Text S1 - The Free Energy Landscape of Small Molecule Unbinding

This file contains the supporting figures and table for this
article.

Figure S1: Time series of DRMS from the X-ray structure for 20
of the 50 runs of BUT at 310 K. The y axis is DRMS in Å and x axis is
time in ns. Figure S2: Time series of distance between centers
of mass of BUT and FKBP active site in 20 of the 50 runs at 310 K. The y
axis is distance in Å and x axis is time in ns. The green or red line
indicates distance at 15 or 10 Å. Figure S3: Scatter plot
of experimental binding energies versus natural logarithm of the unbinding
times extracted from MD at 310 and 350 K. The Pearson correlation
coefficient is −0.84 and −0.83 for 310 and 350 K MD runs,
respectively. The unbinding time and error for each ligand are evaluated by
single-exponential fitting of the cumulative distribution function of
unbinding times using 25 randomly selected MD runs out of 50, and
calculating the average error for the remaining 25 MD runs not used for the
fitting, i.e., the difference between the value predicted by the fitting
curve and the unbinding time measured along the MD trajectory. This
procedure is repeated 100 times for each ligand, and average values of
unbinding time and cross-validated error are shown. Figure S4:
Cut-based FEPs of six ligands at 310 K (black). The distance between centers
of mass of ligand and FKBP active site (green) and the mean first passage
time (red) are also shown with y-axis on the right. Figure S5:
Network representation of the bound states of the six ligands at 310 K. The
largest 25 nodes are marked with numbers and their representatives are shown
in Fig. S6–S11 in Text S1. Figure S6:
Representative poses of the largest 25 nodes of DMSO. Figure
S7: Representative poses of the largest 25 nodes of PENT.
Figure S8: Representative poses of the largest 25 nodes of
BUT. Figure S9: Representative poses of the largest 25 nodes of
DAP. Figure S10: Representative poses of the largest 25 nodes
of DSS. Figure S11: Representative poses of the largest 25
nodes of THI. Figure S12: Cut-based FEPs plotted using as
reference node the most populated node of individual subbasins. These
cut-based FEPs were used to determine the subbasins of the bound state. The
cut-based FEP on the top left corresponds to the one in Figure 2 of the main text. Figure
S13: Simplified network of subbasins in the bound state of BUT.
The nodes are the subbasins identified with the procedure shown in Fig. S12
in Text
S1 except for the black node which represents the unbound state.
The thickness of the links is proportional to the number of the transitions
observed in the 50 MD runs at 310 K. Figure S14: Network
representation of the bound states of the six ligands at 350 K. Only nodes
connected by links of weight 5 or more are shown to avoid overcrowding.
Figure S15: Cut-based FEPs of six ligands at 350 K.
Figure S16: Single-exponential kinetics of unbinding for 6
ligands at 310 K. The plots show the cumulative distribution f(t) of the
unbinding times observed in the 50 MD runs. Note that the unbinding times
obtained by fitting are slightly different from those in Table 1 of the main text
because a cross-validation procedure was used in the latter. Figure
S17: Single-exponential kinetics of unbinding for 6 ligands at
350 K. The plots show the cumulative distribution f(t) of the unbinding
times for 6 ligands at 350 K. The unbinding times range from 1.6 to 5.6 ns,
which is shorter than the corresponding values at 310 K. Figure
S18: Network representations of the bound state for DMSO (top
left), PENT (top right), BUT (middle left), DAP (middle right), DSS (bottom
left), and THI (bottom right). Nodes are colored from red to green according
to the distance of the centers of mass of ligand and FKBP. Figure
S19: Stereoview of the most populated clusters for 6 ligands -
DMSO, PENT, BUT, DAP, DSS and THI (top to bottom). Nodes are colored from
red to green according to the distance of the centers of mass of ligand and
FKBP. Figure S20: Test at 310 K with DMSO. Ten bound state
conformations were randomly chosen from previous MD simulations and ten runs
of 10 ns each with different initial velocities were started for each of
them. Single-exponential kinetics of unbinding is observed and the unbinding
time derived from the plot is 4.2 ns which is similar to the value derived
from the 50 runs started from the X-ray structure of the complex.
Figure S21: Test at 350 K with DMSO. Fifty 5-ns runs with
different velocities were started for each of five randomly oriented poses
of DMSO in the active site of FKBP. Single-exponential kinetics of unbinding
is observed and the unbinding times derived from the plots range from 1.3 to
1.9 ns, which is consistent with the value derived from the 50 runs started
from the X-ray structure of the complex (top, left). Figure
S22: The cFEPs for DMSO (left) and PENT (right) were obtained
using DRMS clustering cutoffs of 0.8 Å, 0.9 Å, 1.0 Å, and
1.5 Å from top to bottom. Figure S23: Diffusivity test
for the clustering of DMSO and THI. The profiles with saving frequency at 4
and 8 ps are similar upon a vertical shift of
ln(), which is
consistent with the diffusive regime. Table S1: Robustness of
TSE definition and Hammond behavior. Each column lists the average distances
between the centers of mass of the ligand and FKBP active site for the
conformations at the TSE. The numbers of TSE nodes and snapshots are shown
in parentheses. Only TSE nodes with weight larger than 5 were used for this
analysis as nodes with very low weight increase the noise.
Note: A movie of the MD simulation of spontaneous unbinding of
BUT from FKBP can be found at http://www.biochem-caflisch.unizh.ch/movie/7/.
(PDF)