Multi-Timescale Dynamics Study of FKBP12 Along the Rapamycin–mTOR Binding Coordinate

• Published in: Journal of molecular biology 2011-01, Vol.405 (2), p.378-394
• Main Authors: Sapienza, Paul J., Mauldin, Randall V., Lee, Andrew L.
• Format: Article
• Language: English
• Subjects: binding proteins; Binding Sites; cell cycle; cell proliferation; cis–trans isomerase; drug dynamics; drugs; enzyme inhibitor; Humans; immunosuppressant; isomerization; Magnetic Resonance Spectroscopy; mammals; Models, Chemical; molecular assembly; nuclear magnetic resonance spectroscopy; Protein Binding; Protein Conformation; Sirolimus - chemistry; Sirolimus - metabolism; Tacrolimus - chemistry; Tacrolimus - metabolism; Tacrolimus Binding Protein 1A - chemistry; Tacrolimus Binding Protein 1A - metabolism; therapeutics; TOR Serine-Threonine Kinases - chemistry; TOR Serine-Threonine Kinases - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2010.10.037

Drugs can affect function in proteins by modulating their flexibility. Despite this possibility, there are very few studies on how drug binding affects the dynamics of target macromolecules. FKBP12 (FK506 binding protein 12) is a prolyl cis–trans isomerase and a drug target. The immunosuppressant drug rapamycin exerts its therapeutic effect by serving as an adaptor molecule between FKBP12 and the cell proliferation regulator mTOR (mammalian target of rapamycin). To understand the role of dynamics in rapamycin-based immunosuppression and to gain insight into the role of dynamics in the assembly of supramolecular complexes, we used 15N, 13C, and 2H NMR spin relaxation to characterize FKBP12 along the binding coordinate that leads to cell cycle arrest. We show that sequential addition of rapamycin and mTOR leads to incremental rigidification of the FKBP12 backbone on the picosecond-nanosecond timescale. Both binding events lead to perturbation of main-chain and side-chain dynamics at sites distal to the binding interfaces, suggesting tight coupling interactions dispersed throughout the FKBP12–rapamycin interface. Binding of the first molecule, rapamycin, quenches microsecond-millisecond motions of the FKBP12 80's loop. This loop provides much of the surface buried at the protein–protein interface of the ternary complex, leading us to assert that preorganization upon rapamycin binding facilitates binding of the second molecule, mTOR. Widespread microsecond-millisecond motions of the backbone persist in the drug-bound enzyme, and we provide evidence that these slow motions represent coupled dynamics of the enzyme and isomerization of the bound drug. Finally, the pattern of microsecond-millisecond dynamics reported here in the rapamycin complex is dramatically different from the pattern in the complex with the structurally related drug FK506. This raises the important question of how two complexes that are highly isomorphic based on high-resolution static models have such different flexibilities in solution. [Display omitted] ► NMR order parameters show that FKBP12 becomes incrementally rigidified upon binding rapamycin and mTOR. ► Rapamycin binding quenches a subset of slow FKBP12 motions. Preorganization is required for mTOR binding. ► Slow motions in the binary and ternary complexes likely result from the coupled isomerization of FKBP12 and the drug. ► Binding of rapamycin and FK506, two highly structurally similar drugs, elicits different slow motions in FKB