9DBO

Crystal structure of a synthetic Fab (R3E9) in complex with the FRB domain of mTOR

Summary for 9DBO

• Entry DOI: 10.2210/pdb9dbo/pdb
• Descriptor: Serine/threonine-protein kinase mTOR, Fab heavy chain, Fab light chain, ... (4 entities in total)
• Functional Keywords: kinase, inhibitor, antibody, signaling protein, transferase-immune system complex, transferase/immune system
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 3
• Total formula weight: 58267.22

Authors

O'Leary, K.M.,Slezak, T.,Kossiakoff, A.A. (deposition date: 2024-08-23, release date: 2025-06-11, Last modification date: 2025-06-18)

Primary citation

O'Leary, K.M.,Slezak, T.,Kossiakoff, A.A.
Conformation-specific synthetic intrabodies modulate mTOR signaling with subcellular spatial resolution.
Proc.Natl.Acad.Sci.USA, 122:e2424679122-e2424679122, 2025

PubMed Abstract: Subcellular compartmentalization is integral to the spatial regulation of mechanistic target of rapamycin (mTOR) signaling. However, the biological outputs associated with location-specific mTOR signaling events are poorly understood and challenging to decouple. Here, we engineered synthetic intracellular antibodies (intrabodies) that are capable of modulating mTOR signaling with genetically programmable spatial resolution. Epitope-directed phage display was exploited to generate high affinity synthetic antibody fragments (Fabs) against the FKBP12-Rapamycin binding site of mTOR (mTOR). We determined high-resolution crystal structures of two unique Fabs that discriminate distinct conformational states of mTOR through recognition of its substrate recruitment interface. By leveraging these conformation-specific binders as intracellular probes, we uncovered the structural basis for an allosteric mechanism governing mTOR complex 1 (mTORC1) stability mediated by subtle structural adjustments within mTOR. Furthermore, our results demonstrated that synthetic binders emulate natural substrates by employing divergent yet complementary hydrophobic residues at defined positions, underscoring the broad molecular recognition capability of mTOR. Intracellular signaling studies showed differential time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by genetically encoded intrabodies, thus supporting a mechanism of inhibition analogous to the natural product rapamycin. Finally, we implemented a feasible approach to selectively modulate mTOR signaling in the nucleus through spatially programmed intrabody expression. These findings establish intrabodies as versatile tools for dissecting the conformational regulation of mTORC1 and should be useful to explore how location-specific mTOR signaling influences disease progression.

PubMed: 40489625
DOI: 10.1073/pnas.2424679122

Experimental method

X-RAY DIFFRACTION (1.55 Å)