Molecular Dynamics Simulations of a Binding Intermediate between FKBP12 and a High-Affinity Ligand.

We characterized a binding intermediate between the protein FKBP12 and one of its high-affinity ligands by means of molecular dynamics simulations. In such an intermediate, which is expected to form at the end-point of the bimolecular diffusional search, short-range interactions between the molecular partners may play a role in the specificity of recognition as well as in the association rate. Langevin dynamics simulations were carried out to generate the intermediate by applying an external biasing force to unbind the ligand from the protein. The intermediate was then refined by seven independent molecular dynamics simulations performed with an explicit solvent model. We found consistent results both for the structure of the protein and for the position of the ligand in the intermediate. The two carbonyl oxygens O2 and O3 of the ligand core region act as two main anchors, making permanent contacts in the intermediate. The transient contacts with the protein are made by the ligand noncore moieties whose structures and mobilities enable many alternative contacts of different types to be formed: π-π molecular overlap and weak hydrogen bonds NH···π, CH···π, and CH···O. Hence, the stability of the ligand at the entrance of the protein binding pocket offers the possibility of fine-tuning a variety of short-range contacts that involve the ligand noncore moieties. Under the hypothesis that the stability of this intermediate is related to the affinity of the ligand, this binding intermediate model comes closest to explaining the role played by the noncore moieties in the affinity of this ligand. Moreover, this model also provides a plausible explanation for how structurally diverse core motifs that all share the carbonyl atoms O2 and O3 bind to FKBP12.