Conformation-specific synthetic intrabodies modulate mTOR signaling with subcellular spatial resolution

Area of Science:

• Molecular Biology
• Cellular Signaling
• Structural Biology

Background:

• Subcellular compartmentalization is crucial for regulating mechanistic target of rapamycin (mTOR) signaling.
• Understanding location-specific mTOR signaling and its biological outcomes remains a challenge.

Purpose of the Study:

• To engineer synthetic intracellular antibodies (intrabodies) for spatially resolved modulation of mTOR signaling.
• To investigate the structural basis of mTOR complex 1 (mTORC1) regulation and its biological consequences.

Main Methods:

• Epitope-directed phage display to generate high-affinity antibody fragments (Fabs) against mTOR's FKBP12-Rapamycin binding site (mTOR^FRB).
• High-resolution crystal structures of Fabs to determine distinct mTOR^FRB conformational states.
• Intracellular signaling studies using genetically encoded intrabodies to assess mTORC1 inhibition and downstream effects.

Main Results:

• Identified novel Fabs that bind to specific mTOR^FRB conformations, revealing an allosteric mechanism for mTORC1 stability.
• Demonstrated that synthetic binders mimic natural substrates, highlighting mTOR^FRB's molecular recognition capabilities.
• Showed differential, time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by intrabodies, analogous to rapamycin.
• Successfully modulated mTOR signaling in the nucleus using spatially programmed intrabody expression.

Conclusions:

• Intrabodies are versatile tools for dissecting mTORC1 conformational regulation with spatial precision.
• This approach enables the study of how location-specific mTOR signaling influences cellular functions and disease.
• Findings provide insights into the structural mechanisms governing mTORC1 activity and regulation.