Biology Reference
In-Depth Information
by using the program DOCK4.0. Residues within a radius of 4 Ǻ around the ATP-
binding pocket of the VicK HATPase_c domain were used for constructing the grids
for the docking screening. Subsequently, the 10,000 compounds with the highest score
as obtained by DOCK search were selected for a second round docking by using the
Autodock 3.05 program, followed by our own filter of drug likeness to eliminate the
non-drug-able molecules. Finally, we manually selected 105 molecules according to
their molecular diversity, shape complementarities, and potential to form hydrogen
bonds in the binding pocket of the VicK HATPase_c domain.
Molecular Modeling of the Interaction between Inhibitors and the Target
Protein
To determine the binding modes, Autodock 3.05 was used for automated docking
analysis. The Lamarchian genetic algorithm (LGA) was applied to deal with the pro-
tein-inhibitor interactions. Some important parameters were set as follows: the initial
number of individuals in population is 50; the elitism value is 1, which automatically
survives into nest generation. The mutation rate is 0.03, which is a probability that
a gene would undergo a random change. The crossover rate, the probability of pro-
portional selection, is 0.80. Every compound was set to have 10 separated GA runs
and finally 10 conformations would be generated. The conformations were clustered
automatically and the conformation with minimum binding free energy in the cluster
with minimum RMSD value was selected as the representative conformation of the
inhibitor.
Cloning, Expression, and Purification of the VicK Protein
The
VicK
gene fragment containing the cytoplasmic signal domains (the HATPase_c
and HisKA domain) of VicK (coding 200-449 aa) was amplified by PCR. The
upstream and the downstream primers were 5'-CGGGATCCGAGCAGGAGA-
AGGAAGAAC-3' and 5'-CGCTCGAGGTCTTCTACTTCATCCTCCCA-3' respec-
tively. Subsequently, the fragment was digested with
EcoR
I and
Xho
I (TaKaRA,
Japan) and ligated into the corresponding sites of pET28a to obtain a recombinant
plasmid pET28/VicK'. After being transformed into
E. coli
strain BL21 (DE3), this re-
combinant plasmid was induced to express the protein of VicK' by 0.2 mM isopropyl-
1-thio-β-D-galactopyranoside (IPTG) at 24°C for 20 hr. Cells were harvested and
sonicated, and then the debris was removed by centrifugation. The fraction containing
the cytoplasmic domain was isolated from the supernatant solution through a His-
tagged column, with a purity of more than 95%, as assessed by gel electrophoresis and
Coomassie Blue staining.
Inhibition Assay for the ATPase Activity
The inhibitory activity of the compounds for the ATPase activity of the VicK' protein
was measured using the Kinase-Glo™ Luminescent Kinase Assay (Promega, Madi-
son, USA). Briefly, 6 μg purified VicK' protein was pre-incubated with a series of
dilutions of compounds in a reaction buffer containing 40 mM Tris-HCl (pH 7.5), 20
mM MgCl
2
and 0.1 mg/ml BSA, at room temperature for 10 min. Then 5 μM ATP
was added for another incubation of 10 min at room temperature, and Kinase-Glo™
