Articles Cite This: ACS Chem. Biol. 2018, 13, 772−781
Biochemical Studies and Molecular Dynamic Simulations Reveal the Molecular Basis of Conformational Changes in DNA Methyltransferase‑1 Fei Ye,†,‡,○ Xiangqian Kong,§,○ Hao Zhang,‡ Yan Liu,‡ Zhiyuan Shao,‡ Jia Jin,† Yi Cai,§ Rukang Zhang,‡ Linjuan Li,‡ Yang W. Zhang,§ Yu-Chih Liu,⊥ Chenhua Zhang,⊥ Wenbing Xie,§ Kunqian Yu,‡ Hong Ding,‡,∇ Kehao Zhao,∥ Shijie Chen,*,‡ Hualiang Jiang,‡ Stephen B. Baylin,*,§ and Cheng Luo*,‡ †
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China Drug Discovery and Design Center, CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China § Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, United States ⊥ Shanghai ChemPartner Co., LTD, Building 5, 998 Halei Road, Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai 201203, P.R. China ∥ China Novartis Institutes for BioMedical Research, Shanghai 201203, China ∇ School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China ‡
S Supporting Information *
ABSTRACT: DNA methyltransferase-1 (DNMT1) plays a crucial role in the maintenance of genomic methylation patterns. The crystal structure of DNMT1 was determined in two different states in which the helix that follows the catalytic loop was either kinked (designated helix-kinked) or well folded (designated helix-straight state). Here, we show that the proper structural transition between these two states is required for DNMT1 activity. The mutations of N1248A and R1279D, which did not affect interactions between DNMT1 and substrates or cofactors, allosterically reduced enzymatic activities in vitro by decreasing kcat/Km for AdoMet. The crystallographic data combined with molecular dynamic (MD) simulations indicated that the N1248A and R1279D mutants bias the catalytic helix to either the kinked or straight conformation. In addition, genetic complementation assays for the two mutants suggested that disturbing the conformational transition reduced DNMT1 activity in cells, which could act additively with existing DNMT inhibitors to decrease DNA methylation. Collectively, our studies provide molecular insights into conformational changes of the catalytic helix, which is essential for DNMT1 catalytic activity, and thus aid in better understanding the relationship between DNMT1 dynamic switching and enzymatic activity.
D
Up to now, several crystal structures of DNMT1 have been reported, and these structures reveal that DNMT1 is a multimodular protein (Figure 1A).9−14 DNMT1 is composed of an N-terminal platform, a replication foci targeting sequence (RFTS), a DNA-binding CXXC domain, two bromo-adjacent homology (BAH) domains, and a C-terminal methyltransferase domain (Figure 1A). The methyltransferase domain of DNMT1 is further divided into two subdomains: the catalytic core and the target recognition domain (TRD) (Figure 1A). On the basis of the conformation of the catalytic core, the crystal structures of DNMT1 can be classified into two distinct states: helix-kinked and helix-straight (Figure S2A, B). In the helix-straight state, where is found the covalent mDNMT1
NA methylation, which is an important epigenetic modification, is required for differentiation, genome stability, genomic imprinting, X-chromosome inactivation, and retrotransposon silencing.1−3 In mammals, DNA methylation often occurs on the carbon-5 position of cytosines within CpG dinucleotides.4,5 DNA methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to cytosine residues in DNA. Mammalian genomes encode three active DNMTs: the de novo methyltransferases DNMT3A and DNMT3B as well as the maintenance methyltransferase DNMT1.6−8 DNMT1, which is highly conserved among different species (Figure S1), shows a preference for hemimethylated DNA and is thus responsible for maintaining cellular DNA methylation patterns.8 Mouse knockout studies have shown that DNMT1 is essential for embryonic development,8 indicating the critical role of DNMT1. © 2018 American Chemical Society
Received: October 16, 2017 Accepted: January 30, 2018 Published: January 30, 2018 772
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology
Figure 1. Helix-straight and -kinked states of mDNMT1. (A) Color-coded domain architecture and numbering of mDNMT1 sequence. (B) Alignment of two states. The helix-kinked and -straight DNMT1 are shown in cyan and magenta, respectively. (C) Role of the key residues in the stabilization of the long straight catalytic helix in helix-straight DNMT1. The residues involved in anchoring helix in a straight conformation are shown as sticks. (D) Role of R1278 and R1279 in the stabilization of the kinked catalytic helix. The residues involved in anchoring helix in the helixkinked conformation are shown as sticks. The hydrogen bonds are depicted as red dashed lines.
(731−1602)/hemi-DNA complex,11 the α-helix following the catalytic loop (residues 1227−1243), which is designated the “catalytic helix” of DNMT1 hereinafter, adopts a straight conformation and extends from residue S1240 to Y1261. The hemimethylated DNA duplex in the helix-straight state is embedded in the catalytic cleft between the catalytic loop and the TRD domain where the catalytic loop penetrates into the DNA minor groove while the TRD domain inserts into the major groove. The reactive cysteine C1229 in the conserved PCQ motif points to the flipped-out cytosine on the DNA target strand (Figure S2A). The helix-kinked state has been observed in the crystal structures of apo-DNMT1 and mDNMT1(650−1602)−DNA complex from both mouse and human.9,10,12,13 In this state, the catalytic helix is interrupted by a kink centered on residues N1248 and S1249, leading to two short helices (S1240−N1248 and L1250−Y1261). The catalytic loop is excluded from the DNA minor groove (Figure S2B). The superimposition of these two states showed that the overall conformation was almost identical, whereas the methyltransferase domain undergoes large conformational changes, especially in the catalytic loop and N-terminal portion of the catalytic helix (residues C1227−S1249) (Figure 1B). In addition, the TRD domain in the helix-kinked state moves away from the DNA-binding site by ∼2−3 Å (Figure 1B).11 In addition to DNMT1, the dynamic region with different conformations can also be found in other DNMTs. Notably, the corresponding sequence of the dynamic region in M.HhaI, the prokaryotic DNA (cytosine-C5)-methyltransferase, contains the active-site loop following the catalytic nucleophile (residue C81). The loop adopts an open conformation in the absence of DNA and a closed conformation after DNA binding
(Figure S3A),15−21 which is similar to DNMT1. Moreover, the corresponding region in DNMT3A is an active site loop that is stabilized by DNMT3L, which stimulates the activity of DNMT3A22,23 via interactions with the C-terminal part of the active site loop (residues G722, L723, and Y724) (Figure S3B).24,25 Significantly, missense mutations at residues G718 and L719 in human DNMT3B, which correspond to residues G722 and L723 in DNMT3A (Figure S3C), are associated with immunodeficiency, centromere instability, and facial anomalies (ICF syndrome).26 Taken together, these dynamic structural properties are conserved in different DNMTs and are involved in the process of enzyme catalysis, suggesting their functional importance in modulating the activity of DNA methylation. Understanding the relationship between DNMT1 dynamic switching and enzymatic activity has great significance. In this study, we conducted a mechanistic investigation on the relationship between DNMT1 conformational transition and catalytic activity. Guided by the structural analysis, enzymatic assays were performed on mutants of several key residues that were involved in maintaining the two different states. The decreased enzymatic activity indicated that both states are important for DNMT1 function. Among those, mutations of N1248 and R1279, which are not involved in either hemimethylated DNA substrate or cofactor binding, allosterically reduced enzymatic activities in vitro by decreasing kcat/Km for AdoMet. Because the crystallographic data showed that both of the mutants adopted helix-kinked conformation, we performed MD simulations to investigate the influence of the two mutants on conformational transition, which suggested that N1248A and R1279D mutations bias the catalytic helix to either the kinked or straight conformation, leading to an 773
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology
Figure 2. DNA binding affinity of mDNMT1 (731−1602) proteins and the catalytic efficiency of mDNMT1 (731−1602) proteins for hemimethylated DNA and AdoMet (SAM). (A) Methylation activities of mDNMT1 and its mutants that disturb the helix-straight conformation (red panel) or the helix-kinked conformation (blue panel). Methylation activities were monitored via reactions with a hemi-mCpG DNA substrate. (B) Methylation kinetics of mDNMT1 WT, N1248A, and R1279D mutants for hemi-mCG DNA duplexes (left panel) or SAM (right panel). The N1248A and R1279D mutants showed a related decrease in the catalytic activity toward both the hemimethylated 14-nucleotide oligomer DNA and SAM. The data were fit by nonlinear regression of the Michaelis−Menten equation to obtain the Michaelis−Menten parameters listed in Tables S2 and S3. (C, D) DNA binding affinity of mDNMT1 (731−1602) proteins measured by fluorescence polarization (FP) assays. FAM-14-mer duplexes were incubated with increasing amounts of mDNMT1 proteins. Wild-type (WT) and mutant mDNMT1 show similar binding affinities for hemimethylated substrates or fully methylated products. (E, F) SPR curves for mDNMT1 binding with (E) AdoMet and (F) AdoHcy are shown. The concentrations of AdoMet/AdoHcy injected over the NTA chip are indicated. These assays yielded KD values for mDNMT1 binding with AdoMet/AdoHcy.
■
impaired structural transition process. Furthermore, genetic complementation assays for the two mutants indicated that disturbing the conformational transition reduced DNMT1 activity in cells, which can act additively with existing DNMT inhibitors to decrease DNA methylation. Taken together, these findings expand our general understanding of the conformational transitions of DNMT1 and provide insight into the allosteric regulation of the DNMTs family.
RESULTS AND DISCUSSION
Disturbing Either of the Two States of DNMT1 Leads to Reduced Enzymatic Activity in Vitro. First, we analyzed two different crystal structures of DNMT1. The crystal structure of helix-kinked DNMT1 revealed that the long straight helix broke into two shorter helices at residues N1248 and S1249 (Figure S2B), suggesting the two residues act as a “hinge” in the catalytic helix. Along with other residues 774
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology
Figure 3. Crystal structures of N1248A and R1279D mutants. (A, left panel) Superimposition of the N1248A mutant (orange) and wild-type apoDNMT1 (PDB ID: 3PT9, cyan). (right panel) Close-up view of interactions between the catalytic loop and the rigid part of the methyltransferase domain in the N1278A mutant. (B, left panel) Superimposition of the R1279D mutant (magenta) and wild-type apo-DNMT1 (PDB ID: 3PT9, cyan). (right panel) Close-up view of interactions between the catalytic loop and the rigid part of the methyltransferase domain in the R1279D mutant.
stabilization of DNMT1, enzyme kinetics and binding assays were performed on N1248A and R1279D mutants. The enzyme kinetic assays indicated that kcat for hemimethylated substrates dropped from 0.56 ± 0.02 h−1 for the wild-type DNMT1 to 0.111 ± 0.007 and 0.3229 ± 0.0005 h−1 for N1248A and R1279D, respectively, demonstrating that the mutant proteins showed a related decrease in preference for hemimethylated 14-nucleotide oligomer DNA (Figure 2B, Table S1). However, the ratio (kcat/Km) of N1248A or R1279D was comparable to that of wild-type DNMT1, indicating that these mutations do not weaken the efficiency of reactions using hemimethylated substrates. Enzymatic assays have also been performed on different mutants to investigate the influence of activities and preferences for AdoMet. In contrast to their effect on hemimethylated DNA substrates, the N1248A and R1279D mutations showed a distinct decrease in catalytic efficiency for AdoMet. The ratio (kcat/Km) decreased from 0.99 ± 0.09 h−1 μM−1 for wild-type DNMT1 to 0.25 ± 0.02 and 0.576 ± 0.008 h−1 μM−1 for the N1248A and R1279D mutants, respectively (Figure 2B and Table S2). Then, the fluorescence polarization assays were conducted to measure interactions between DNMT1 and DNA. As shown in Figure 2C and D, hemimethylated DNA and fully methylated DNA were labeled by FAM. Among the WT and mutant DNMT1 proteins, the equilibrium dissociation constant (KD) between each protein and DNA was similar, which suggested
observed from the crystal structure of the helix-straight DNMT1 (residues S1233, N1236, and S1244), these residues may be important for maintaining the long straight helix (Figure 1C). The crystal structure of the helix-kinked state showed that R1278 and R1279 act as an anchor between the catalytic loop and the rigid part of the methyltransferase domain (Figure 1D). The corresponding residues in human DNMT1 are residues K1275 and R1276 (Figure S1), suggesting that the conserved positive charge of these residues plays an important role in the helix-kinked state of DNMT1. Because the catalytic helix is adjacent to the active site and the catalytic cysteine switches as the helix swings (Figure S2), these residues that maintain either the kinked or straight helix conformations may play a role in regulating DNMT1 enzymatic activity. Guided by the structural analysis, we mutated a number of residues mentioned above to monitor their impact on DNA methylation activity. As shown in Figure 2A, all single mutants showed reduced activities. Among these, residues N1248, S1249, R1278, and R1279 are not involved in either hemimethylated DNA substrate or cofactor binding. These findings suggested that both of the helix-straight and -kinked states were important for DNMT1 enzymatic activity and that disturbing either of the two states could allosterically decrease methylation rates in vitro. N1248A and R1279D Mutations Decrease the kcat/Km for AdoMet. For investigating the effects of disturbing the 775
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology that the N1248A and R1279D mutations did not affect the binding affinity of the enzyme for hemimethylated substrates or fully methylated products (Figure 2C and D). Moreover, to more precisely validate the interaction between DNMT1 and the cofactors, the surface plasmon resonance (SPR)-based binding assay was performed. As shown in Figure 2E, the KD between wild-type DNMT1 and AdoMet was around 5.75 μM, whereas N1248A and R1279D mutants were 6.61 and 3.78 μM, respectively. On the other hand, the KD between wild-type DNMT1 and S-adenosylhomocysteine (AdoHcy) was ∼0.47 μM, which was similar to the KD between the mutants and AdoHcy (Figure 2F). All these results demonstrated neither mutation influenced the binding affinity for AdoMet or AdoHcy. In addition, we observed that the mutations of C1229, M1235, and R1237, which are critical for catalytic reaction or substrate binding, essentially abolished the catalytic activity of DNMT1, consistent with the observation by a previous study (Figure 2A, Figure S4).11 Taken together, neither of the two mutations weakened interactions between DNMT1 and substrates or cofactors, which is reasonable because the two residues are not involved in either substrate or cofactor binding. The decreased catalytic activity of N1248A and R1279D mainly resulted from reduced catalytic efficiency for AdoMet. Crystal Structures of N1248A and R1279D Mutants. To further investigate whether the mutations could influence the conformation of DNMT1, we determined the crystal structure of N1248A (731−1602) of mouse DNMT1 at 2.10 Å resolution (Figure 3A, Table 1, PDB ID: 5GUT). An overlay of the mutant and wild-type apo-DNMT1 yielded a small RMSD value (0.346 Å), indicating that the N1248A mutant adopted a helix-kinked conformation. Despite the mutation of N1248, the conformation of the key residues that are important for helixkinked state were almost identical (Figure 3A). The N1248A mutant was bound to AdoHcy (Figure S5A), and two Cys3Hiscoordinated Zn2+ ions stabilized the BAH1 and TRD domain (Figure S5B and C). Aligning the different crystal structures in helix-kinked mDNMT1 revealed different bending angles for the catalytic helix (Figure S5D), validating the flexibility of this domain. Additionally, the 3.08 Å structure of the R1279D mutant (residues 731−1602) of mDNMT1 in the free-state was also determined (Figure 3B, Table 1, PDB ID: 5GUV). Two Zn2+ ions coordinated in a Cys3His manner were observed in BAH1 and TRD in the structure of R1279D (Figure S6A and B). Although the protein also adopts the helix-kinked state, the mutation of arginine to aspartic acid leads to weaker interactions between the catalytic loop and the rigid part of the methyltransferase domain (Figure 3B), implying that the R1279D mutation might bias the catalytic helix toward the straight conformation. Structural superimposition of different mDNMT1 structures showed that the TRD domain swings in different states (Figure S6C). Taken together, the crystal structures suggested that N1248A and R1279D mutants underwent a minor conformational changes and slightly affected the conformation of DNMT1. N1248A and R1279D Mutations Disturb the Proper Structural Transition of DNMT1 between the Two States. Because the N1248A and R1279D mutants both adopted the helix-kinked conformation (Figure 3) and neither of the mutations affected the binding affinity of the enzyme for substrates or cofactors (Figure 2C−F), we proposed that the well-balanced transition between these two states are important for DNMT1 catalysis and that the decreased enzymatic
Table 1. Crystallographic Statistics for Structure Determination of mDNMT1 (731−1602) Mutants
wavelength (Å) resolution range (Å) space group unit cell total reflections unique reflections multiplicity completeness (%) mean I/sigma (I) R-merge R-meas reflections used in refinement reflections used for R-free R-work R-free number of nonhydrogen atoms macromolecules ligands protein residues RMS (bonds) RMS (angles) Ramachandran favored (%) Ramachandran allowed (%) Ramachandran outliers (%) rotamer outliers (%) clash score average B-factor macromolecules ligands solvent
mDNMT1(731−1602) N1248A mutant
mDNMT1(731−1602) R1279D mutant
0.9788 44.36−2.099 (2.174− 2.099) P 21 21 21 75.424, 78.554, 164.544, 90, 90, 90 773582 (69097) 57583 (4526) 13.4 (12.4) 97 (98) 22.28 (4.92) 0.1071 (0.6067) 0.1114 (0.6326) 55934 (4526)
0.9791 61.6−3.078 (3.188− 3.078) P 41 21 2 79.039, 79.039, 393.276, 90, 90, 90 164426 (1615) 24155 (224) 6.8 (7.2) 99.2 (98.2) 13.8 (3.6) 0.147 (0.611) 0.167 (0.645) 24047 (2347)
2839 (242)
1233 (120)
0.1996 (0.2381) 0.2305 (0.2704) 6962
0.2143 (0.2558) 0.2686 (0.3371) 6542
6431 52 794 0.012 1.80 96
6479 2 820 0.013 1.80 95
4.2
5.4
0
0
1.7 9.64 40.35 40.32 63.17 38.27
1.7 11.73 45.99 46.08 56.46 36.39
activities of the mutants may result from impaired structural transition. To investigate the specific conformational transition in DNMT1, we performed a 2 μs MD simulation on the protein structure extracted from the covalent mDNMT1 (731− 1602)/hemi-DNA complex,11 which adopts the helix-straight state. The RMS fluctuation (RMSF) profile, which reflects the mobility of a certain residue around its average position, indicated that large fluctuations occurred in several segments (Figure 4A): the 850−861 and 1110−1140 segments, which are just the disordered parts in the crystal structures, the catalytic loop and N-terminal of catalytic helix (1227−1249 segment), which adopt dramatically distinct conformations in different states as well as the TRD domain (1336−1551 segment), suggesting the RMSF profile is consistent with the crystal structures (Figure 4A). The B-factor (thermal factor) in the crystal structure of helix-straight DNMT1, which is used as a measure for any type of displacement of an atom from its mean position, was extracted and transformed to RMSF using the formula B = 8π2·RMSF2/3 (Figure 4A). Furthermore, the snapshots isolated from the trajectory indicates that C1229 shifted away from the active conformation and gradually flipped 776
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology
Figure 4. Conformational transition of helix-straight DNMT1. (A) Residue dynamics was obtained by averaging atomic fluctuations over the 1 μs MD simulation for helix-straight DNMT1. The highly flexible domains including the 850−861 and 1110−1140 segments, which are just the disordered parts in the crystal structures (red), the catalytic loop, and N-terminal of the catalytic helix (1227−1249 segment, magenta) and the TRD domain (1336−1551 segment, green). (B, top) Secondary structures of the catalytic helix as a function of time for helix-straight DNMT1 in trajectory as calculated using DSSP. The structures were analyzed every 100 ps. (bottom) Snapshot structures of DNMT1 extracted from the trajectory in which the secondary structure was obtained using DSSP analysis. (C, top panel) Energy landscapes for the conformational transition of wild-type DNMT1 (first 1 μs trajectory, right panel), the N1248A mutant (1 μs trajectory, middle panel), and the R1279D mutant (1 μs trajectory, right panel). Reaction coordinates were defined according to PC1 and PC2 obtained from PCA based on the 2 μs trajectory of well-helix-straight DNMT1. (bottom panel) Typical conformations of the first and third local energy minima during the trajectory of wild-type DNMT1 (1 μs), which are similar to the values for the trajectory of the N1248A mutant (1 μs) and R1279D mutant (1 μs).
to another orientation (Figure S7A). The consistency between the theoretical and experimental RMS fluctuation (RMSF), as well as the shift of C1229, validated the reliability of our model. To identify the most significant motions in helix-straight DNMT1, we performed principal component analysis (PCA)27 based on the MD trajectory. PCA considers each motion as the variance of atomic fluctuations around the average structure. The first mode (PC1, Movie S1) that represented the largest atomic fluctuation in the sampled conformations consisted of
two predominant motions (Figure S7B). First, the catalytic helix underwent a dramatic structural change that results in the helix-kinked conformation. Second, the TRD domain gradually shifted away from the catalytic cleft. Movements of the catalytic loop and TRD domain led to an increase in the width of the catalytic cleft where substrate DNA is expected to fit (Figure S7C), which was consistent with crystal structures (Figure S7D). Additionally, to examine the detailed conformational transition of the DNMT1, we calculated the transformation of 777
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology
Figure 5. Impairing the conformational transition decreases DNMT1 activity in cells and augments the efficiency of DAC in reducing DNA methylation. (A) Western blot analysis of DNMT1 protein levels in the HCT116 DNMT1 hypomorph cells expressing either WT or mutant DNMT1 along with depletion of the endogenous truncated protein. The basal levels of endogenous DNMT1 in HCT116 and hypomorph cells (MT1 hypo) are also shown. (B, C) WT and mutant DNMT1 were assessed for their ability to maintain DNA methylation upon the depletion of endogenous truncated protein in the hypomorph cells by both (B) 5-mC dot blots and (C) 5-mC ELISA. Two-fold serial dilutions (from 400 to 12.5 ng) of genomic DNA were analyzed in the dot blot assay (B). The ELISA data were normalized to the results from WT DNMT1-containing cells and presented as the mean ± SD (n = 3). ***P < 0.001 versus WT DNMT1. HCT116 DKO1 cells were used as controls for genome-wide depletion of DNA methylation. (D) 5-mC ELISA analysis of the DNA methylation level for both DAC- and mock-treated cells expressing either WT or mutant DNMT1 along with the depletion of the endogenous protein. The measurements were normalized to those from mock-treated cells and presented as the mean ± SD (n = 3). ***P < 0.001 versus mock treatment; ns: not significant.
flexibility of the straight catalytic helix, whereas the R1279D mutation led to greater stability. As mentioned above, we performed principal component analysis (PCA) based on the 2 μs MD trajectory of wild-type helix-straight DNMT1 (Figure S7B), and the first two principal components were PC1 and PC2. Here, we constructed rough energy landscapes for the conformational transition of wild-type DNMT1 (first 1 μs trajectory) and the two mutants projected onto PC1 and PC2; every frame of the structure generated by MD simulations corresponded to two coordinates (Figure 4C, Figure S9). All of the energy landscapes consisted of several peaks; the conformations of each peak were selected based on the corresponding coordinates. Basically, the first peak corresponds well to the helix-straight state, which served as the starting structure. Concomitant with conformational changes, DNMT1 moved to the top of the third peak to achieve a state very similar to that of the helix-kinked DNMT1. The energy landscapes suggested that the two mutants of DNMT1 influenced the conformation distributions during the structural transition; N1248A mutant biased DNMT1 toward the helix-kinked conformation, whereas the R1279D mutant preferred the helix-straight conformation (Figure 4C, Figure S9). Consequently, a proper dynamic equilibrium between the two states of DNMT1 is required for catalysis; the N1248A and R1279D mutants decreased enzymatic activity by disturbing the well-balanced structural transition. Impaired Conformational Transition Can Decrease DNA Methylation in Cells. To explore the functional role of
the secondary structure of catalytic helix along the trajectory of simulation by DSSP28 (Figure 4B). The profile of the secondary structure transformation and snapshots of the handle domain extracted from the MD trajectory revealed that the helical structure of residues 1249−1261 was stable during the simulation, which was also in accordance with the crystallology observations (Figure 1B). However, the N-terminal part of the catalytic helix (residues 1240−1248) underwent a helical unfolding/refolding process. Finally, the long catalytic helix was consequently broken into two shorter helices at the positions of residues N1248 and S1249, resulting in a conformation very similar to that of the helix-kinked state (Figure 4B). Taken together, all these results showed that DNMT1 might spontaneously undergo a conformational transition from the helix-straight state to the helix-kinked conformation in the absence of DNA. Next, to investigate the influence of the two mutants on conformational transition, we carried out 1 μs MD simulations on constructed N1248A and R1279D mutants, which both adopted the helix-straight conformation. The RMSD profile combined with secondary structure transformation indicated that the straight catalytic helix of the N1248A mutant was less stable than that of the R1279D mutant (Figure S8A, B). Approximately 32% of the helical structure of segment 1242− 1249 in N1248A mutant showed helical unfolding; in contrast, the ratio of unfolded helix in R1279D was only 2% (Figure S8C), implying that the N1248A mutation led to greater 778
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology
conserved dynamic structural properties in different DNMTs, these findings potentially reflect molecular basis for the dynamic process shared in the whole DNMT family in general and thus aid in better understanding the relationship between DNMTs dynamic switching and enzymatic activity. Intriguingly, our data also showed that disrupting the conformational transition of the catalytic helix can complement current DNA demethylation agents to reduce DNA methylation. However, the C1226W mutant, which changes the catalytic core of DNMT1, showed no DNA methylation changes. This suggests that the conformational transition site may serve as an allosteric site to develop DNMT1 inhibitors with a novel mode of action (MOA). These inhibitors may block the normal conformational transition and thus inhibit DNMT1 activity allosterically, which is similar to the IDH-1 mutant selective inhibitors that bound to Seg-2 of this protein and precluded the loop-helix transition necessitated by its enzymatic turnover.35,36 As we know, most DNMT1 inhibitors lack specificity and lead to toxic side-effects. Contrast to the binding pockets for AdoMet (Figure S3D), the sequence of the active-site loop (catalytic loop and catalytic helix in DNMT1) varies in different DNMTs (Figure S3C). Thus, targeting this site will possibly improve the probability of finding selective inhibitors. Therefore, our finding suggests that regulation of the dynamic catalytic helix may serve as a novel approach for developing specific DNMT1 inhibitors to avoid the nonselectivity of AdoMet analogues. Overall, our research provides a solid framework in general for understanding the molecular mechanisms of the entire DNMT family, which is potentially valuable for the development of novel inhibitors for gene promoter DNA hypermethylation found in most human cancers.
the conformational transition in maintaining DNA methylation in cells, we performed a genetic complementation assay in the HCT116 DNMT1 hypomorph cells.29 Compared with parent HCT116 cells, the bulk of DNA methylation in the hypomorph cells is retained by a truncated DNMT1 form that leaves ∼15% of the protein remaining.30,31 As shown in Figure 5, the level of truncated DNMT1 in the hypomorph cells was further depleted by an effective shRNA32 that was specific for the endogenous protein (Figure 5A), resulting in a significant reduction in the overall DNA methylation (Figure 5B, C). The introduction of wild-type DNMT1, rather than the catalytically deficient C1226W mutant,33,34 maintains or even slightly increases the level of the DNA methylation relative to the parental hypomorph cells upon the depletion of the endogenous protein (Figure 5B, C). Similar findings were also observed in a recent study performed in human embryonic stem cells in which the wild-type DNMT1, but not the C1226W mutant, could rescue cell death and global DNA demethylation after endogenous DNMT1 knockout.34 We then studied the functional consequences of the DNMT1 N1245A and R1276D mutants, which were equivalent to the N1248A and R1279D mutants in mDNMT1. Both DNMT1 mutants led to apparent decreases in overall 5-mC levels compared with those of the WT protein, whereas the N1245A mutant had a slightly stronger effect (∼35%) than the R1279D mutant (∼25%), consistent with the in vitro enzymatic observations (Figure 2A). Notably, the decreased activity of the DNMT1 mutants can probably not be attributed to differential protein expression given that the levels of the exogenous WT and mutant proteins were comparable to those of endogenous DNMT1 in the HCT116 cells (Figure 5A). Collectively, the genetic complementation data indicated that disturbing the conformational transition partially impaired DNMT1 function in cells. The agent 5-deoxy-aza-cytidine (DAC) is clinically used as a DNMT inhibitor. We hypothesized that combining our DNMT1 mutants that affect the conformation of the protein with DNA demethylation agents might additively reduce DNA methylation. Indeed, compared with the moderate decrease in DNA methylation caused by DNMT1 mutants alone and by DAC treatment of WT DNMT1 cells, a much lower DNA methylation level was achieved in cells harboring either DNMT1 mutant after 48 h DAC exposure (Figure 5D). By contrast, virtually no DNA methylation changes were observed in cells expressing a mutant fully inactive for DNMT1 catalytic activity. These observations suggested that targeting the conformational transition of the catalytic helix could complement current DNA demethylating agents to reduce DNA methylation in cancer cells.
■ ■
METHODS
A description of the methods used in this work is located in the Supporting Information.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications Web site at DOI: Methods, Figures S1−S9, Table S1−S2 and movie S1(PDF) The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00890. Methods details, sequence alignment, helix-straight and -kinked states of mDNMT1, structures of M.HhaI and DNMT3a/DNMT3L complex, mDNMT1-mediated cytosine methylation kinetics, crystal structures of the N1248A and R1279D mutants of mDNMT1 (731− 1602), conformational change during 1 μs MD trajectory of helix-straight DNMT1, catalytic helix biasing, DNMT1-catalyzed enzymatic methylation assays, and crystallographic statistics (PDF) Motions corresponding to the first component (PC1) obtained by PCA on the simulation trajectory of helixstraight DNMT1 (AVI)
■
CONCLUSIONS In this study, we conducted a mechanistic investigation of the dynamic transition of DNMT1 based on a combination of in vitro enzymatic assay, crystallography, long-term MD simulations, and cell-based assays. Our findings showed that both the helix-straight and -kinked states are essential for enzyme catalysis of DNMT1. N1248A and R1279D mutants, which did not affect the binding affinity between DNMT1 and substrates or cofactors, allosterically reduced enzymatic activity in vitro and in cells by disturbing the proper structural transition of DNMT1 between the two states. Thereby, these two residues could be used as tools to study the detailed mechanism of the helix transition in modulating DNMT1 activity. Given the
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. *E-mail:
[email protected]. 779
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology ORCID
(11) Song, J., Teplova, M., Ishibe-Murakami, S., and Patel, D. J. (2012) Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 335, 709−712. (12) Zhang, Z. M., Liu, S., Lin, K., Luo, Y., Perry, J. J., Wang, Y., and Song, J. (2015) Crystal Structure of Human DNA Methyltransferase 1. J. Mol. Biol. 427, 2520−2531. (13) Cheng, J., Yang, H., Fang, J., Ma, L., Gong, R., Wang, P., Li, Z., and Xu, Y. (2015) Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation. Nat. Commun. 6, 7023. (14) Kanada, K., Takeshita, K., Suetake, I., Tajima, S., and Nakagawa, A. (2017) Conserved threonine 1505 in the catalytic domain stabilizes mouse DNA methyltransferase 1. J. Biochem., 271−278. (15) O’Gara, M., Roberts, R. J., and Cheng, X. (1996) A structural basis for the preferential binding of hemimethylated DNA by HhaI DNA methyltransferase,. J. Mol. Biol. 263, 597−606. (16) O’Gara, M., Zhang, X., Roberts, R. J., and Cheng, X. (1999) Structure of a binary complex of HhaI methyltransferase with Sadnosyl-l-methionine formed in the presence of a short non-specific DNA oligonucleotide. J. Mol. Biol. 287, 201−209. (17) Cheng, X., Kumar, S., Posfai, J., Pflugrath, J. W., and Roberts, R. J. (1993) Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine. Cell 74, 299−307. (18) Shieh, F. K., and Reich, N. O. (2007) AdoMet-dependent methyl-transfer: Glu119 is essential for DNA C5-cytosine methyltransferase M.HhaI. J. Mol. Biol. 373, 1157−1168. (19) Shieh, F. K., Youngblood, B., and Reich, N. O. (2006) The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI,. J. Mol. Biol. 362, 516−527. (20) Youngblood, B., Shieh, F. K., De Los Rios, S., Perona, J. J., and Reich, N. O. (2006) Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI. J. Mol. Biol. 362, 334−346. (21) Jin, L., Ye, F., Zhao, D., Chen, S., Zhu, K., Zheng, M., Jiang, R. W., Jiang, H., and Luo, C. (2014) Metadynamics simulation study on the conformational transformation of HhaI methyltransferase: an induced-fit base-flipping hypothesis. BioMed Res. Int. 2014, 304563. (22) Chedin, F., Lieber, M. R., and Hsieh, C. L. (2002) The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a,. Proc. Natl. Acad. Sci. U. S. A. 99, 16916−16921. (23) Kareta, M. S., Botello, Z. M., Ennis, J. J., Chou, C., and Chedin, F. (2006) Reconstitution and mechanism of the stimulation of de novo methylation by human DNMT3L. J. Biol. Chem. 281, 25893−25902. (24) Jia, D., Jurkowska, R. Z., Zhang, X., Jeltsch, A., and Cheng, X. (2007) Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature 449, 248−251. (25) Guo, X., Wang, L., Li, J., Ding, Z., Xiao, J., Yin, X., He, S., Shi, P., Dong, L., Li, G., Tian, C., Wang, J., Cong, Y., and Xu, Y. (2014) Structural insight into autoinhibition and histone H3-induced activation of DNMT3A. Nature 517, 640−644. (26) Xu, G. L., Bestor, T. H., Bourc’his, D., Hsieh, C. L., Tommerup, N., Bugge, M., Hulten, M., Qu, X., Russo, J. J., and Viegas-Pequignot, E. (1999) Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402, 187−191. (27) Amadei, A., Linssen, A. B., and Berendsen, H. J. (1993) Essential dynamics of proteins. Proteins: Struct., Funct., Genet. 17, 412−425. (28) Kabsch, W., and Sander, C. (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577−2637. (29) Rhee, I., Jair, K. W., Yen, R. W., Lengauer, C., Herman, J. G., Kinzler, K. W., Vogelstein, B., Baylin, S. B., and Schuebel, K. E. (2000) CpG methylation is maintained in human cancer cells lacking DNMT1. Nature 404, 1003−1007. (30) Egger, G., Jeong, S., Escobar, S. G., Cortez, C. C., Li, T. W., Saito, Y., Yoo, C. B., Jones, P. A., and Liang, G. (2006) Identification of DNMT1 (DNA methyltransferase 1) hypomorphs in somatic knockouts suggests an essential role for DNMT1 in cell survival. Proc. Natl. Acad. Sci. U. S. A. 103, 14080−14085.
Xiangqian Kong: 0000-0002-9596-7825 Cheng Luo: 0000-0003-3864-8382 Author Contributions ○
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The computation resources were supported by Computer Network Information Center, Chinese Academy of Sciences and Tianjin Supercomputing Center. We thank the staffs from BL17U1 beamline at Shanghai Synchrotron Radiation Facility (SSRF) and from BL19U1 beamline of National Facility for Protein Science Shanghai (NFPS) at SSRF for assistance during data collection. We are extremely grateful to the National Centre for Protein Science Shanghai (Shanghai Science Research Center, Protein Expression and Purification system) for their instrument support and technical assistance. We gratefully acknowledge financial support from National Key R&D Program of China (2017YFB0202600 to H.D.), the National Natural Science Foundation of China (21472208 and 81625022 to C.L., 81703415 and 81430084 to S.C.), CAS Strategic Priority Research Program (XDA12020304 and XDA12020353 to C.L.), and Zhejiang Province Natural Science Foundation (LY18H300008 to F.Y.). This work is also sponsored by the Shanghai Sailing Program (17YF1423100 to S.C.), National Institutes of Environmental Health Sciences (NIEHS) grants (RO1 ES011858 to S.B.B.), the Hodson Trust (S.B.B.), China Postdoctoral Science Foundation (2016M601676 to S.C.), and National Key Scientific Instrument & Equipment Development Program of China (2012YQ03026010 to C.L.).
■
REFERENCES
(1) Bird, A. (2002) DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6−21. (2) Li, E. (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3, 662−673. (3) Reik, W., and Lewis, A. (2005) Co-evolution of X-chromosome inactivation and imprinting in mammals. Nat. Rev. Genet. 6, 403−410. (4) Cheng, X., and Blumenthal, R. M. (2008) Mammalian DNA methyltransferases: a structural perspective. Structure 16, 341−350. (5) Law, J. A., and Jacobsen, S. E. (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204−220. (6) Okano, M., Xie, S., and Li, E. (1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19, 219−220. (7) Aoki, A., Suetake, I., Miyagawa, J., Fujio, T., Chijiwa, T., Sasaki, H., and Tajima, S. (2001) Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 29, 3506−3512. (8) Li, E., Bestor, T. H., and Jaenisch, R. (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915−926. (9) Song, J., Rechkoblit, O., Bestor, T. H., and Patel, D. J. (2011) Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036−1040. (10) Takeshita, K., Suetake, I., Yamashita, E., Suga, M., Narita, H., Nakagawa, A., and Tajima, S. (2011) Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1). Proc. Natl. Acad. Sci. U. S. A. 108, 9055−9059. 780
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
Articles
ACS Chemical Biology (31) Chen, T., Hevi, S., Gay, F., Tsujimoto, N., He, T., Zhang, B., Ueda, Y., and Li, E. (2007) Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells. Nat. Genet. 39, 391−396. (32) Cai, Y., Tsai, H. C., Yen, R. C., Zhang, Y. W., Kong, X., Wang, W., Xia, L., and Baylin, S. B. (2017) Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells. Genome Res. 27, 533−544. (33) Clements, E. G., Mohammad, H. P., Leadem, B. R., Easwaran, H., Cai, Y., Van Neste, L., and Baylin, S. B. (2012) DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes. Nucleic Acids Res. 40, 4334−4346. (34) Liao, J., Karnik, R., Gu, H., Ziller, M. J., Clement, K., Tsankov, A. M., Akopian, V., Gifford, C. A., Donaghey, J., Galonska, C., Pop, R., Reyon, D., Tsai, S. Q., Mallard, W., Joung, J. K., Rinn, J. L., Gnirke, A., and Meissner, A. (2015) Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells. Nat. Genet. 47, 469− 478. (35) Okoye-Okafor, U. C., Bartholdy, B., Cartier, J., Gao, E. N., Pietrak, B., Rendina, A. R., Rominger, C., Quinn, C., Smallwood, A., Wiggall, K. J., Reif, A. J., Schmidt, S. J., Qi, H., Zhao, H., Joberty, G., Faelth-Savitski, M., Bantscheff, M., Drewes, G., Duraiswami, C., Brady, P., Groy, A., Narayanagari, S. R., Antony-Debre, I., Mitchell, K., Wang, H. R., Kao, Y. R., Christopeit, M., Carvajal, L., Barreyro, L., Paietta, E., Makishima, H., Will, B., Concha, N., Adams, N. D., Schwartz, B., McCabe, M. T., Maciejewski, J., Verma, A., and Steidl, U. (2015) New IDH1 mutant inhibitors for treatment of acute myeloid leukemia. Nat. Chem. Biol. 11, 878−886. (36) Deng, G., Shen, J., Yin, M., McManus, J., Mathieu, M., Gee, P., He, T., Shi, C., Bedel, O., McLean, L. R., Le-Strat, F., Zhang, Y., Marquette, J. P., Gao, Q., Zhang, B., Rak, A., Hoffmann, D., Rooney, E., Vassort, A., Englaro, W., Li, Y., Patel, V., Adrian, F., Gross, S., Wiederschain, D., Cheng, H., and Licht, S. (2015) Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule. J. Biol. Chem. 290, 762−774.
781
DOI: 10.1021/acschembio.7b00890 ACS Chem. Biol. 2018, 13, 772−781
