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Updated: Sep 3, 2025
A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction
Published on: August 27, 2019
A Curvilinear-Path Umbrella Sampling Approach to Characterizing the Interactions Between Rapamycin and Three FKBP12
Dhananjay C Joshi1, Charlie Gosse2, Shu-Yu Huang1
1Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan.
Abstract:
Rapamycin is an immunosuppressant macrolide that exhibits anti-proliferative properties through inhibiting the mTOR kinase. In fact, the drug first associates with the FKBP12 enzyme before interacting with the FRB domain of its target. Despite the availability of structural and thermodynamic information on the interaction of FKBP12 with rapamycin, the energetic and mechanistic understanding of this process is still incomplete. We recently reported a multiple-walker umbrella sampling simulation approach to characterizing the protein-protein interaction energetics along curvilinear paths. In the present paper, we extend our investigations to a protein-small molecule duo, the FKBP12•rapamycin complex. We estimate the binding free energies of rapamycin with wild-type FKBP12 and two mutants in which a hydrogen bond has been removed, D37V and Y82F. Furthermore, the underlying mechanistic details are analyzed. The calculated standard free energies of binding agree well with the experimental data, and the roles of the hydrogen bonds are shown to be quite different for each of these two mutated residues. On one hand, removing the carboxylate group of D37 strongly destabilizes the association; on the other hand, the hydroxyl group of Y82 is nearly unnecessary for the stability of the complex because some nonconventional, cryptic, indirect interaction mechanisms seem to be at work.
Insights
This study investigates the binding mechanism of rapamycin with FKBP12 using advanced simulations. It reveals distinct roles for hydrogen bonds in stabilizing the FKBP12-rapamycin complex, with D37 being crucial and Y82 less so.
Area of Science:
- Biochemistry
- Computational Biology
- Pharmacology
Background:
- Rapamycin is an immunosuppressant macrolide that inhibits mTOR kinase.
- Rapamycin binds to FKBP12 before interacting with its target.
- The energetic and mechanistic details of FKBP12-rapamycin interaction require further elucidation.
Purpose of the Study:
- To extend computational simulations to protein-small molecule interactions, specifically the FKBP12•rapamycin complex.
- To estimate binding free energies of rapamycin with wild-type FKBP12 and two mutants (D37V, Y82F).
- To analyze the mechanistic details and the role of hydrogen bonds in the binding process.
Main Methods:
- Utilized a multiple-walker umbrella sampling simulation approach.
- Applied simulations to characterize protein-small molecule interaction energetics.
- Calculated standard free energies of binding for wild-type and mutant FKBP12.
Main Results:
- Calculated binding free energies closely matched experimental data.
- Identified distinct roles for hydrogen bonds involving D37 and Y82.
- Removal of D37's carboxylate group significantly destabilized the complex, while Y82's hydroxyl group was less critical.
Conclusions:
- The study provides a detailed energetic and mechanistic understanding of the FKBP12•rapamycin complex.
- Hydrogen bonds play differential roles in stabilizing the complex, with D37 being essential and Y82 having indirect stabilizing mechanisms.
- Computational simulations are effective for characterizing protein-small molecule interactions.
