Biochemical Studies and Molecular Dynamic Simulations Reveal the Molecular Basis of Conformational Changes in DNA Methyltransferase-1

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 k/ K 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.

Molecular Processes Connecting DNA Methylation Patterns with DNA Methyltransferases and Histone Modifications in Mammalian Genomes

“…Finally, we describe recent advances regarding the methylation of non-CpG sites, methylation of adenine residues in human cells, and methylation of mitochondrial DNA. For more detailed reviews on DNA methylation patterns and functions of DNA methylation, refer to [ 9 , 10 , 11 , 12 , 13 ], and for reviews describing the enzymology of DNMTs to [ 14 , 15 , 16 ]. This review will not focus on DNA methylation in other species like plants, fungi, or arthropods, where many (though not all) of the basic processes are conserved, but additional phenomena are observed.…”

Structural Basis of DNMT1- and DNMT3A-Mediated DNA Methylation

“…3E). Indeed, a subsequent study indicates that disruption of this conformational transition leads to impaired enzymatic activity of DNMT1 [39], highlighting the importance of this conformational switch in DNMT1-mediated DNA methylation.…”

Adaptive evolution in DNMT2 supports its role in the dipteran immune response

“…Incidentally, past studies indicate these four regions contribute significantly towards DNMT2's MTase activity with regards to substrate binding and catalytic activity (Goll, et al 2006). Furthermore, high clustering of sites in the TRD region is significant, given that they (AaDNMT2: 208, DmDNMT2: 261L) are located in a catalytically critical region that is known to penetrate the major groove of the nucleic acid substrate (Ye, et al 2018). Finally, for both orthologs, a large proportion of sites present at the N-terminus (AaDNMT2: 44G,55S,105I, DmDNMT2: 23G,44G,55S,66A) and the TRD (AaDNMT2: 147H, 222C, DmDNMT2: 220H, 223Q, 245T, 252S) were found to be present on the solvent accessible surface ( Figure 2B,C).…”

“…In order to better understand IPOD's cellular function, we performed a domain analyses using Pfam and InterPro. We identified a DUF4766 (PF15973) domain (Residues: 82 -232) present in all orthologs, and InterPro suggested nearly 90% of the protein (Residues: 33 -349) contains a non-cytoplasmic domain, with a smaller signal peptide domain (Residues: 1 -32) present at the N-terminal end (Posterior Probability Score > 0.99) (Goll, et al 2006;Ye, et al 2018). Notably, we found nearly 28% (97/397) of the total protein length to be made of glycine residues, which are associated with a high degree of disordered structure.…”

“…and is predicted to interact with the nucleic acid substrate based on past simulation studies using mammalian DNMT1 (Ye, et al 2018). These differences also give rise to altered surface charge distribution between DmDNMT2 and AaDNMT2, further signifying potential differences in inter-molecular associations and/or target specificity between these orthologs.…”