Ten-Eleven Translocation 2 5-hydroxymethylcytosine (TET2-5hmc)

Marcus Hong '21 and Chris Rutter '20

Contents:

I. Introduction

Model View:

What are TET enzymes?:

Ten-eleven translocation, or TET, enzymes are a type of DNA-modifying protein involved in the demethylation of 5-methylcytosine (5mC). Specifically, TET2-5hmc catalyzes the oxidation of 5mC to yield 5-hydroxymethyl cytosine (5hmC)1. This can subsequently form 5-formylcytosine (5fC) then 5-carboxylcytosine (5caC), both of which can be converted to unmodified C by Terminal deoxynucleotidyl transferase (TdT)2. TET2-5hmc catalysis is thus a necessary precursor to the completed demethylation of 5mC to an unmodified C.1

Why do we care about demethylation?:

DNA methylation/demethylation processes are thought to play important roles in gene repression, most notably in cancer prevention. Mutated TET2 genes are associated with the early stages of various cancers such as myeloid leukemia and melanoma1, meaning that the demethylation made possible by TET2-5hmc is thought to be an important element in tumour suppression2. Prior to the Tahiliani et al. identification of TET1 as having potential to oxidize the 5-methyl group of thymine3, DNA methylation was seen as irreversible and fixable only through DNA replication repair mechanisms2. This is an especially notable problem when we consider the nature of 5-C methylation, which makes cytosines look remarkably similar to thymines1 . Thus, 5mCs often go undetected by base excision repair when matched to an adenine, meaning DNA repair mechanisms are far less reliable in this circumstance. It is now clear that methylation-demethylation pathways are intricately tied in with cancer, and it is likely that the 5mC similarity with thymine plays an associated role. In addition, understanding the cancer linkage promises therapeutic applications.

Now that I care, what structural elements should I look for in this complex?:

TET enzyme is a DNA-modifying enzyme, meaning the most important element of the complex is the interaction between protein and DNA. The crystal structure will reveal key insights into understanding the mechanism of TET-mediated oxidation of the 5-methyl group. These insights are gained primarily through looking at the catalytic region, which is characterized by specific domains, binding sites, and secondary structure.

II. Basic Overview

The crystalline structure of TET2-5hmc seen in Hu et al. consists of a reduced, but catalytically active, form of TET2 bound to a double-stranded DNA molecule containing one methyl-CpG site . The enzyme consists of Cys-N , Cys-C , and the Double Stranded Beta Helix ( DSBH) domains. Residues 1128-1131, 1136-1143, 1925-1936, 1464-1481, the GSLinker1 (dotted line in image) in TET2, and bases C11�, T12, and A12� in the DNA were excluded from the crystals due to lack of electron density1.

III. THE DSBH Core and Catalytic Cavity

We begin this story with the core catalytic region of TET2-5hmc , which like all TET proteins is located in the C-terminus. This region consists of a cysteine-rich domain, a double-stranded Beta-helix domain in complex with the DNA duplex ( DSBH, or a jelly-roll motif) , and binding sites for cofactors Fe (II) and 2-oxoglutarate (2-OG) . The crystalline DSBH contains a central core made up of two adjacent antiparallel Beta sheets (B12 and B13 ) next to Fe (II) and N-oxalylglycine ( , which is an anolog of 2-OG), both of which are located in the center of the complex. The prealtered 5-methyl group of the cytosine orients to face the Fe (II) and NOG catalytic elements, and oxidation can proceed if NOG is replaced by 2-oxoglutarate. Fe (II) is maintained by H1382, D1384, and H1881 . NOG interactions include the stabilization of 1-carboxylate by residue R1261 and 5-carboxylate by H1416, R1896, and S1898 . Due to their high importance in catalysis, Fe (II) and NOG related elements tend to be highly conserved.

The core is further supported by a combination of the DSBH and Cys-rich domains. The Cys-rich domain can be further classified into two subdomains, Cys-rich N-terminal (Cys-N) and Cys-rich C-terminal (Cys-C) , which the DSBH core. Cys-N of alpha helix 1 and a four-stranded Beta sheet (B2-5) joined to a Beta sheet of the DSBH core through alpha-3. Notable elements of Cys-C of Beta-6 connecting L1 and L2 and helices alpha-2 and alpha-3 directly adjacent to the DSBH core. The Cys-rich domain is highly conserved due to their importance in protein stability and catalytic coordination. Three additional Beta sheets occupy the bottom of the DSBH core, whereas the top of the DSBH core houses the DNA1.

Three Zinc are located in separate corners of TET2-5hmc. Zn 1 is coordinated by C1135, C1133, H1219, and C1221, and contributes to TET2 enzymatic activity through increased structure stability. Zn 2 and Zn 3 are coordinated by the Cys-rich and DSBH domains and contribute to catalysis through stabilization of reactions and DNA recognition by TET2. Zn 2 is managed through three DSBH core loops, Cys-N , and Cys-C interactions. Specifically, Zn 2 is coordinated by H1380, C1273, C1271, and C1193 from the DSBH core loop. Zn 3 coordination with H1912, C1298, C1289, and C1358 stabilize L2, which is vital for the TET2-5hmc interaction with DNA. Zinc coordination is perhaps the most important and conserved aspect of TET2-5hmc , which is consistent with many cancer-causing TET2-5hmc mutations being products of Zinc -involved residue mutations 1.

IV. DNA/Methyl-CpG - TET2-5hmC Interactions

Having looked at the interactions in the catalytic site, we next move into the DNA interacting with the enzyme. After all, the protein must first bind the correct area with sufficient affinity if it is to catalyze. The methylated cytosine interacts with the catalytic cavity only after being flipped out of the inner helix, and does so through inserting into a groove created by two loops from the Cys-C subdomain (L1 and L2). L1 (residues 1256-1273) supports DNA, while L2 (residues 1288-1312) inserts into the minor groove opposite of L1 1.

The DNA binding portion of TET2-5hmc is made up of primarily basic and hydrophobic residues, which are capable of Van Der Waal interactions and hydrogen bonding to both help the DNA to stick and to stabilize protein structure. Excluding the mC6 base portion, DNA contact with TET2-5hmc is made extensively through polar interactions between polar residues and base phosphate groups. This is seen in the between mC6 and G7 phosphate groups with residues R1262 and S1290. are also formed between the phosphate groups of the G8, and T9 bases and the Cys-C residues K1299, and S1303. As the only protein-DNA H-bonds present outside of the mC6 base involve interactions with phosphate groups in the DNA backbone, it clear that there is no sequence-specific hydrogen bonding present besides mC6. This makes sense, as methylated cytosines are not confined to specific DNA sequences.

Nonpolar interactions are also important in TET2-5hmc function. interactions between residues W1291, M1293, Y1294, Y1295, and R1302 create a segment which inserts into the minor groove of DNA between C5:G5� and G8:C8�. This segment is important in that it contains residues M1293 and Y1294, which push out and replace G6� from the default base stacking position. In addition, the minor groove binding induces a bend in the DNA, which is a common theme of minor groove binding. Specifically, the DNA bends by roughly 40 degrees1 , which allows mC6 to be flipped out and inserted into the catalytic component of TET2-5hmc by weakening the G6�:mC6 interaction. When these nonpolar residues are mutated out, TET2-5hmc function goes away, meaning that this is an essential part of TET2-5hmc function1.

In terms of actually recognizing the methyl-CpG dinucleotide, H-bonds again come into play. mC6 is recognized by 2 H-bonds between the base nitrogens N3 and N4 and highly conserved residues H1904 and N1387 . Base-stacking interactions help reinforce this recognition. Residue Y1902 and the pyrimidine base of mC6 strengthen this interaction via interactions . H-bonds are also a recurrent theme, as seen with the ribose ring C-5 oxygen of mC6 and residue R12611. The methyl group itself is relatively inactive in recognition, which can be interpreted as both unsurprising and surprising. On one hand, the methyl must be free to undergo hydroxylation, but on the other hand the type of base edited is not involved in recognition, meaning 5C would interact with similar affinity despite not being able to participate in catalytic activity by TET2-5hmc. While these are the mC6-specific reactions, it is also important to note that the surrounding mC7 and G6� bases all participate in base-stacking interactions which reinforce DNA adhesion. Without the mC7:G7' adjacent base pair, or the complementary G6� base, significant decreases in enzymatic activity result1.

V. Implications

The crystal structure of the TET2-5hmc - DNA Complex consists of Cys-N , Cys-C , and DSBH domains, and gives clear insights into the interactions involved in binding and 5mC demethylation. These interactions are important because TET2 mutations involving Fe (II) /NOG chelating, zinc chelating, and DNA interactions, all essential for proper hydroxylation and thus demethylation, have been linked to several cancers1. Because of this, TET2 holds promising therapeutic and diagnostic potential, and is currently a hot-button topic in oncological research.

VI. References

1. Hu, L. et al., 2013. Crystal Structure of TET2-DNA Complex: Insight into TET-Mediated 5mC Oxidation. Cell, 155(7), 1545-1555. doi:10.1016/j.cell.2013.11.020

2. Rasmussen, K. D., and Helin, K. 2016. Role of TET enzymes in DNA methylation, development, and cancer.Genes and Development , 30(7), 733-750. doi:10.1101/gad.276568.115. .

3. Tahiliani, M. et al., 2009. Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1. Science , 324(5929), 930-935. doi:10.1126/science.1170116

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