title: Structural insight into substrate preference for TET-mediated oxidation.
abstract: DNA methylation is an important epigenetic modification. Ten-eleven translocation (TET) proteins are involved in DNA demethylation through iteratively oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Here we show that human TET1 and TET2 are more active on 5mC-DNA than 5hmC/5fC-DNA substrates. We determine the crystal structures of TET2-5hmC-DNA and TET2-5fC-DNA complexes at 1.80 Å and 1.97 Å resolution, respectively. The cytosine portion of 5hmC/5fC is specifically recognized by TET2 in a manner similar to that of 5mC in the TET2-5mC-DNA structure, and the pyrimidine base of 5mC/5hmC/5fC adopts an almost identical conformation within the catalytic cavity. However, the hydroxyl group of 5hmC and carbonyl group of 5fC face towards the opposite direction because the hydroxymethyl group of 5hmC and formyl group of 5fC adopt restrained conformations through forming hydrogen bonds with the 1-carboxylate of NOG and N4 exocyclic nitrogen of cytosine, respectively. Biochemical analyses indicate that the substrate preference of TET2 results from the different efficiencies of hydrogen abstraction in TET2-mediated oxidation. The restrained conformation of 5hmC and 5fC within the catalytic cavity may prevent their abstractable hydrogen(s) adopting a favourable orientation for hydrogen abstraction and thus result in low catalytic efficiency. Our studies demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. Therefore, TET proteins are evolutionarily tuned to be less reactive towards 5hmC and facilitate the generation of 5hmC as a potentially stable mark for regulatory functions.
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Lulu
authors.initials: L
authors.lastname: Hu
authors.affiliation: Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Junyan
authors.initials: J
authors.lastname: Lu
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Jingdong
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Qinhui
authors.initials: Q
authors.lastname: Rao
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Ze
authors.initials: Z
authors.lastname: Li
authors.affiliation: Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Hou
authors.affiliation: Laboratory of Structural Biology, Tsinghua University, Beijing 100084, China.|MOE Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Lou
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gong
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Mengjie
authors.initials: M
authors.lastname: Liu
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Sun
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Xiaotong
authors.initials: X
authors.lastname: Yin
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Xiangshi
authors.initials: X
authors.lastname: Tan
authors.affiliation: Department of Chemistry, University of California-Riverside, Riverside, California 92521-0403, USA.
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of California-Riverside, Riverside, California 92521-0403, USA.
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Theoretical Chemistry Institute, Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Fang
authors.affiliation: Theoretical Chemistry Institute, Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Cui
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
authors.affiliation: Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.|Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
authors.forename: Chuan
authors.initials: C
authors.lastname: He
authors.affiliation: Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Hualiang
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Luo
authors.affiliation: Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.|State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Xu
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism / Q000031:analogs & derivatives; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D049308:Dioxygenases; D006801:Humans; D006860:Hydrogen Bonding; D006899:Mixed Function Oxygenases; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C560973:5-formylcytosine; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; D049308:Dioxygenases; C541081:TET2 protein, human