdbqt \
-o autodock_protein/inbound_ligand_without_zn/protein_inbound_ligand_without_zn.gpf -p npts=40,40,40 -p gridcenter=9.97,9.359,26.102

./autogrid4 -p autodock_protein/inbound_ligand_without_zn/protein_inbound_ligand_without_zn.gpf -l autodock_protein/inbound_ligand_without_zn/protein_inbound_ligand_without_zn.glg

vina --ligand autodock_protein/inbound_ligand_without_zn/4nq6_ligand.pdbqt --maps autodock_protein/hydrogen_added_4nq6_no_zn --scoring ad4 \
 --exhaustiveness 32 --out inbound_ligand_without_zn/4nq6_ligand_out.pdbqt | tee inbound_ligand_without_zn/4nq6_ligand_out.log

without zinc purpurogalin ligand

pythonsh autodockZN_files/prepare_gpf.py -l autodock_protein/pur_no_zn/pur.pdbqt -r autodock_protein/hydrogen_added_4nq6_no_zn.pdbqt \
-o autodock_protein/pur_no_zn/pur_ligand_without_zn.gpf -p npts=40,40,40 -p gridcenter=9.97,9.359,26.102

./autogrid4 -p autodock_protein/pur_no_zn/pur_ligand_without_zn.gpf -l autodock_protein/pur_no_zn/pur_ligand_without_zn.glg

vina --ligand autodock_protein/pur_no_zn/pur.pdbqt --maps autodock_protein/hydrogen_added_4nq6_no_zn --scoring ad4 \
 --exhaustiveness 32 --out autodock_protein/pur_no_zn/pur_ligand_without_zn_out.pdbqt | tee autodock_protein/pur_no_zn/pur_ligand_without_zn_out.log

without zinc clavulanate ligand

pythonsh autodockZN_files/prepare_gpf.py \
-l autodock_protein/ref_potassium_clavulanate_no_zn/clavulanate.pdbqt -r autodock_protein/hydrogen_added_4nq6_no_zn.pdbqt \
-o autodock_protein/ref_potassium_clavulanate_no_zn/clavulanate.gpf -p npts=40,40,40 -p gridcenter=9.97,9.359,26.102

./autogrid4 -p autodock_protein/ref_potassium_clavulanate_no_zn/clavulanate.gpf -l autodock_protein/ref_potassium_clavulanate_no_zn/clavulanate_ligand_without_zn.glg

vina --ligand autodock_protein/ref_potassium_clavulanate_no_zn/clavulanate.pdbqt --maps autodock_protein/hydrogen_added_4nq6_no_zn --scoring ad4 \
 --exhaustiveness 32 --out autodock_protein/ref_potassium_clavulanate_no_zn/clavulanate.pdbqt_ligand_without_zn_out.pdbqt | tee autodock_protein/ref_potassium_clavulanate_no_zn/clavulanate.pdbqt_ligand_without_zn_out.log

RMSD calculation of co-crystal and docked conformer in pymol

```py pymol # load the co-crystal as reference load 4nq6_ligand.pdbqt,crystal # load the docked structure

load 4nq6_ligand_out.pdbqt, docked

load autodoc_protein/hydrogen_added_4nq6_no_zn.pdb,protein_no_zn load autodoc_protein/hydrogen_added_4nq6.pdb, protein

print(cmd.count_states("docked"))

```python
python
cmd.split_states("docked")
f = open("rmsd_crystal.txt","w")
for state in range(1,cmd.count_states("docked") +1):
    cmd.create(f"protein_complex_%.4d"%state,f"protein or docked_%.4d"%state)
    r = cmd.rms(f"docked_%.4d"%state,"crystal")
    print(f"RMSD of conformer %s is %s"%(state,r))
    f.write(f"RMSD of conformer %s is %s\n"%(state,r))
    cmd.save(f"protein_complex_%.4d.pdb"%state,f"protein_complex_%.4d"%state)
    cmd.delete(f"protein_complex_%.4d"%state)
f.close()

cmd.delete("docked_*")
python end

pdb 4nq6 |Conformers| RMSD score of co-crystal ligand with zinc ($\AA$) | without zinc ($\AA$) |Docking score with zinc ($kcal mol^-1 $)| Docking score without zinc ($kcal mol^-1 $) | |—-|—-|—-|—-|—-| |1|0.825|0.909|-5.19|-4.926| |2|0.954|1.040|-5.165|-4.914| |3|0.925|0.824|-5.002|-4.825| |4|0.785|0.783|-4.857|-4.557| |5|0.684|0.685|-4.818|-4.485| |6|0.782|0.867|-4.618|-4.456| |7|0.925|0.384|-4.476|-4.299| |8|0.616|0.559|-4.452|-4.251| |9|0.703|0.706|-4.409|-4.078|

Image of the crystal and docked structure

The above table highlights the rmsd profile of the docked structure with the co-crystal. In the following image, docked co-crystal is shown in green stick representaion, superimposed to the crystal structre. The ligand bound with and without zinc has simialr pose but certain, salt-bridge and metal-coordination is lost during docking.

.

docking with reference compound potassium clavulanate and purpurogalin.

the docking results of both compunds are similar intems of docking score. The best pose of reference compound docked to receptor is shown below where clavulanate is shown in light blue stick representation, the docking score of the reference compound with and without zinc are -4.401,-4.272 respectively.

The docking result of the purpurogalin to the receptor is shown below where purpurogalin is shown in light blue stick representation, the docking score of the reference compound with and without zinc are -4.846,-4.631 respectively. .

From interaction analysis, many interactions with the surrounding residues are conserved but certain saltbridges in the crystallographic structure is not observed. This can be investigaed in molecular dynamics.