construct the PMF (Fig.4). US protocol has become a routine
method for the study of events involving nonequilibrium states,
such as conformational changes, binding/unbinding processes, or
even chemical reactions when coupled with QM/MM hybrid
methods.
In this guideline, we used US method in order to study the
unbinding process of the biaryl-triazolopyridine derivative bound
to p38 crystal structure. While we provide a detailed description of
the methodology, the proposed parameters of this particular study
are not necessarily the optimized ones. To make sure that the
calculation will provide useful and coherent results, the different
parameters of US calculations have to be verified first:- The choice of the restrained variableζis the crucial step for the
preparation of US calculation. The nature of the restrained
variable (distance, angle, dihedral, or linear combination of
them, etc.) has to be carefully chosen, to represent in the best
manner the event to sample. In this example, on p38 kinase in
complex with an inhibitor, the distance between one residue of
the Hinge region (THR104) and the ligand has been consid-
ered as the restrained variableζfor the unbinding process. This
distance is measured between two points: for the threonine
residue, the center of mass of the three atoms of the backbone
is chosen. Indeed, the backbone of the protein is less flexible
than its side chains. The second center of mass is calculated
using the two six-membered rings of the inhibitor. The oscilla-
tor strength related to the bias is also a parameter to consider
and is dependent of the event to study and the nature of
ligand–receptor interactions. In this case, the strength is arbi-
trarily fixed to 20 kcal/mol (seeNote 16).
Fig. 4Scheme of the US principle. Left: Representation of the evolution of the energy along the restrained
variableζ. Each window is centered on a fixed value ofζth. Right: potential of mean force (PMF) obtained with
the use of the WHAM algorithm. The sampling distributions obtained for each window are transformed into
free energy profile
418 Sonia Ziada et al.