Rapamycin, a macrolide that possesses immunosuppressive and anti-proliferative properties, has also been used as an artificial trigger to modulate genetically engineered reaction networks, and its derivatives have been approved for clinical use between in graft transplant and oncology. FRB is the rapamycine binding domain of the mTOR kinase, which belongs to the mTORC1/mTORC2 complexes. Despite of the vast knowledge accumulated since the 90’s, there are still many pending questions. We have combined single-molecule force spectroscopy (SMFS), molecular dynamics (MD), and nuclear magnetic resonance (NMR) to acquire a multi-timescale view of the interactions among the FKBP12-rapamycin-FRB tenary complex. We will demonstrate how the structural changes in the drug-resistant mTOR mutant influence the binding of rapamycine, thereby identifying the key interactions for maintaining the tenary complex. Specifically, how different mutants in the two proteins FKBP12 and FRB affect the interactions in this tenary complex were investigated with these approaches in a cohesive manner. We extensively applied our recently developed curvilinear-path umbrella sampling (CPUS) approach to estimate the standard free energy of binding and to characterize the curvilinear dissociation physical trajectories of this tenary complex. We anticipate that our investigations will be beneficiary for future design of rapamycin analogs (rapalogs).

Structures and dynamics of FKBP-rapamycine-mTOR binding domain charaterizaed by single-molecule force spectroscopy, molecular dynamics simulations and nuclear magnetic resonace

ACS Fall 2021

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