Complementary Steric Engineering at the Protein-Ligand Interface for Analogue-Sensitive TET Oxygenases

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• Ten-eleven translocation (TET) enzymes are crucial for mammalian genome regulation through DNA demethylation.
• Over 60 human C-H oxygenases exist, making it challenging to determine individual enzyme functions.
• Targeting specific TET enzymes is vital for understanding their roles in cellular processes.

Purpose of the Study:

• To develop a method for specifically perturbing the activity of individual TET enzymes.
• To engineer analogue-sensitive TET2 variants for conditional inhibition.
• To establish a general platform for studying 2-ketoglutarate (2KG)-dependent oxygenases.

Main Methods:

• Rational engineering of the TET2-2-ketoglutarate (2KG) interface to create variants with expanded active sites.
• Identification and engineering of a bulky gatekeeper residue for TET proteins.
• Development of orthogonal mutant-inhibitor pairs using N-oxalylglycine (NOG) derivatives.
• Application of cell-permeable NOG analogues to inhibit TET2 mutants in intact human cells.

Main Results:

• Successfully engineered TET2 variants sensitive to specific NOG derivatives.
• Demonstrated orthogonal inhibition of TET2 mutants, allowing conditional modulation of cytosine methylation.
• Showcased the generality of the approach for other TET proteins.
• Applied the system to probe TET-mediated transcriptional activity in cells.

Conclusions:

• Developed a versatile platform for creating analogue-sensitive 2KG-dependent oxygenases.
• Enabled precise control over TET enzyme activity to study their functions in various signaling pathways.
• Provided a new tool for dissecting the roles of specific C-H oxygenases in genome regulation and cellular processes.