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

Ten-eleven translocation (TET) enzymes employ O2, earth-abundant iron, and 2-ketoglutarate (2KG) to perform iterative C–H oxidation of 5-methylcytosine in DNA to control expression of the mammalian genome. Given that more than 60 such C–H oxygenases are present in humans, determining context-depende...

Full description

Saved in:
Bibliographic Details
Published in:Journal of the American Chemical Society 2018-08, Vol.140 (32), p.10263-10269
Main Authors: Sudhamalla, Babu, Wang, Sinan, Snyder, Valerie, Kavoosi, Sam, Arora, Simran, Islam, Kabirul
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
DNA Methylation
HEK293 Cells
Humans
Ligands
Mixed Function Oxygenases - genetics
Mixed Function Oxygenases - metabolism
Protein Conformation
Protein Engineering
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Ten-eleven translocation (TET) enzymes employ O2, earth-abundant iron, and 2-ketoglutarate (2KG) to perform iterative C–H oxidation of 5-methylcytosine in DNA to control expression of the mammalian genome. Given that more than 60 such C–H oxygenases are present in humans, determining context-dependent functions of each of these enzymes is a pivotal challenge. In an effort to tackle the problem, we developed analogue-sensitive TET enzymes to perturb the activity of a specific member. We rationally engineered the TET2–2KG interface to develop TET2 variants with an expanded active site that can be specifically inhibited by the N-oxalylglycine (NOG) derivatives carrying a complementary steric “bump”. Herein, we describe the identification and engineering of a bulky gatekeeper residue for TET proteins, characterize the orthogonal mutant–inhibitor pairs, and show generality of the approach. Employing cell-permeable NOG analogues, we show that the TET2 mutant can be specifically inhibited to conditionally modulate cytosine methylation in chromosomal DNA in intact human cells. Finally, we demonstrate application of the orthogonal mutant–inhibitor pair to probe transcriptional activity of a specific TET member in cells. Our work provides a general platform for developing analogue-sensitive 2KG-dependent oxygenases to unravel their functions in diverse signaling processes.
ISSN:0002-7863
1520-5126
1520-5126
DOI:10.1021/jacs.8b05283