To test reproducibility, both VD3 molecules were extracted from the crystallographic
complex (PDB 2GJ5) and VD3 was docked back to the whole protein through BD. Table
IS shows the first five lowest binding free energies, all placed VD3 within site A with
binding free energies ranging between -5.67 to -7.63 kcal mol-1, similar results than that
reported elsewhere using combining docking and MDSs,54 and confirming the ability of
this approach to recognize the calyx as the main binding site.
Pose
Estimated Binding Free
energy (kcal/mol)
1
-7.63 ±0.5
2
-7.22 ±0.1
3
-6.38 ±0.2
4
-5.78 ±0.4
5
-5.67 ±0.2
Table IS. Binding free energy of VD3 associated to site A of βlg monomer obtained by BD.
As it was not found the secondary binding site (site B) by using the procedure above
mentioned, another protocol was used to reveal a secondary binding site, in which the target
was the monomer with site A already occupied with a VD3 molecule. By following this
protocol, there were observed seven binding poses which were distributed along three
different parts of the protein surface due to the fact that site A was already occupied (Table
IIS).
Among these conformations, the first binding pose placed VD3 in between β-strand I and
the α-helix (site B), pose 2 close to the entrance of the protein calyx (site C), and pose 3 in
between betweenβ-strands F-G and α-helix (figure 2B), result in agreement at least for
binding poses 1 and 2 with those reported elsewhere.54 Interestingly, the first binding pose
1
correspond to that of site B of the crystallographic complex, results that reveal the ability of
docking to identify potential protein regions for a second binding site, however, it is worth
to note that from the seven binding poses, the three corresponding to VD3 bounded to site
B showed opposite orientation, binding either tail-first or head-first towards βLG with
RMSD of 2.008 (28 to 28 atoms), 1.831 (26 to 26 atoms), and 0.884 (25 to 25 atoms) with
respect to the crystallographic structure (figure 2C), with binding energies ranging between
-6.0 and -7.36 kcal mol-1, similar values than that reported elsewhere for site B but using
AutoDock Vina.54
Pose
1
2
Estimated Binding Free energy
(kcal/mol)
-7.36 ±0.2
-6.36 ±0.1
3
4
5
6
7
-6.18 ±0.5
-5.98 ±0.2
-5.96 ±0.3
-5.94 ±0.5
-5.42 ±0.1
Observations
betweenβ-strand I and the α-helix (site B)
At the cavity entrance forming contacts with
residues at AB-EF and GH loops (site C)
betweenβ-strands G-H and the α-helix
siteB
betweenβ-strands G-H and the α-helix
siteB
betweenβ-strands G-H and the α-helix
Table IIS. Binding free energy of VD3 associated to the external surface of βlg monomer,
obtained by BD, whereas the calyx is already occupied with a VD3 molecule. The locations
are shown in Figure 2B.
In the βlg dimeric complex with VD3 as site A, there were observed 10 binding poses that
were distributed along three different parts of the protein surface (Table IIIS). The first
binding pose placed VD3 at site C as observed for the βlg monomer, the second at site B1
and the third at site B2 (Table IIIS), these two latter binding poses with lower binding free
energies ranging between -3.39 and -4.58 kcal mol-1 than the βlg monomer.
2
Pose
1
2
3
4
5
6
7
8
9
10
Estimated Binding Free
energy (kcal/mol)
-4.58 ±0.2
-4.57 ±0.1
-4.46 ±0.3
-3.92 ±0.2
-3.90 ±0.1
-3.87 ±0.5
-3.75 ±0.3
-3.67 ±0.2
-3.39 ±0.1
-3.20 ±0.1
Observacions
site C
site B1
site C (chain A)
site C (chain A)
site C (chain B)
site C (chain B)
site C (chain B)
site B2
site C (chain A)
site B1
Table IIIS. Binding free energy of VD3 associated to the external surface of βlg dimer
obtained by BD, whereas site A1 and A2 are already occupied with VD3 molecules. The
locations are shown in Figure 2E.
3