Human DNMT1 transition state structure Quan Dua, Zhen Wanga, and Vern L. Schramma,1 aDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461 Contributed by Vern L. Schramm, February 2, 2016 (sent for review November 13, 2015; reviewed by Albert Jeltsch and Dinshaw J. Patel) Human DNA methyltransferase 1 (DNMT1) maintains the epige- detailed chemical insights into the catalytic mechanisms of enzymes netic state of DNA by replicating CpG methylation signatures from acting mostly on small molecules and has led to the design of some parent to daughter strands, producing heritable methylation patterns of the most powerful enzyme inhibitors (15, 16). Enzymes in epi- through cell divisions. The proposed catalytic mechanism of DNMT1 genetic regulations often involve large and complex substrates, involves nucleophilic attack of Cys1226 to cytosine (Cyt) C6, methyl creating experimental challenges in both KIE measurements and transfer from S-adenosyl-L-methionine (SAM) to Cyt C5, and proton computational models. Nonetheless, TS analysis can be applied to abstraction from C5 to form methylated CpG in DNA. Here, we report complex enzyme systems, including the 50S ribosomes (17), as long the subangstrom geometric and electrostatic structure of the major as the chemical steps can be interrogated with the appropriate transition state (TS) of the reaction catalyzed by human DNMT1. Ex- isotope labels. perimental kinetic isotope effects were used to guide quantum me- Here, we measured 10 experimental KIEs to investigate the chanical calculations to solve the TS structure. Methyl transfer occurs 1226 TS and catalytic mechanisms of human DNMT1. By combining after Cys attack to Cyt C6, and the methyl transfer step is chem- these experimental KIE values with QM calculations, we established ically rate-limiting for DNMT1. Electrostatic potential maps were com- the subangstrom TS structure for human DNMT1. Our results also pared for the TS and ground states, providing the electronic basis for show methyl transfer to be the major chemical barrier in the reaction interactions between the protein and reactants at the TS. Understand- coordinate, rather than the Cys attack, β-elimination from the C5- ing the TS of DNMT1 demonstrates the possibility of using similar position, or departure of the 5-methyl Cyt from the catalytic site Cys. analysis to gain subangstrom geometric insight into the complex re- The work demonstrates an experimental approach to analyze the TS actions of epigenetic modifications. structures of complex epigenetic enzymes, for unraveling their cata- lytic mechanisms, and for advancing target-specific drug designs. DNA methyltransferase | S-adenosyl-L-methionine | CpG methylation | 5-methylcytosine | transition state Results Isotope Labeling of DNA and SAM as DNMT1 Substrates. A library of uman DNA methyltransferases (DNMTs) catalyze the for- six hemimethylated DNA and eight SAM substrates with site- Hmation of 5-methylcytosine (5mC) at CpG sites on DNA, a specific isotope labels was synthesized to measure the respective key epigenetic mark present in the human genome (1). DNA KIEs. The isotopic labels were placed in chemical bond positions methylation is involved in transcriptional silencing, cellular differ- such that all atoms directly involved in the chemical steps of entiation, genomic imprinting, and X-chromosome inactivation. In DNMT1-catalyzed reaction were represented. Six isotopically la- addition, hypermethylation of CpG islands at gene promoter re- beled dCTPs were prepared as the building blocks for the DNA gions has been associated with carcinogenesis (2). Maintenance of 2 14 13 14 3 14 3 substrates [5- H, 5′- C]-, [5- C, 5′- C]-, [6- H]-, [6- C]-, [5′- H2]-, DNA methylation patterns is conducted by human DNMT1, a and [5′-14C]-dCTPs through coupled reactions using up to 14 dif- multidomain protein of 1,616 amino acids. The C-terminal meth- ferentenzymes(18)(SI Appendix,Figs.S1andS2). Each iso- yltransferase domain shows sequence similarities to the bacterial topically labeled dCTP was incorporated into a 26-bp DNA by methyltransferases (3). Crystal structures of mouse and human in vitro replication using Klenow fragment extension (Fig. 2 A–C). DNMT1 complexed with different substrates have provided a The DNA molecules synthesized as labeled reactants all contained structural basis for DNMT1-mediated maintenance DNA methyl- ation (4, 5). Domain interactions and large conformational changes Significance are responsible for properly positioning hemimethylated DNA within the active site and catalyze methyl transfer from S-adenosyl- L-methionine (SAM) to DNA. Site-directed mutations have offered DNA methyltransferase 1 (DNMT1) is the major enzyme re- insights into the structure–function relationship of DNMTs (6, 7), sponsible for maintenance of DNA CpG methylation marks in but their transition state (TS) structures have remained unknown. human cells. The enzyme is a validated target for cancer, but DNMT1 has been proposed to follow a catalytic mechanism current treatments are mutagenic. Knowledge of the transition shared by bacterial DNA-(cytosine C5)-methyltransferases (4, 8– state (TS) structure of DNMT1 will inform the chemical reaction 10): nucleophilic attack of cytosine (Cyt) C6 by Cys1226 of DNMT1, mechanism and provide information for TS analog design. Here, methyl transfer from SAM to Cyt C5, and β-elimination of H5 to we report the subangstrom geometric and electrostatic character produce 5mC in the final step (Fig. 1). Recent quantum mechanics of the TS for the DNMT1 methylation of hemimethylated DNA. (QM)/molecular mechanics (MM) and molecular dynamics (MD) The experimental and computational TS analysis indicates methyl transfer is the rate-limiting chemical step for the reaction. Methyl simulations of the bacterial M.HhaI methyltransferase suggested group transfer can be characterized as a loose nucleophilic sub- that Cys1226 attack is concerted with methyl transfer (11, 12), and stitution TS. TS analysis of DNMT1 demonstrates an approach to that β-elimination of H5 is the rate-limiting step (12). The com- understand a complex epigenetic enzyme. bination of kinetic isotope effects (KIEs) and computational chemistry can test predicted reaction mechanisms and can provide Author contributions: Q.D., Z.W., and V.L.S. designed research; Q.D. and Z.W. performed a model of the TS structure. research; Q.D. contributed new reagents/analytic tools; Q.D., Z.W., and V.L.S. analyzed Enzymes catalyze reactions by forming short-lived TSs from data; and Q.D., Z.W., and V.L.S. wrote the paper. their reactants held in Michaelis complexes (13). The lifetime of a Reviewers: A.J., Universität Stuttgart; and D.J.P., Memorial Sloan–Kettering Cancer Center. −14 chemical TS is typically around 10 s, on the time scale of chemical The authors declare no conflict of interest. bond vibrations. No spectroscopic method is generally available to 1To whom correspondence should be addressed. Email: [email protected]. observe the chemical structure of TSs directly for enzymatic reac- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tions (14). TS analysis based on experimental KIEs has provided 1073/pnas.1522491113/-/DCSupplemental. 2916–2921 | PNAS | March 15, 2016 | vol. 113 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1522491113 Downloaded by guest on September 26, 2021 Fig. 1. DNA methylation catalyzed by DNMT1. (A) Proposed catalytic mechanism for DNMT1 in- volves three chemical TSs (TS1, TS2, and TS3). SAH, S-adenosylhomocysteine. Cys attack at TS1 brings a negative charge (−1) to the Cyt ring, whereas Cys withdrawal after TS3 restores the aromaticity of the Cyt. (B) Based on the KIE analysis presented here, methyl transfer is chemically rate-limiting for DNMT1 and has a higher energy barrier than the thiol-attack and β-elimination steps. Small forward commit- ments demonstrate the chemical steps to have a higher energy barrier than the binding and release of substrates. one hemimethylated CpG site, in which the unmethylated to the rates of substrate binding and product release. These rate 2′-deoxycytidine (dC) residue is enriched with specific isotopes. similarities (called commitment factors) can obscure the values This substrate design provides a single methylation site per DNA of the intrinsic chemical isotope effects and must be quantitated for methyltransferase; therefore, the observed KIEs are not to permit calculation of intrinsic isotope effects. We measured complicated by processive DNA methylations. In addition, the forward commitment factor (Cf) values for DNA and SAM eight species of isotopically labeled SAM substrates were syn- by isotope trapping experiments. These experiments used pulse– thesized enzymatically from labeled ATP and methionine using chase analysis to trace radiolabeled product formation over the SAM synthetase (SI Appendix,Fig.S3), including [Me-13C, 8-14C]-, course of the reaction (SI Appendix, Fig. S4 and Table S3). The 14 3 14 3 36 14 3 [Me- C]-, [Me- H3]-, [5′- C]-, [5′- H2]-, [ S, 8- C]-, [1′- H]-, Cf values for DNA and SAM bound to DNMT1 were found to be and [8-14C]-SAMs. The 36S-labeled SAM was synthesized from small and insignificant (0.016 and 0.0013, respectively). The 36 BIOCHEMISTRY elemental S (19). The percentages of enrichment for stable heavy small Cf values establish that DNA and SAM bind to and release isotopes (i.e., 2H, 13C, 36S) in our isotopically labeled reactants were from DNMT1 63 and 770 times, respectively, before each cata- measured by MS. Those values, together with the lists of isotopic lytic turnover and are not highly committed to the chemical starting materials, are summarized in SI Appendix, Tables S1 and S2. steps. Small Cf values demonstrate that DNMT1-catalyzed methyl transfer is much slower than substrate binding and DNA and SAM Show Low Substrate Commitments in Vitro. In enzy- release steps. DNA binding requires Cyt base-flipping by matic reactions, the rate of chemical bond changes can be similar DNMT1, and with dsDNA, multiple excursions into the catalytic Fig. 2. Development of chemical tools for KIE measurement on DNMT1.