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Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.

Hashimoto H, Pais JE, Zhang X, Saleh L, Fu ZQ, Dai N, Corrêa IR, Zheng Y, Cheng X - Nature (2013)

Bottom Line: Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1.The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC.The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.

ABSTRACT
Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

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Comparison of NgTet1 and AlkBa–b, Structures of NgTet1 and AlkB aligned in a similar orientation. c–d, NgTet1 (c) and AlkB (d) are shown in relatively similar orientations. The surface charge at neutral pH is displayed as blue for positive, red for negative, and white for neutral. e, Superimposition of NgTet1 (5mC) and AlkB (3mC) in the active sites. The metal ions (M) are shown as balls and NOG or αKG (in the back) as sticks. f–g, Co-variation between the location of the target base (5mC in NgTet1 and 3mC in AlkB) and the NOG/αKG-interacting arginine (R224 of NgTet1 and R210 of AlkB).
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Figure 3: Comparison of NgTet1 and AlkBa–b, Structures of NgTet1 and AlkB aligned in a similar orientation. c–d, NgTet1 (c) and AlkB (d) are shown in relatively similar orientations. The surface charge at neutral pH is displayed as blue for positive, red for negative, and white for neutral. e, Superimposition of NgTet1 (5mC) and AlkB (3mC) in the active sites. The metal ions (M) are shown as balls and NOG or αKG (in the back) as sticks. f–g, Co-variation between the location of the target base (5mC in NgTet1 and 3mC in AlkB) and the NOG/αKG-interacting arginine (R224 of NgTet1 and R210 of AlkB).

Mentions: The αKG dioxygenase family8,9 includes members of the AlkB-like DNA/RNA repair enzymes10. We compared the complex structure of NgTet1-DNA-NOG-Mn2+ to that of Escherichia coli AlkB-DNA-αKG-Mn2+ (Fig. 3) and its human homolog ABH2 (Extended Data Fig. 6)11,12 (the only other dioxygenases acting on nucleic acids structurally characterized in complex with DNA). The structures of NgTet1 and AlkB can be superimposed via the core elements of the jelly-roll fold (colored in Fig. 3a–b). Both enzymes contain the hairpin loop (L1) after strand β5 and the active-site loop (L2) prior to strand β7. Besides the N-terminal and C-terminal additions (Extended Data Fig. 6a), NgTet1 has, within the core region, extra helices α5 and α6, immediately after the kinked helix α4 (owing to Pro72 located in the middle of the helix). In the places of h3 and h7, two 310-helices unique to NgTet1 (Fig. 3a), AlkB has two additional β-strands, adjacent to β5 of the major sheet and β11 of the minor sheet, respectively (Fig. 3b). Unique to AlkB is an additional 12-residue-long loop (L3) prior to strand β5 making DNA backbone contacts, whereas the corresponding loop L3 in NgTet1 is a 4-residue short loop containing an invariant Lys137 among the eight NgTet proteins (Extended Data Fig. 1c).


Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.

Hashimoto H, Pais JE, Zhang X, Saleh L, Fu ZQ, Dai N, Corrêa IR, Zheng Y, Cheng X - Nature (2013)

Comparison of NgTet1 and AlkBa–b, Structures of NgTet1 and AlkB aligned in a similar orientation. c–d, NgTet1 (c) and AlkB (d) are shown in relatively similar orientations. The surface charge at neutral pH is displayed as blue for positive, red for negative, and white for neutral. e, Superimposition of NgTet1 (5mC) and AlkB (3mC) in the active sites. The metal ions (M) are shown as balls and NOG or αKG (in the back) as sticks. f–g, Co-variation between the location of the target base (5mC in NgTet1 and 3mC in AlkB) and the NOG/αKG-interacting arginine (R224 of NgTet1 and R210 of AlkB).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4364404&req=5

Figure 3: Comparison of NgTet1 and AlkBa–b, Structures of NgTet1 and AlkB aligned in a similar orientation. c–d, NgTet1 (c) and AlkB (d) are shown in relatively similar orientations. The surface charge at neutral pH is displayed as blue for positive, red for negative, and white for neutral. e, Superimposition of NgTet1 (5mC) and AlkB (3mC) in the active sites. The metal ions (M) are shown as balls and NOG or αKG (in the back) as sticks. f–g, Co-variation between the location of the target base (5mC in NgTet1 and 3mC in AlkB) and the NOG/αKG-interacting arginine (R224 of NgTet1 and R210 of AlkB).
Mentions: The αKG dioxygenase family8,9 includes members of the AlkB-like DNA/RNA repair enzymes10. We compared the complex structure of NgTet1-DNA-NOG-Mn2+ to that of Escherichia coli AlkB-DNA-αKG-Mn2+ (Fig. 3) and its human homolog ABH2 (Extended Data Fig. 6)11,12 (the only other dioxygenases acting on nucleic acids structurally characterized in complex with DNA). The structures of NgTet1 and AlkB can be superimposed via the core elements of the jelly-roll fold (colored in Fig. 3a–b). Both enzymes contain the hairpin loop (L1) after strand β5 and the active-site loop (L2) prior to strand β7. Besides the N-terminal and C-terminal additions (Extended Data Fig. 6a), NgTet1 has, within the core region, extra helices α5 and α6, immediately after the kinked helix α4 (owing to Pro72 located in the middle of the helix). In the places of h3 and h7, two 310-helices unique to NgTet1 (Fig. 3a), AlkB has two additional β-strands, adjacent to β5 of the major sheet and β11 of the minor sheet, respectively (Fig. 3b). Unique to AlkB is an additional 12-residue-long loop (L3) prior to strand β5 making DNA backbone contacts, whereas the corresponding loop L3 in NgTet1 is a 4-residue short loop containing an invariant Lys137 among the eight NgTet proteins (Extended Data Fig. 1c).

Bottom Line: Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1.The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC.The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.

ABSTRACT
Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

Show MeSH
Related in: MedlinePlus