PMC 20230815 pmc.key 5173035 CC BY no 2 2 10.18632/oncotarget.9692 5173035 27259995 9692 40965 27 DNA N6-adenine methyltransferase M1.HpyAVI substrate recognition AdoMet-binding Helicobacter pylori Immunology and Microbiology Section Immune response Immunity This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 40977 surname:Ma;given-names:Bo surname:Ma;given-names:Ji surname:Liu;given-names:Dong surname:Guo;given-names:Ling surname:Chen;given-names:Huiling surname:Ding;given-names:Jingjin surname:Liu;given-names:Wei surname:Zhang;given-names:Hongquan TITLE front 7 2016 0 Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori 0.9988349 experimental_method cleaner0 2023-09-15T14:48:22Z MESH: Biochemical and structural characterization 0.9984956 protein_type cleaner0 2023-09-15T14:48:27Z MESH: DNA N6-adenine methyltransferase 0.99830234 species cleaner0 2023-09-15T14:48:33Z MESH: Helicobacter pylori ABSTRACT abstract 108 DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes. Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively. The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme. Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity. Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity. ptm MESH: cleaner0 2023-09-15T15:12:13Z DNA N6-methyladenine 0.99842924 taxonomy_domain cleaner0 2023-09-15T14:48:39Z DUMMY: bacteria 0.998017 ptm cleaner0 2023-09-15T14:55:25Z MESH: N6-methyladenine protein PR: cleaner0 2023-09-15T15:44:16Z M1.HpyAVI 0.99685204 protein_type cleaner0 2023-09-15T14:48:27Z MESH: DNA N6-adenine methyltransferase 0.99692714 species cleaner0 2023-09-15T14:48:33Z MESH: Helicobacter pylori 0.998652 evidence cleaner0 2023-09-18T09:27:59Z DUMMY: crystal structures 0.9985555 protein_state cleaner0 2023-09-15T14:49:21Z DUMMY: cofactor-free 0.9989319 protein_state cleaner0 2023-09-15T14:49:26Z DUMMY: AdoMet-bound 0.9984761 evidence cleaner0 2023-09-18T09:28:02Z DUMMY: structures protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.9982943 protein_type cleaner0 2023-09-15T14:49:44Z MESH: AdoMet-dependent MTase 0.99887353 site cleaner0 2023-09-15T14:49:57Z SO: DNA binding regions 0.99915063 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases 0.9985891 experimental_method cleaner0 2023-09-15T14:50:02Z MESH: Site-directed mutagenesis 0.9995345 residue_name_number cleaner0 2023-09-15T14:50:05Z DUMMY: D29 0.9995165 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.99952316 residue_name_number cleaner0 2023-09-15T14:50:15Z DUMMY: P41 0.9979963 protein_state cleaner0 2023-09-18T09:00:19Z DUMMY: highly flexible structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.9976624 protein_type cleaner0 2023-09-15T14:48:27Z MESH: DNA N6-adenine methyltransferase INTRO title_1 1269 INTRODUCTION INTRO paragraph 1282 DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes. Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression. DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence. 0.5646959 ptm cleaner0 2023-09-15T14:50:29Z MESH: DNA methylation 0.9983785 taxonomy_domain cleaner0 2023-09-15T14:51:00Z DUMMY: prokaryotes 0.998254 taxonomy_domain cleaner0 2023-09-15T14:51:04Z DUMMY: eukaryotes chemical CHEBI: cleaner0 2023-09-15T15:14:12Z DNA ptm MESH: cleaner0 2023-09-15T14:50:29Z DNA methylation 0.99836695 protein_type cleaner0 2023-09-15T14:51:11Z MESH: DNA methyltransferases 0.9990213 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.99902046 chemical cleaner0 2023-09-15T14:50:44Z CHEBI: S-adenosyl-L- methionine 0.99917704 chemical cleaner0 2023-09-15T14:50:48Z CHEBI: AdoMet chemical CHEBI: cleaner0 2023-09-15T15:14:24Z DNA chemical CHEBI: cleaner0 2023-09-18T08:33:18Z DNA INTRO paragraph 1763 Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases. C5-cytosine MTases, for example, methylate C5 of cytosine (m5C). In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer. By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown. N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC). N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages. The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions. Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species. DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila. 0.995378 protein_type cleaner0 2023-09-15T14:51:22Z MESH: DNA MTases chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl chemical CHEBI: cleaner0 2023-09-15T15:14:33Z DNA 0.99841994 protein_type cleaner0 2023-09-15T14:51:35Z MESH: C5-cytosine MTases 0.9554958 residue_name cleaner0 2023-09-15T14:51:51Z SO: cytosine 0.5558117 ptm cleaner0 2023-09-15T14:53:44Z MESH: m5C 0.9986461 taxonomy_domain cleaner0 2023-09-15T14:51:05Z DUMMY: eukaryotes 0.42158782 ptm cleaner0 2023-09-15T14:53:44Z MESH: m5C 0.9946444 species cleaner0 2023-09-15T14:54:51Z MESH: human 0.9984837 taxonomy_domain cleaner0 2023-09-15T14:52:58Z DUMMY: prokaryotic 0.9975991 protein_type cleaner0 2023-09-15T14:52:43Z MESH: DNA cytosine MTase 0.998495 protein_type cleaner0 2023-09-15T14:52:53Z MESH: N4-cytosine MTases 0.9925321 taxonomy_domain cleaner0 2023-09-15T14:53:07Z DUMMY: thermophilic 0.9845115 taxonomy_domain cleaner0 2023-09-15T14:53:10Z DUMMY: mesophilic 0.99733055 taxonomy_domain cleaner0 2023-09-15T14:48:40Z DUMMY: bacteria chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.662408 residue_name cleaner0 2023-09-15T14:51:52Z SO: cytosine 0.92875 ptm cleaner0 2023-09-15T14:53:58Z MESH: 4mC ptm MESH: cleaner0 2023-09-18T09:27:38Z N4 methylation 0.99867094 taxonomy_domain cleaner0 2023-09-15T14:53:03Z DUMMY: bacterial chemical CHEBI: cleaner0 2023-09-15T15:14:44Z DNA taxonomy_domain DUMMY: cleaner0 2023-09-15T14:56:28Z bacteriophages 0.99827415 protein_type cleaner0 2023-09-15T14:53:24Z MESH: N6-adenine MTases 0.9711873 residue_name cleaner0 2023-09-15T14:54:08Z SO: adenine 0.98714834 ptm cleaner0 2023-09-15T14:54:26Z MESH: 6mA 0.9985636 taxonomy_domain cleaner0 2023-09-15T14:51:01Z DUMMY: prokaryotes chemical CHEBI: cleaner0 2023-09-15T15:14:54Z DNA 0.96919215 ptm cleaner0 2023-09-15T14:54:26Z MESH: 6mA 0.99854434 taxonomy_domain cleaner0 2023-09-18T09:27:43Z DUMMY: eukaryotic chemical CHEBI: cleaner0 2023-09-18T08:33:46Z DNA 0.9743464 ptm cleaner0 2023-09-15T14:54:26Z MESH: 6mA 0.84729904 taxonomy_domain cleaner0 2023-09-15T14:54:37Z DUMMY: Chlamydomonas 0.99722385 species cleaner0 2023-09-15T14:54:42Z MESH: C.elegans 0.9626723 taxonomy_domain cleaner0 2023-09-15T14:54:46Z DUMMY: Drosophila INTRO paragraph 3177 All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems. The R-M systems are composed of two enzymes displaying opposing activities. “R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage. The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages. The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements. In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification. By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets. ptm MESH: cleaner0 2023-09-18T08:31:49Z methylation 0.99844944 taxonomy_domain cleaner0 2023-09-15T14:51:01Z DUMMY: prokaryotes 0.99861634 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.9980486 protein_type cleaner0 2023-09-15T14:56:19Z MESH: restriction endonuclease chemical CHEBI: cleaner0 2023-09-15T15:15:09Z DNA 0.99867034 protein_type cleaner0 2023-09-15T14:56:23Z MESH: modification methyltransferase 0.99824834 taxonomy_domain cleaner0 2023-09-15T14:48:40Z DUMMY: bacteria 0.99335164 taxonomy_domain cleaner0 2023-09-15T14:56:28Z DUMMY: bacteriophages chemical CHEBI: cleaner0 2023-09-15T15:15:18Z DNA 0.99397075 protein_type cleaner0 2023-09-15T14:56:38Z MESH: DNA adenine or cytosine methyltransferase 0.99784696 protein_type cleaner0 2023-09-15T14:56:20Z MESH: restriction endonuclease 0.9003789 protein_type cleaner0 2023-09-15T14:56:45Z MESH: DNA adenine or cytosine methyltransferase INTRO paragraph 4158 To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes. All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base. Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order. According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε). N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species. For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on. 0.9985929 taxonomy_domain cleaner0 2023-09-15T14:53:03Z DUMMY: bacterial 0.9986365 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.9897859 experimental_method cleaner0 2023-09-18T10:02:45Z MESH: structurally characterized 0.9991573 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases 0.9947482 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved structure_element SO: cleaner0 2023-09-18T08:36:18Z catalytic domain 0.9982283 site cleaner0 2023-09-18T09:35:08Z SO: active site chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.9962698 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet structure_element SO: cleaner0 2023-09-18T08:34:46Z target (DNA)-recognition domain structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD chemical CHEBI: cleaner0 2023-09-15T15:15:33Z DNA chemical CHEBI: cleaner0 2023-09-15T15:15:43Z DNA 0.9656195 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: Conserved 0.99657625 evidence cleaner0 2023-09-15T14:58:17Z DUMMY: structures structure_element SO: cleaner0 2023-09-18T08:35:22Z I-X 0.99850804 protein_type cleaner0 2023-09-15T14:58:34Z MESH: cytosine MTases structure_element SO: cleaner0 2023-09-18T08:35:02Z I-VIII structure_element SO: cleaner0 2023-09-18T08:35:13Z X 0.9986151 protein_type cleaner0 2023-09-15T14:58:13Z MESH: adenine MTases 0.8387143 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved 0.9985342 protein_type cleaner0 2023-09-15T14:58:44Z MESH: exocyclic amino MTases 0.99915075 protein_type cleaner0 2023-09-15T15:00:07Z MESH: α 0.9991696 protein_type cleaner0 2023-09-15T15:00:15Z MESH: β 0.9992083 protein_type cleaner0 2023-09-15T15:00:25Z MESH: γ 0.9991721 protein_type cleaner0 2023-09-15T15:00:32Z MESH: ζ 0.99910283 protein_type cleaner0 2023-09-15T15:00:40Z MESH: δ 0.9991536 protein_type cleaner0 2023-09-15T15:00:49Z MESH: ε protein_type MESH: cleaner0 2023-09-15T14:59:23Z N6-adenine and N4-cytosine MTases 0.99840933 taxonomy_domain cleaner0 2023-09-15T14:53:03Z DUMMY: bacterial 0.9991761 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases 0.999046 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases structure_element SO: cleaner0 2023-09-18T08:36:17Z catalytic domain structure_element SO: cleaner0 2023-09-18T08:36:35Z C-terminal domain protein PR: cleaner0 2023-09-15T15:44:49Z M.TaqI protein PR: cleaner0 2023-09-15T15:45:05Z M.MboIIA protein PR: cleaner0 2023-09-15T15:45:22Z M.RsrI structure_element SO: cleaner0 2023-09-18T08:36:51Z helix bundle 0.99908864 protein cleaner0 2023-09-15T15:02:49Z PR: EcoDam INTRO paragraph 5553 DNA methylation is thought to influence bacterial virulence. DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila. Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action. Dam was considered a promising target for antimicrobial drug development. ptm MESH: cleaner0 2023-09-15T14:50:29Z DNA methylation 0.9987413 taxonomy_domain cleaner0 2023-09-15T14:53:03Z DUMMY: bacterial 0.99834085 protein_type cleaner0 2023-09-18T08:37:53Z MESH: DNA adenine methyltransferase 0.9982779 species cleaner0 2023-09-15T15:03:43Z MESH: Salmonella typhimurium 0.9983058 species cleaner0 2023-09-15T15:03:47Z MESH: Aeromonas hydrophila ptm MESH: cleaner0 2023-09-15T15:07:59Z DNA adenine methylation ptm MESH: cleaner0 2023-09-15T15:16:42Z DNA adenine methylation 0.9145081 protein_type cleaner0 2023-09-15T15:16:58Z MESH: Dam INTRO paragraph 6031 Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide. It is a major pathogen of gastritis and peptic ulcer diseases as well as a cancer-causing factor for gastric cancer. H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma. H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems. H. pylori 26695, for example, has 23 R-M systems. Methyltransferases were suggested to be involved in H. pylori pathogenicity. M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system. This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme. M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase. It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences. Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori. The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD. Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI. In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition. 0.9981073 species cleaner0 2023-09-15T14:48:33Z MESH: Helicobacter pylori 0.9708807 taxonomy_domain cleaner0 2023-09-15T15:03:55Z DUMMY: Gram-negative bacterium 0.9986656 species cleaner0 2023-09-15T14:54:51Z MESH: human 0.9977002 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.9980526 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.9977798 taxonomy_domain cleaner0 2023-09-15T14:48:40Z DUMMY: bacteria 0.9855281 species cleaner0 2023-09-15T15:05:22Z MESH: H. pylori 26695 0.99871564 protein_type cleaner0 2023-09-18T09:24:53Z MESH: Methyltransferases 0.99808294 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori protein PR: cleaner0 2023-09-15T15:46:13Z M1.HpyAVI 0.9983508 protein_type cleaner0 2023-09-18T09:24:59Z MESH: DNA adenine MTase 0.9968161 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI protein PR: cleaner0 2023-09-15T15:45:45Z M2.HpyAVI 0.8533181 protein_type cleaner0 2023-09-18T09:25:16Z MESH: restriction enzyme protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.995255 gene cleaner0 2023-09-15T15:05:36Z GENE: hp0050 0.9986347 protein_type cleaner0 2023-09-18T08:31:18Z MESH: N6-adenine methyltransferase 0.998701 protein_type cleaner0 2023-09-15T15:05:42Z MESH: β-class MTase 0.97984064 chemical cleaner0 2023-09-15T15:07:36Z CHEBI: 5′-GAGG-3′, 0.9948631 chemical cleaner0 2023-09-15T15:07:34Z CHEBI: 5′-GGAG-3′ 0.997119 chemical cleaner0 2023-09-15T15:07:32Z CHEBI: 5′-GAAG-3′ 0.65228903 residue_name cleaner0 2023-09-15T15:07:27Z SO: adenines ptm MESH: cleaner0 2023-09-18T08:31:49Z methylation 0.64126253 residue_name cleaner0 2023-09-15T15:07:28Z SO: adenines 0.9987367 protein_type cleaner0 2023-09-15T15:05:54Z MESH: N6-adenine MTases ptm MESH: cleaner0 2023-09-18T08:31:49Z methylation 0.99674714 chemical cleaner0 2023-09-15T15:07:10Z CHEBI: 5′-GAAG-3′ protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.9982695 species cleaner0 2023-09-18T09:25:33Z MESH: H.pylori 0.9978259 chemical cleaner0 2023-09-15T15:07:08Z CHEBI: 5′-GAGG-3′ 0.8282734 evidence cleaner0 2023-09-15T15:07:00Z DUMMY: structure 0.9972285 evidence cleaner0 2023-09-18T09:28:07Z DUMMY: crystal structure protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.9966917 species cleaner0 2023-09-15T15:05:22Z MESH: H. pylori 26695 0.99867177 protein_type cleaner0 2023-09-18T09:25:21Z MESH: N6-adenine MTase 0.99710625 evidence cleaner0 2023-09-15T15:07:02Z DUMMY: structure 0.9983472 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.99784255 evidence cleaner0 2023-09-15T15:07:04Z DUMMY: structure structure_element SO: cleaner0 2023-09-18T08:28:01Z loop structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.9985078 experimental_method cleaner0 2023-09-15T15:06:45Z MESH: structural and biochemical analyses 0.99314356 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved 0.99952173 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 0.99875796 site cleaner0 2023-09-18T09:35:12Z SO: catalytic site 0.9995128 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 protein_type MESH: cleaner0 2023-09-18T08:31:07Z methyltransferase protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.99849313 protein_state cleaner0 2023-09-18T09:25:08Z DUMMY: non-conserved 0.999527 residue_name_number cleaner0 2023-09-15T14:50:16Z DUMMY: P41 RESULTS title_1 8207 RESULTS RESULTS title_2 8215 Overall structure 0.99722517 evidence cleaner0 2023-09-18T09:28:13Z DUMMY: structure RESULTS paragraph 8233 Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment. The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (>300 mM), which prompted that M1.HpyAVI is likely a halophilic protein. 0.99891526 protein_state cleaner0 2023-09-15T15:08:14Z DUMMY: full-length protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.9889261 species cleaner0 2023-09-15T15:08:26Z MESH: Escherichia coli 0.99712944 chemical cleaner0 2023-09-15T15:47:04Z CHEBI: sodium chloride protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI protein_state DUMMY: cleaner0 2023-09-15T15:08:57Z halophilic RESULTS paragraph 8575 The cofactor-free and AdoMet-bound proteins were crystallized at different conditions. Both structures were determined by means of molecular replacement, and refined to 3.0 Å and 3.1 Å, respectively. Statistics of X-ray data collection and structure refinement were summarized in Table 1. 0.99866265 protein_state cleaner0 2023-09-15T14:49:21Z DUMMY: cofactor-free 0.99892694 protein_state cleaner0 2023-09-15T14:49:26Z DUMMY: AdoMet-bound 0.9983777 experimental_method cleaner0 2023-09-15T15:09:11Z MESH: crystallized 0.99722373 evidence cleaner0 2023-09-18T09:28:17Z DUMMY: structures 0.9987483 experimental_method cleaner0 2023-09-15T15:09:13Z MESH: molecular replacement 0.9252138 experimental_method cleaner0 2023-09-15T15:09:15Z MESH: X-ray data collection 0.9931567 experimental_method cleaner0 2023-09-15T15:09:17Z MESH: structure refinement T1.xml T1 TABLE table_title_caption 8866 Data collection and structure refinement statistics of M1.HpyAVI 0.9951065 evidence cleaner0 2023-09-18T09:28:27Z DUMMY: structure refinement statistics 0.9775794 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI T1.xml T1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="box" rules="all"><thead><tr><th align="left" valign="middle" rowspan="1" colspan="1"/><th align="left" valign="middle" rowspan="1" colspan="1">M1.HpyAVI</th><th align="left" valign="middle" rowspan="1" colspan="1">M1.HpyAVI-AdoMet complex</th></tr></thead><tbody><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Data collection</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Wavelength (Å)</td><td align="left" valign="middle" rowspan="1" colspan="1">1.0000</td><td align="left" valign="middle" rowspan="1" colspan="1">0.97772</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Space group</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>P</italic>4₃2₁2</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>P</italic>6₅</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Unit-cell parameters (Å, ˚)</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>a</italic> = <italic>b</italic> = 69.73, <italic>c</italic> = 532.75<break/><italic>α = β = γ</italic> = 90</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>a = b</italic> = 135.60, <italic>c</italic> = 265.15<break/><italic>α = β</italic> = 90, <italic>γ</italic> = 120</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Resolution range (Å) <xref ref-type="table-fn" rid="tfn_001">^a</xref></td><td align="left" valign="middle" rowspan="1" colspan="1">49.09-3.00 (3.09-3.00)</td><td align="left" valign="middle" rowspan="1" colspan="1">48.91-3.10 (3.18-3.10)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Unique reflections <xref ref-type="table-fn" rid="tfn_001">^a</xref></td><td align="left" valign="middle" rowspan="1" colspan="1">27243</td><td align="left" valign="middle" rowspan="1" colspan="1">49833</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Multiplicity <xref ref-type="table-fn" rid="tfn_001">^a</xref></td><td align="left" valign="middle" rowspan="1" colspan="1">3.7 (3.8)</td><td align="left" valign="middle" rowspan="1" colspan="1">5.6 (4.0)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Completeness (%)<xref ref-type="table-fn" rid="tfn_001">^a</xref></td><td align="left" valign="middle" rowspan="1" colspan="1">98.7 (98.9)</td><td align="left" valign="middle" rowspan="1" colspan="1">99.7 (97.8)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Mean <italic>I/δ</italic> (<italic>I</italic>) <xref ref-type="table-fn" rid="tfn_001">^a</xref></td><td align="left" valign="middle" rowspan="1" colspan="1">12.1 (3.4)</td><td align="left" valign="middle" rowspan="1" colspan="1">14.0 (1.9)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Solvent content (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">58.67</td><td align="left" valign="middle" rowspan="1" colspan="1">61.96</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic>_merge <xref ref-type="table-fn" rid="tfn_001">^a</xref></td><td align="left" valign="middle" rowspan="1" colspan="1">0.073 (0.378)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.106 (0.769)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Structure refinement</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic>_work</td><td align="left" valign="middle" rowspan="1" colspan="1">0.251</td><td align="left" valign="middle" rowspan="1" colspan="1">0.221</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic>_free</td><td align="left" valign="middle" rowspan="1" colspan="1">0.308</td><td align="left" valign="middle" rowspan="1" colspan="1">0.276</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">R.m.s.d., bond lengths (Å)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.007</td><td align="left" valign="middle" rowspan="1" colspan="1">0.007</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">R.m.s.d., bond angles (˚)</td><td align="left" valign="middle" rowspan="1" colspan="1">1.408</td><td align="left" valign="middle" rowspan="1" colspan="1">1.651</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Ramachandran plot</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Favoured region (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">89.44</td><td align="left" valign="middle" rowspan="1" colspan="1">91.44</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Allowed region (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">9.58</td><td align="left" valign="middle" rowspan="1" colspan="1">7.11</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Outliers (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.99</td><td align="left" valign="middle" rowspan="1" colspan="1">1.45</td></tr></tbody></table> 8931 M1.HpyAVI M1.HpyAVI-AdoMet complex Data collection Wavelength (Å) 1.0000 0.97772 Space group P43212 P65 Unit-cell parameters (Å, ˚) a = b = 69.73, c = 532.75α = β = γ = 90 a = b = 135.60, c = 265.15α = β = 90, γ = 120 Resolution range (Å) a 49.09-3.00 (3.09-3.00) 48.91-3.10 (3.18-3.10) Unique reflections a 27243 49833 Multiplicity a 3.7 (3.8) 5.6 (4.0) Completeness (%)a 98.7 (98.9) 99.7 (97.8) Mean I/δ (I) a 12.1 (3.4) 14.0 (1.9) Solvent content (%) 58.67 61.96 Rmergea 0.073 (0.378) 0.106 (0.769) Structure refinement Rwork 0.251 0.221 Rfree 0.308 0.276 R.m.s.d., bond lengths (Å) 0.007 0.007 R.m.s.d., bond angles (˚) 1.408 1.651 Ramachandran plot Favoured region (%) 89.44 91.44 Allowed region (%) 9.58 7.11 Outliers (%) 0.99 1.45 protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI complex_assembly GO: cleaner0 2023-09-15T15:10:21Z M1.HpyAVI-AdoMet 0.9458426 evidence cleaner0 2023-09-18T09:28:30Z DUMMY: R.m.s.d 0.94231135 evidence cleaner0 2023-09-18T09:28:33Z DUMMY: R.m.s.d T1.xml T1 TABLE table_footnote 9745 Values in parentheses are statistics of the highest resolution shell. RESULTS paragraph 9815 Four and eight protein monomers resided in the asymmetric units of the two crystal structures. Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models. Details of invisible amino acids are given in Table S1. 0.99846953 oligomeric_state cleaner0 2023-09-15T15:10:43Z DUMMY: monomers 0.9979135 evidence cleaner0 2023-09-15T15:10:35Z DUMMY: crystal structures structure_element SO: cleaner0 2023-09-18T08:30:32Z loops 0.99736017 residue_range cleaner0 2023-09-15T15:10:46Z DUMMY: 32-61 0.9974658 residue_range cleaner0 2023-09-15T15:10:49Z DUMMY: 152-172 0.997647 evidence cleaner0 2023-09-15T15:10:39Z DUMMY: structures 0.9979949 evidence cleaner0 2023-09-15T15:10:37Z DUMMY: electron density RESULTS paragraph 10156 The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 Å on 191 Cα atoms. The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core. Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side. All these conserved structural motifs form a typical α/β Rossmann fold. The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity. The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition. The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII. The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA. This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence. 0.9968401 evidence cleaner0 2023-09-18T09:28:38Z DUMMY: structures 0.9982009 evidence cleaner0 2023-09-18T08:20:46Z DUMMY: RMSD protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.9979481 evidence cleaner0 2023-09-18T09:28:41Z DUMMY: structures 0.9984342 protein_type cleaner0 2023-09-15T14:49:44Z MESH: AdoMet-dependent MTase structure_element SO: cleaner0 2023-09-18T08:25:14Z β-sheet structure_element SO: cleaner0 2023-09-18T08:25:33Z α-helices 0.9966743 evidence cleaner0 2023-09-18T09:28:43Z DUMMY: structures 0.99891853 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases structure_element SO: cleaner0 2023-09-18T08:30:04Z helices structure_element SO: cleaner0 2023-09-18T08:24:59Z αA structure_element SO: cleaner0 2023-09-18T08:25:44Z αB structure_element SO: cleaner0 2023-09-18T08:25:55Z αZ structure_element SO: cleaner0 2023-09-18T08:25:14Z β-sheet structure_element SO: cleaner0 2023-09-18T08:24:28Z αD structure_element SO: cleaner0 2023-09-18T08:26:06Z αE structure_element SO: cleaner0 2023-09-18T08:26:17Z αC 0.99812 protein_state cleaner0 2023-09-15T15:25:40Z DUMMY: conserved structure_element SO: cleaner0 2023-09-18T08:27:27Z α/β Rossmann fold structure_element SO: cleaner0 2023-09-18T08:29:40Z catalytic motif structure_element SO: cleaner0 2023-09-18T08:27:09Z DPPY structure_element SO: cleaner0 2023-09-18T08:28:01Z loop structure_element SO: cleaner0 2023-09-18T08:24:28Z αD structure_element SO: cleaner0 2023-09-18T08:24:17Z β4 0.99921036 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.96160835 site cleaner0 2023-09-18T09:35:16Z SO: cavity structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.99757195 residue_range cleaner0 2023-09-15T15:26:15Z DUMMY: 136-166 structure_element SO: cleaner0 2023-09-18T08:28:43Z β7 structure_element SO: cleaner0 2023-09-18T08:25:55Z αZ 0.9987655 protein_state cleaner0 2023-09-18T09:30:35Z DUMMY: highly diverse 0.9991721 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases chemical CHEBI: cleaner0 2023-09-15T15:28:47Z DNA structure_element SO: cleaner0 2023-09-18T08:29:16Z hairpin loop 0.9974844 residue_range cleaner0 2023-09-15T15:26:17Z DUMMY: 101-133 structure_element SO: cleaner0 2023-09-18T08:28:56Z β6 structure_element SO: cleaner0 2023-09-18T08:28:43Z β7 0.9946524 chemical cleaner0 2023-09-18T09:36:48Z CHEBI: DNA structure_element SO: cleaner0 2023-09-18T08:27:47Z minor groove protein PR: cleaner0 2023-09-15T15:45:05Z M.MboIIA protein PR: cleaner0 2023-09-15T15:45:22Z M.RsrI protein PR: cleaner0 2023-09-15T15:47:39Z M.pvuII 0.7994238 protein_state cleaner0 2023-09-18T09:30:40Z DUMMY: missing structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.99738026 residue_range cleaner0 2023-09-15T15:26:20Z DUMMY: 33-58 0.9981224 evidence cleaner0 2023-09-18T09:28:48Z DUMMY: structure protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI structure_element SO: cleaner0 2023-09-18T08:28:26Z loop I protein PR: cleaner0 2023-09-15T15:44:50Z M.TaqI 0.99823296 evidence cleaner0 2023-09-18T09:29:16Z DUMMY: structure protein_state DUMMY: cleaner0 2023-09-15T15:26:42Z without DNA structure_element SO: cleaner0 2023-09-18T08:28:01Z loop protein_state DUMMY: cleaner0 2023-09-18T09:29:06Z well ordered evidence DUMMY: cleaner0 2023-09-15T15:48:16Z M.TaqI-DNA complex structure ptm MESH: cleaner0 2023-09-15T14:50:29Z DNA methylation 0.65298414 residue_name cleaner0 2023-09-18T10:02:22Z SO: adenine chemical CHEBI: cleaner0 2023-09-15T15:28:58Z DNA oncotarget-07-40965-g001.jpg F1 FIG fig_title_caption 11634 Overall structure of M1.HpyAVI 0.9969964 evidence cleaner0 2023-09-18T09:29:21Z DUMMY: structure protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI oncotarget-07-40965-g001.jpg F1 FIG fig_caption 11665 A. Free form B. AdoMet-bound form. Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY. Rainbow coloring from blue through green to red indicates the N- to C-terminal position of the residues in the model. The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases. The AdoMet molecule is shown in green. 0.99928844 protein_state cleaner0 2023-09-15T15:30:17Z DUMMY: Free 0.9989874 protein_state cleaner0 2023-09-15T14:49:26Z DUMMY: AdoMet-bound protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.99276835 protein_type cleaner0 2023-09-15T14:49:44Z MESH: AdoMet-dependent MTase structure_element SO: cleaner0 2023-09-18T08:25:13Z β-sheet structure_element SO: cleaner0 2023-09-18T08:25:33Z α-helices structure_element SO: cleaner0 2023-09-18T08:24:59Z αA structure_element SO: cleaner0 2023-09-18T08:25:43Z αB structure_element SO: cleaner0 2023-09-18T08:25:54Z αZ structure_element SO: cleaner0 2023-09-18T08:24:28Z αD structure_element SO: cleaner0 2023-09-18T08:26:06Z αE structure_element SO: cleaner0 2023-09-18T08:26:16Z αC 0.9992446 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.99239594 protein_state cleaner0 2023-09-15T15:31:27Z DUMMY: bound in 0.9498381 site cleaner0 2023-09-15T15:31:30Z SO: cavity 0.9990746 protein_state cleaner0 2023-09-15T15:31:32Z DUMMY: conserved structure_element SO: cleaner0 2023-09-18T08:27:08Z DPPY structure_element SO: cleaner0 2023-09-18T08:25:33Z α-helices structure_element SO: cleaner0 2023-09-18T08:26:57Z β-strands 0.99922407 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases 0.9991905 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet RESULTS title_2 12286 Dimeric state of M1.HpyAVI in crystal and solution 0.9984648 oligomeric_state cleaner0 2023-09-15T15:50:01Z DUMMY: Dimeric protein PR: cleaner0 2023-09-15T15:44:17Z M1.HpyAVI 0.9944754 evidence cleaner0 2023-09-15T15:48:43Z DUMMY: crystal 0.8232841 experimental_method cleaner0 2023-09-18T10:03:07Z MESH: solution RESULTS paragraph 12337 Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state. Increasing number of dimeric DNA MTases, however, has been identified from later studies. For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution. In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures. Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding. 0.99734926 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases protein PR: cleaner0 2023-09-15T15:49:01Z M.BamHI protein PR: cleaner0 2023-09-15T15:49:16Z M.EcoRI 0.99874026 oligomeric_state cleaner0 2023-09-15T15:49:20Z DUMMY: monomer 0.82037216 chemical cleaner0 2023-09-18T09:36:54Z CHEBI: DNA 0.9990522 protein_type cleaner0 2023-09-18T09:25:47Z MESH: MTase 0.9989624 protein_state cleaner0 2023-09-18T08:41:12Z DUMMY: hemimethylated ptm MESH: cleaner0 2023-09-18T08:31:49Z methylation 0.99889743 protein_state cleaner0 2023-09-18T08:41:17Z DUMMY: fully methylated 0.9988433 oligomeric_state cleaner0 2023-09-15T15:50:01Z DUMMY: dimeric 0.9972397 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.9688514 protein cleaner0 2023-09-15T15:49:40Z PR: M.DpnII 0.90760726 protein cleaner0 2023-09-15T15:45:22Z PR: M.RsrI 0.94044906 protein cleaner0 2023-09-15T15:49:49Z PR: M.KpnI 0.99051577 protein cleaner0 2023-09-15T15:45:05Z PR: M.MboIIA 0.9987752 oligomeric_state cleaner0 2023-09-15T15:49:57Z DUMMY: dimers 0.9991013 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases 0.98035747 protein cleaner0 2023-09-15T15:45:05Z PR: M.MboIIA 0.88078374 protein cleaner0 2023-09-15T15:45:22Z PR: M.RsrI 0.9984231 protein cleaner0 2023-09-18T09:27:51Z PR: TTH0409 0.998779 oligomeric_state cleaner0 2023-09-15T15:49:57Z DUMMY: dimers 0.9989133 evidence cleaner0 2023-09-15T15:50:19Z DUMMY: crystal structures 0.9965406 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.9439273 protein cleaner0 2023-09-15T15:50:11Z PR: M.CcrMI 0.99796015 species cleaner0 2023-09-15T15:50:16Z MESH: Bacillus amyloliquefaciens 0.9990553 protein_type cleaner0 2023-09-18T09:25:59Z MESH: MTase 0.9988293 oligomeric_state cleaner0 2023-09-15T15:49:53Z DUMMY: dimer 0.9987846 oligomeric_state cleaner0 2023-09-15T15:49:20Z DUMMY: monomer 0.854749 chemical cleaner0 2023-09-18T09:36:57Z CHEBI: DNA RESULTS paragraph 13063 According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures. Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein. In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C). An area of ~1900 Å2 was buried at the dimeric interface, taking up ca 17% of the total area. The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96. In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3). protein_state DUMMY: cleaner0 2023-09-18T09:00:14Z conserved 0.6177626 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI protein_type MESH: cleaner0 2023-09-15T15:50:56Z β-subgroup 0.99775726 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved structure_element SO: cleaner0 2023-09-18T08:26:35Z NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A 0.99900466 site cleaner0 2023-09-18T09:25:55Z SO: dimerization interface 0.99882424 evidence cleaner0 2023-09-18T09:29:28Z DUMMY: crystal structures 0.9157514 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved 0.6214757 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.8981352 oligomeric_state cleaner0 2023-09-18T08:42:08Z DUMMY: dimerization 0.99884087 oligomeric_state cleaner0 2023-09-15T15:49:54Z DUMMY: dimer protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99886215 evidence cleaner0 2023-09-18T09:29:32Z DUMMY: crystal structures 0.9988851 oligomeric_state cleaner0 2023-09-15T15:10:43Z DUMMY: monomers 0.99883354 site cleaner0 2023-09-15T15:51:39Z SO: dimeric interface 0.99888295 oligomeric_state cleaner0 2023-09-15T15:50:01Z DUMMY: dimeric 0.99459386 bond_interaction cleaner0 2023-09-18T08:41:48Z MESH: hydrogen bonds 0.9927003 bond_interaction cleaner0 2023-09-18T08:41:53Z MESH: salt bridges 0.9994772 residue_name_number cleaner0 2023-09-18T09:35:50Z DUMMY: R86 0.99945253 residue_name_number cleaner0 2023-09-18T09:35:55Z DUMMY: D93 0.999468 residue_name_number cleaner0 2023-09-18T09:35:59Z DUMMY: E96 0.99881965 oligomeric_state cleaner0 2023-09-15T15:49:54Z DUMMY: dimer 0.99808276 evidence cleaner0 2023-09-18T09:29:35Z DUMMY: structure protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99878925 protein_type cleaner0 2023-09-15T15:51:20Z MESH: β-class MTases 0.5326209 protein cleaner0 2023-09-15T15:51:28Z PR: M1.MboIIA 0.827632 protein cleaner0 2023-09-15T15:45:22Z PR: M.RsrI 0.9987478 protein cleaner0 2023-09-18T08:21:05Z PR: TTHA0409 0.5131915 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9987937 oligomeric_state cleaner0 2023-09-15T15:49:54Z DUMMY: dimer oncotarget-07-40965-g002.jpg F2 FIG fig_title_caption 14040 M1.HpyAVI exists as dimer in crystal and solution protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.9986904 oligomeric_state cleaner0 2023-09-15T15:49:54Z DUMMY: dimer 0.99385875 evidence cleaner0 2023-09-18T09:29:40Z DUMMY: crystal oncotarget-07-40965-g002.jpg F2 FIG fig_caption 14090 A. A conserved interface area of β-class MTases is defined in M1.HpyAVI. Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta. D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution. FPLC system coupled to a Superdex 75 10/300 column. Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein. 0.9954254 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved 0.9813417 site cleaner0 2023-09-18T09:35:21Z SO: interface area 0.9987238 protein_type cleaner0 2023-09-15T15:52:31Z MESH: β-class MTases 0.944242 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.6439788 oligomeric_state cleaner0 2023-09-18T08:42:07Z DUMMY: Dimerization 0.9992505 protein_state cleaner0 2023-09-18T09:30:50Z DUMMY: free 0.9820862 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.99895936 protein_state cleaner0 2023-09-18T09:31:01Z DUMMY: cofactor-bound 0.9916945 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.99879223 oligomeric_state cleaner0 2023-09-15T15:10:43Z DUMMY: monomers 0.99919957 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.9989366 experimental_method cleaner0 2023-09-18T08:42:13Z MESH: Gel-filtration analysis 0.950566 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9988373 oligomeric_state cleaner0 2023-09-15T15:49:54Z DUMMY: dimer 0.9948841 experimental_method cleaner0 2023-09-18T08:42:17Z MESH: FPLC 0.8600793 evidence cleaner0 2023-09-18T08:43:07Z DUMMY: Elution profiles 0.9417641 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI RESULTS paragraph 14635 To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column. The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D). 0.82748175 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9987332 oligomeric_state cleaner0 2023-09-15T15:50:01Z DUMMY: dimeric evidence DUMMY: cleaner0 2023-09-18T08:42:55Z molecular mass RESULTS paragraph 14919 Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution. This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis. These treatments probably changed protein conformation somehow and also the oligomeric state. 0.62250036 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9988067 oligomeric_state cleaner0 2023-09-15T15:49:54Z DUMMY: dimer 0.99615896 evidence cleaner0 2023-09-18T08:43:10Z DUMMY: crystal 0.9988349 protein_type cleaner0 2023-09-18T09:26:04Z MESH: β-class MTases 0.99878055 experimental_method cleaner0 2023-09-18T08:43:14Z MESH: dynamic light scattering 0.99791723 experimental_method cleaner0 2023-09-18T08:43:18Z MESH: DLS 0.9989257 experimental_method cleaner0 2023-09-18T10:03:15Z MESH: gel-filtration chromatography 0.5806133 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9988205 oligomeric_state cleaner0 2023-09-18T08:43:31Z DUMMY: monomeric 0.7840338 chemical cleaner0 2023-09-18T09:37:03Z CHEBI: arginine 0.79573244 chemical cleaner0 2023-09-18T08:44:00Z CHEBI: arginine 0.98972815 chemical cleaner0 2023-09-18T09:37:06Z CHEBI: glycerol RESULTS title_2 15513 Structure comparisons 0.9956933 experimental_method cleaner0 2023-09-18T08:45:16Z MESH: Structure comparisons RESULTS paragraph 15535 As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup. Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively. The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures. By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures. Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected. Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible. 0.99873894 protein_type cleaner0 2023-09-18T09:26:08Z MESH: β-class N6 adenine MTase 0.9134107 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.99740237 evidence cleaner0 2023-09-18T09:29:44Z DUMMY: structure 0.9921482 protein cleaner0 2023-09-15T15:45:05Z PR: M.MboIIA 0.99176246 protein cleaner0 2023-09-15T15:45:22Z PR: M.RsrI 0.99879396 experimental_method cleaner0 2023-09-18T08:45:12Z MESH: Superimposition 0.8726051 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9975291 evidence cleaner0 2023-09-18T08:45:56Z DUMMY: RMSDs structure_element SO: cleaner0 2023-09-18T08:23:58Z TRD 0.9977891 residue_range cleaner0 2023-09-18T10:05:30Z DUMMY: 133-163 0.93727803 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI structure_element SO: cleaner0 2023-09-18T08:25:33Z α-helices structure_element SO: cleaner0 2023-09-18T08:26:58Z β-strands structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.9748257 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI protein_state DUMMY: cleaner0 2023-09-18T09:35:00Z lacking structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.9817461 chemical cleaner0 2023-09-18T09:37:11Z CHEBI: DNA structure_element SO: cleaner0 2023-09-18T08:44:36Z major groove 0.69469416 chemical cleaner0 2023-09-18T09:37:15Z CHEBI: DNA 0.9986234 protein_state cleaner0 2023-09-18T09:00:19Z DUMMY: highly flexible structure_element SO: cleaner0 2023-09-18T08:28:01Z loop structure_element SO: cleaner0 2023-09-18T08:24:17Z β4 structure_element SO: cleaner0 2023-09-18T08:24:28Z αD 0.9978159 residue_range cleaner0 2023-09-18T10:05:34Z DUMMY: 33-58 0.95462805 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9977138 evidence cleaner0 2023-09-18T09:29:48Z DUMMY: structures 0.99794513 evidence cleaner0 2023-09-18T09:29:51Z DUMMY: structures 0.9923938 protein cleaner0 2023-09-15T15:45:05Z PR: M.MboIIA 0.9922559 protein cleaner0 2023-09-15T15:45:22Z PR: M.RsrI 0.9986303 experimental_method cleaner0 2023-09-18T08:45:06Z MESH: Sequence alignment 0.9674974 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.99730754 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.48058376 protein_state cleaner0 2023-09-18T09:31:09Z DUMMY: flexible oncotarget-07-40965-g003.jpg F3 FIG fig_title_caption 16816 Structural comparisons between M1.HpyAVI and other DNA MTases 0.954988 experimental_method cleaner0 2023-09-18T08:46:09Z MESH: Structural comparisons protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.998433 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases oncotarget-07-40965-g003.jpg F3 FIG fig_caption 16878 A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange. protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI protein PR: cleaner0 2023-09-15T15:45:05Z M.MboIIA protein PR: cleaner0 2023-09-15T15:45:22Z M.RsrI 0.9991122 protein cleaner0 2023-09-18T08:21:05Z PR: TTHA0409 0.9992655 protein cleaner0 2023-09-18T08:21:39Z PR: DpnM protein PR: cleaner0 2023-09-15T15:01:22Z M.TaqI protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI protein_state DUMMY: cleaner0 2023-09-18T09:32:38Z long disorder structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.9337187 protein_state cleaner0 2023-09-18T09:31:23Z DUMMY: structure-rich structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.9921058 protein cleaner0 2023-09-15T15:45:05Z PR: M.MboIIA protein PR: cleaner0 2023-09-15T15:45:22Z M.RsrI 0.99877506 protein cleaner0 2023-09-18T08:21:05Z PR: TTHA0409 structure_element SO: cleaner0 2023-09-18T08:46:25Z DNA-binding domain 0.99922466 protein cleaner0 2023-09-18T08:21:39Z PR: DpnM 0.9894016 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.97637516 protein cleaner0 2023-09-15T15:45:05Z PR: M.MboIIA 0.84092015 protein cleaner0 2023-09-15T15:45:22Z PR: M.RsrI 0.9985129 protein cleaner0 2023-09-18T08:21:05Z PR: TTHA0409 structure_element SO: cleaner0 2023-09-18T08:24:17Z β4 structure_element SO: cleaner0 2023-09-18T08:24:28Z αD 0.99199146 protein cleaner0 2023-09-15T15:45:05Z PR: M.MboIIA 0.98690724 protein cleaner0 2023-09-15T15:45:22Z PR: M.RsrI structure_element SO: cleaner0 2023-09-18T08:46:25Z DNA-binding domain 0.9993131 protein cleaner0 2023-09-18T08:21:39Z PR: DpnM structure_element SO: cleaner0 2023-09-18T08:36:35Z C-terminal domain 0.99063236 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI RESULTS paragraph 17375 N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D). Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1). protein_type MESH: cleaner0 2023-09-18T08:23:01Z N6-adenine and N4-cytosine MTases 0.9988351 experimental_method cleaner0 2023-09-18T10:03:21Z MESH: Structural comparison 0.9828641 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.99869984 protein_type cleaner0 2023-09-18T08:20:39Z MESH: β-class N4 cytosine MTase 0.9993332 protein cleaner0 2023-09-18T08:21:04Z PR: TTHA0409 0.998705 evidence cleaner0 2023-09-18T08:20:45Z DUMMY: RMSD structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.9986004 evidence cleaner0 2023-09-18T08:20:49Z DUMMY: structures structure_element SO: cleaner0 2023-09-18T08:25:33Z α-helices RESULTS paragraph 17863 M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM). Both comparisons gave RMSDs above 3.0 Å (Figure 3E and 3F). These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition. 0.71178216 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9987031 protein_type cleaner0 2023-09-15T15:05:55Z MESH: N6-adenine MTases protein_type MESH: cleaner0 2023-09-18T08:21:34Z α-class 0.99924123 protein cleaner0 2023-09-18T08:21:39Z PR: DpnM protein_type MESH: cleaner0 2023-09-18T08:21:58Z γ-class 0.992579 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI 0.9981804 evidence cleaner0 2023-09-18T08:45:56Z DUMMY: RMSDs 0.9785749 protein_state cleaner0 2023-09-18T09:32:42Z DUMMY: lack structure_element SO: cleaner0 2023-09-18T08:22:41Z counterpart loop structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.9628337 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.60606277 chemical cleaner0 2023-09-18T09:37:19Z CHEBI: DNA RESULTS paragraph 18281 Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups. This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme. 0.9905677 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.8527761 protein_state cleaner0 2023-09-18T09:32:19Z DUMMY: long disordered structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.84751034 protein_state cleaner0 2023-09-18T09:32:57Z DUMMY: secondary structure-rich structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD protein_type MESH: cleaner0 2023-09-18T08:23:19Z β-class N6 adenine or N4 cytosine MTases 0.99855304 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.99791336 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori RESULTS title_2 18646 AdoMet-binding pocket 0.99897456 site cleaner0 2023-09-18T08:47:32Z SO: AdoMet-binding pocket RESULTS paragraph 18668 The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases. A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures. Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose. In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet. Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B). The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure. 0.9991107 site cleaner0 2023-09-18T09:35:26Z SO: cofactor binding pocket 0.9654598 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9948056 residue_range cleaner0 2023-09-18T10:06:12Z DUMMY: 7-9 0.9951399 residue_range cleaner0 2023-09-18T10:06:15Z DUMMY: 29-31 0.9965372 residue_range cleaner0 2023-09-18T10:06:17Z DUMMY: 165-167 0.99617857 residue_range cleaner0 2023-09-18T10:06:20Z DUMMY: 216-218 0.9353764 residue_number cleaner0 2023-09-18T10:07:00Z DUMMY: 221 0.99897015 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved 0.9982679 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.9966004 bond_interaction cleaner0 2023-09-18T08:48:01Z MESH: hydrogen bond 0.99955183 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 structure_element SO: cleaner0 2023-09-18T08:29:41Z catalytic motif structure_element SO: cleaner0 2023-09-18T08:27:09Z DPPY 0.83229464 protein_state cleaner0 2023-09-18T09:33:02Z DUMMY: bound 0.99907744 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.99916065 protein_type cleaner0 2023-09-18T09:26:15Z MESH: MTase 0.9978751 evidence cleaner0 2023-09-18T09:26:20Z DUMMY: structures 0.9995264 residue_name_number cleaner0 2023-09-18T08:48:33Z DUMMY: D8 0.99946505 residue_name_number cleaner0 2023-09-18T08:48:38Z DUMMY: A9 0.99714154 bond_interaction cleaner0 2023-09-18T08:47:57Z MESH: hydrogen-bonds 0.99749845 chemical cleaner0 2023-09-18T09:37:32Z CHEBI: purine 0.99953866 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 0.997235 bond_interaction cleaner0 2023-09-18T08:47:51Z MESH: hydrogen bonding 0.9980957 chemical cleaner0 2023-09-18T09:37:35Z CHEBI: ribose 0.9994992 residue_name_number cleaner0 2023-09-18T08:48:43Z DUMMY: H168 0.99952984 residue_name_number cleaner0 2023-09-18T08:48:47Z DUMMY: T200 0.9995173 residue_name_number cleaner0 2023-09-18T08:48:51Z DUMMY: S198 0.99901843 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.99861515 experimental_method cleaner0 2023-09-18T08:50:14Z MESH: Superposition 0.95827633 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9965333 evidence cleaner0 2023-09-18T09:26:22Z DUMMY: structures 0.53536797 protein_state cleaner0 2023-09-18T09:26:30Z DUMMY: rather conserved 0.9970794 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI protein_state DUMMY: cleaner0 2023-09-18T09:26:54Z bound 0.9969217 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI 0.9959515 protein_state cleaner0 2023-09-18T09:26:34Z DUMMY: absence of 0.99926144 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved chemical CHEBI: cleaner0 2023-09-15T14:50:49Z AdoMet structure_element SO: cleaner0 2023-09-18T08:48:19Z FXGXG 0.9978817 evidence cleaner0 2023-09-18T09:29:55Z DUMMY: structure oncotarget-07-40965-g004.jpg F4 FIG fig_title_caption 19568 Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding 0.9989014 experimental_method cleaner0 2023-09-18T10:03:29Z MESH: Structural and biochemical analyses 0.8775193 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved 0.99950695 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 0.9995067 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 0.9987552 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet oncotarget-07-40965-g004.jpg F4 FIG fig_caption 19686 A. The cofactor-binding cavity of M1.HpyAVI. Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked. B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange). The AdoMet terminal carboxyl of M.TaqI reveals different orientations. C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST). The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet. The bound fraction is shown on the y-axis against the protein concentration. AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM). The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated. Measurements were made with 40% LED and 40% laser power at 25°C. D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay. [3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background. E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta). Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4). 0.9989493 site cleaner0 2023-09-18T09:35:31Z SO: cofactor-binding cavity protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99707735 bond_interaction cleaner0 2023-09-18T08:41:49Z MESH: hydrogen bonds 0.99910104 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.9961052 bond_interaction cleaner0 2023-09-18T08:48:02Z MESH: hydrogen bond 0.9977174 experimental_method cleaner0 2023-09-18T08:50:13Z MESH: Superposition 0.9991123 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.9963967 evidence cleaner0 2023-09-18T09:30:00Z DUMMY: structures protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99905735 protein cleaner0 2023-09-18T08:21:39Z PR: DpnM 0.9953942 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI 0.99904925 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.9956592 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI 0.99713755 evidence cleaner0 2023-09-18T08:54:33Z DUMMY: Cofactor binding affinity 0.99916446 protein_state cleaner0 2023-09-18T08:49:40Z DUMMY: wt 0.9976549 protein_state cleaner0 2023-09-18T08:49:45Z DUMMY: mutants protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99877167 experimental_method cleaner0 2023-09-18T08:50:04Z MESH: microscale thermophoresis 0.998623 experimental_method cleaner0 2023-09-18T08:50:08Z MESH: MST 0.9986141 evidence cleaner0 2023-09-18T08:54:37Z DUMMY: binding affinity protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.8690669 protein_state cleaner0 2023-09-18T09:33:12Z DUMMY: unlabeled 0.9990721 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.9992286 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.9387604 experimental_method cleaner0 2023-09-18T08:50:19Z MESH: titrated protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99916697 protein_state cleaner0 2023-09-18T08:49:41Z DUMMY: wt 0.99836916 protein_state cleaner0 2023-09-18T08:49:50Z DUMMY: mutant 0.99854326 evidence cleaner0 2023-09-18T08:54:41Z DUMMY: dissociation constant 0.99862623 evidence cleaner0 2023-09-18T08:54:44Z DUMMY: KD 0.9983845 evidence cleaner0 2023-09-18T09:30:15Z DUMMY: isotherm protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99194276 protein_state cleaner0 2023-09-18T08:49:41Z DUMMY: wt mutant MESH: cleaner0 2023-09-18T08:52:11Z M1.HpyAVI-D8A mutant MESH: cleaner0 2023-09-18T08:52:32Z M1.HpyAVI-D29A mutant MESH: cleaner0 2023-09-18T08:52:53Z M1.HpyAVI-H168A mutant MESH: cleaner0 2023-09-18T08:51:07Z M1.HpyAVI-S198A mutant MESH: cleaner0 2023-09-18T08:51:26Z M1.HpyAVI-T200A mutant MESH: cleaner0 2023-09-18T08:53:13Z M1.HpyAVI-E216A 0.9817704 protein_type cleaner0 2023-09-18T08:53:26Z MESH: DNA methyltransferase 0.9427799 protein_state cleaner0 2023-09-18T09:33:25Z DUMMY: wide type 0.87695163 protein_state cleaner0 2023-09-18T08:49:46Z DUMMY: mutants 0.9975476 experimental_method cleaner0 2023-09-18T08:53:49Z MESH: radioactive assay chemical CHEBI: cleaner0 2023-09-18T08:54:19Z [3H]-methyl 0.78140366 chemical cleaner0 2023-09-18T09:37:41Z CHEBI: DNA 0.9070639 chemical cleaner0 2023-09-18T09:37:45Z CHEBI: 5′-GAGG-3′ 0.99856466 experimental_method cleaner0 2023-09-18T08:50:14Z MESH: Superposition protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99369144 protein cleaner0 2023-09-15T15:45:05Z PR: M.MboIIA 0.9863444 protein cleaner0 2023-09-15T15:45:23Z PR: M.RsrI 0.99943274 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 0.9994393 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 0.9114434 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved 0.9983665 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases RESULTS paragraph 21373 To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay. As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all. 0.69778824 experimental_method cleaner0 2023-09-18T08:55:21Z MESH: single mutants 0.99535275 experimental_method cleaner0 2023-09-18T10:03:36Z MESH: replacing 0.99935883 residue_name_number cleaner0 2023-09-18T08:48:34Z DUMMY: D8 0.9994481 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 0.9994443 residue_name_number cleaner0 2023-09-18T08:48:43Z DUMMY: H168 0.99945134 residue_name_number cleaner0 2023-09-18T08:48:51Z DUMMY: S198 0.9994473 residue_name_number cleaner0 2023-09-18T08:48:47Z DUMMY: T200 0.99935776 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 0.9922931 residue_name cleaner0 2023-09-18T08:59:05Z SO: alanine 0.9984741 evidence cleaner0 2023-09-18T09:30:20Z DUMMY: ligand binding affinity 0.9987993 experimental_method cleaner0 2023-09-18T08:50:05Z MESH: microscale thermophoresis 0.9986884 experimental_method cleaner0 2023-09-18T08:50:09Z MESH: MST 0.99891734 protein_state cleaner0 2023-09-18T08:55:07Z DUMMY: wild type 0.8193448 protein_state cleaner0 2023-09-18T08:49:46Z DUMMY: mutants 0.9980161 evidence cleaner0 2023-09-18T08:54:45Z DUMMY: KD 0.9979571 mutant cleaner0 2023-09-18T08:55:40Z MESH: D29A 0.99816173 mutant cleaner0 2023-09-18T08:55:42Z MESH: E216A 0.95574015 protein_state cleaner0 2023-09-18T08:49:46Z DUMMY: mutants 0.8913477 evidence cleaner0 2023-09-18T08:55:36Z DUMMY: protein-AdoMet affinity RESULTS paragraph 21808 The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding. Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI. The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E). E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet. Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction. To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants. As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement. The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity. 0.99543846 bond_interaction cleaner0 2023-09-18T08:41:49Z MESH: hydrogen bonds 0.99947375 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 0.9994604 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 0.9984509 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.99554175 experimental_method cleaner0 2023-09-18T08:55:56Z MESH: Mutation chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99947006 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 0.93916696 site cleaner0 2023-09-18T08:56:00Z SO: catalytic active site 0.99586785 structure_element cleaner0 2023-09-18T08:27:09Z SO: DPPY 0.9994399 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 0.99761105 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved chemical CHEBI: cleaner0 2023-09-18T08:35:41Z amino acid 0.998909 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases 0.9994332 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 structure_element SO: cleaner0 2023-09-18T08:56:11Z β2 0.51602983 chemical cleaner0 2023-09-18T09:37:48Z CHEBI: ribose 0.9988117 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.99830014 experimental_method cleaner0 2023-09-18T10:03:41Z MESH: Replacement 0.9955804 residue_name cleaner0 2023-09-18T08:59:05Z SO: alanine 0.9938001 bond_interaction cleaner0 2023-09-18T08:41:49Z MESH: hydrogen bonds 0.9766973 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl experimental_method MESH: cleaner0 2023-09-18T10:04:00Z [3H]AdoMet radiological assay chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.87604487 protein_state cleaner0 2023-09-18T08:49:46Z DUMMY: mutants 0.9986948 experimental_method cleaner0 2023-09-18T10:04:05Z MESH: radiological assay 0.9986708 experimental_method cleaner0 2023-09-18T08:50:09Z MESH: MST 0.998423 mutant cleaner0 2023-09-18T08:57:02Z MESH: D29A 0.99846244 mutant cleaner0 2023-09-18T08:57:03Z MESH: E216A 0.98877406 protein_state cleaner0 2023-09-18T08:49:46Z DUMMY: mutants chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl protein_state DUMMY: cleaner0 2023-09-18T08:49:46Z mutants 0.6297416 protein_type cleaner0 2023-09-18T08:31:07Z MESH: methyltransferase RESULTS paragraph 22847 As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases. We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation. We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay. We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B). G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket. Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity. Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding. The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C). In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding. structure_element SO: cleaner0 2023-09-18T08:59:23Z FXGXG 0.99913895 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved chemical CHEBI: cleaner0 2023-09-15T14:50:49Z AdoMet 0.99815035 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.7042762 protein_state cleaner0 2023-09-18T08:49:46Z DUMMY: mutants 0.73182875 structure_element cleaner0 2023-09-18T10:07:45Z SO: FMGSG 0.9881989 residue_name cleaner0 2023-09-18T08:59:05Z SO: alanine chemical CHEBI: cleaner0 2023-09-18T08:35:41Z amino acid 0.9988029 mutant cleaner0 2023-09-18T10:07:56Z MESH: F195A 0.99854386 protein_state cleaner0 2023-09-18T08:49:50Z DUMMY: mutant 0.99794036 evidence cleaner0 2023-09-18T08:58:37Z DUMMY: ligand binding affinity chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl protein_state DUMMY: cleaner0 2023-09-18T08:49:46Z mutants 0.9985948 experimental_method cleaner0 2023-09-18T08:50:09Z MESH: MST 0.91924936 experimental_method cleaner0 2023-09-18T10:04:32Z MESH: radiological assay 0.99950063 residue_name_number cleaner0 2023-09-18T08:58:29Z DUMMY: G197 0.9926398 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.998075 experimental_method cleaner0 2023-09-18T08:58:52Z MESH: mutagenesis 0.9994849 residue_name_number cleaner0 2023-09-18T09:36:05Z DUMMY: M196 0.99947757 residue_name_number cleaner0 2023-09-18T09:36:10Z DUMMY: G199 0.99952686 residue_name_number cleaner0 2023-09-18T08:58:29Z DUMMY: G197 0.99806124 protein_state cleaner0 2023-09-18T09:00:14Z DUMMY: conserved 0.9982574 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.9896696 experimental_method cleaner0 2023-09-18T10:04:41Z MESH: replacing 0.99589795 residue_name cleaner0 2023-09-18T08:59:04Z SO: alanine 0.9990523 site cleaner0 2023-09-18T08:58:39Z SO: cofactor-binding pocket 0.9981822 experimental_method cleaner0 2023-09-18T08:58:53Z MESH: Mutagenesis 0.9973271 residue_name cleaner0 2023-09-18T08:59:09Z SO: glycine 0.9975895 protein cleaner0 2023-09-18T08:57:53Z PR: M.EcoKI 0.991685 protein cleaner0 2023-09-18T08:58:06Z PR: M.EcoP15I 0.52265966 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.99767673 experimental_method cleaner0 2023-09-18T10:04:55Z MESH: mutational study 0.999522 residue_name_number cleaner0 2023-09-18T08:58:33Z DUMMY: F195 0.99891686 mutant cleaner0 2023-09-18T10:08:03Z MESH: F195A 0.9988581 protein_state cleaner0 2023-09-18T08:49:50Z DUMMY: mutant 0.99871886 experimental_method cleaner0 2023-09-18T10:05:01Z MESH: structural analysis 0.995095 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.9995129 residue_name_number cleaner0 2023-09-18T08:58:34Z DUMMY: F195 0.99672353 bond_interaction cleaner0 2023-09-18T10:06:54Z MESH: π-stacking interaction 0.99898654 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.9984652 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet 0.8519888 protein_state cleaner0 2023-09-18T08:58:44Z DUMMY: bound in 0.99786323 site cleaner0 2023-09-18T08:58:41Z SO: pocket protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.9978897 experimental_method cleaner0 2023-09-18T08:58:52Z MESH: mutagenesis 0.9979746 protein cleaner0 2023-09-18T08:58:16Z PR: M.EcoRV 0.9917243 chemical cleaner0 2023-09-15T14:50:49Z CHEBI: AdoMet RESULTS title_2 24211 Potential DNA-binding sites 0.99896115 site cleaner0 2023-09-18T09:35:35Z SO: DNA-binding sites RESULTS paragraph 24239 The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58). The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA. As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD. 0.9988854 site cleaner0 2023-09-18T09:35:39Z SO: DNA binding region 0.8993177 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI structure_element SO: cleaner0 2023-09-18T08:29:18Z hairpin loop 0.997762 residue_range cleaner0 2023-09-18T10:06:30Z DUMMY: 101-133 structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.997704 residue_range cleaner0 2023-09-18T10:06:32Z DUMMY: 136-166 0.99897075 protein_state cleaner0 2023-09-18T09:00:19Z DUMMY: highly flexible structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.9976094 residue_range cleaner0 2023-09-18T10:06:36Z DUMMY: 33-58 structure_element SO: cleaner0 2023-09-18T08:29:18Z hairpin loop structure_element SO: cleaner0 2023-09-18T08:28:57Z β6 structure_element SO: cleaner0 2023-09-18T08:28:43Z β7 0.9982666 protein_state cleaner0 2023-09-18T09:00:13Z DUMMY: conserved structure_element SO: cleaner0 2023-09-18T08:59:48Z HRRY 0.8395535 structure_element cleaner0 2023-09-18T08:27:48Z SO: minor groove 0.9990257 protein_state cleaner0 2023-09-18T09:33:30Z DUMMY: bound 0.9979765 chemical cleaner0 2023-09-18T09:37:53Z CHEBI: DNA structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.94398093 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.99851084 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.99915683 protein_state cleaner0 2023-09-18T09:00:06Z DUMMY: disordered structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD RESULTS paragraph 24784 In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either. This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT). The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases. These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis. 0.99875844 protein_state cleaner0 2023-09-18T09:00:19Z DUMMY: highly flexible structure_element SO: cleaner0 2023-09-18T08:28:01Z loop structure_element SO: cleaner0 2023-09-18T08:27:09Z DPPY 0.66211873 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9986974 evidence cleaner0 2023-09-18T09:01:34Z DUMMY: electron density structure_element SO: cleaner0 2023-09-18T08:30:32Z loops 0.99905926 protein_state cleaner0 2023-09-15T14:49:26Z DUMMY: AdoMet-bound 0.9975859 evidence cleaner0 2023-09-18T09:01:28Z DUMMY: structures 0.9956278 protein cleaner0 2023-09-15T15:47:39Z PR: M.PvuII 0.9992005 protein cleaner0 2023-09-18T08:21:39Z PR: DpnM 0.99534625 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.9867502 chemical cleaner0 2023-09-18T09:37:56Z CHEBI: DNA evidence DUMMY: cleaner0 2023-09-18T09:33:56Z protein-DNA complex structures 0.99605507 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI 0.99500823 protein cleaner0 2023-09-18T09:00:42Z PR: M.HhaI 0.996617 protein cleaner0 2023-09-18T09:00:50Z PR: M.HaeIII protein_state DUMMY: cleaner0 2023-09-18T09:01:07Z well-ordered structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.99599034 evidence cleaner0 2023-09-18T09:01:37Z DUMMY: structures 0.7158421 residue_name cleaner0 2023-09-18T09:01:23Z SO: adenine 0.9972713 bond_interaction cleaner0 2023-09-18T08:48:02Z MESH: hydrogen bond structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.9991862 protein_type cleaner0 2023-09-15T14:49:50Z MESH: MTases protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI RESULTS title_2 25548 Key residue for wider spectrum of substrate recognition RESULTS paragraph 25604 Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment. Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A). Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants. As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI. 0.6295532 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9984703 protein_type cleaner0 2023-09-18T09:27:01Z MESH: N6 adenine MTase 0.989359 residue_name cleaner0 2023-09-18T09:38:21Z SO: adenine 0.9972758 chemical cleaner0 2023-09-18T09:02:21Z CHEBI: 5′-GAGG-3′ 0.9922677 chemical cleaner0 2023-09-18T09:02:24Z CHEBI: 5′-GGAG-3′ 0.9940481 residue_name cleaner0 2023-09-15T15:07:28Z SO: adenines 0.99715126 chemical cleaner0 2023-09-18T09:38:05Z CHEBI: 5′-GAAG-3′ 0.99270916 residue_name cleaner0 2023-09-18T09:38:31Z SO: adenine 0.9974624 chemical cleaner0 2023-09-18T09:02:26Z CHEBI: 5′-GAGG-3′ protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI chemical CHEBI: cleaner0 2023-09-18T09:01:59Z DNA protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.99622726 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.9988635 experimental_method cleaner0 2023-09-18T09:03:12Z MESH: multiple sequence alignment 0.9983783 experimental_method cleaner0 2023-09-18T10:05:06Z MESH: sequence comparison 0.9984338 experimental_method cleaner0 2023-09-18T10:05:10Z MESH: structural analysis 0.9994925 residue_name_number cleaner0 2023-09-15T14:50:16Z DUMMY: P41 0.99946815 residue_name_number cleaner0 2023-09-18T09:03:55Z DUMMY: N111 0.9995011 residue_name_number cleaner0 2023-09-18T09:03:59Z DUMMY: K165 0.99950707 residue_name_number cleaner0 2023-09-18T09:04:03Z DUMMY: T166 0.9943739 experimental_method cleaner0 2023-09-18T10:05:15Z MESH: replaced 0.99705803 residue_name cleaner0 2023-09-18T10:02:27Z SO: serine 0.997026 residue_name cleaner0 2023-09-18T10:02:33Z SO: threonine 0.9970084 residue_name cleaner0 2023-09-18T10:02:35Z SO: threonine 0.9967733 residue_name cleaner0 2023-09-18T10:02:37Z SO: valine experimental_method MESH: cleaner0 2023-09-18T09:03:01Z [3H]AdoMet radiological assay chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.99777615 protein_state cleaner0 2023-09-18T09:34:03Z DUMMY: wide type 0.98206836 protein_state cleaner0 2023-09-18T08:49:46Z DUMMY: mutants chemical CHEBI: cleaner0 2023-09-18T09:02:08Z DNA 0.99718624 chemical cleaner0 2023-09-18T09:02:15Z CHEBI: 5′-GAGG-3′ 0.9764722 chemical cleaner0 2023-09-18T09:02:17Z CHEBI: 5′-GAAG-3′, 0.9746132 protein_state cleaner0 2023-09-18T08:49:46Z DUMMY: mutants chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.9992687 protein_state cleaner0 2023-09-18T08:49:41Z DUMMY: wt protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.9540184 chemical cleaner0 2023-09-18T09:02:19Z CHEBI: 5′-GGAG-3′, chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.99837804 mutant cleaner0 2023-09-18T09:03:42Z MESH: P41S 0.99891484 protein_state cleaner0 2023-09-18T08:49:50Z DUMMY: mutant 0.9990494 protein_state cleaner0 2023-09-18T08:55:08Z DUMMY: wild type protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI oncotarget-07-40965-g005.jpg F5 FIG fig_title_caption 26782 Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI 0.99885774 experimental_method cleaner0 2023-09-18T08:45:06Z MESH: Sequence alignment 0.99821293 experimental_method cleaner0 2023-09-18T09:04:20Z MESH: structural analysis 0.97248703 experimental_method cleaner0 2023-09-18T09:04:22Z MESH: radioactive methyl transfer activity protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI oncotarget-07-40965-g005.jpg F5 FIG fig_caption 26931 A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues. Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow). Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004). B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively. Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min. The experiments were repeated for three times and data were corrected by subtraction of the background. 0.9984727 experimental_method cleaner0 2023-09-18T08:45:06Z MESH: Sequence alignment protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.997293 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.99947685 residue_name_number cleaner0 2023-09-15T14:50:16Z DUMMY: P41 0.99948114 residue_name_number cleaner0 2023-09-18T09:03:56Z DUMMY: N111 0.99947375 residue_name_number cleaner0 2023-09-18T09:03:59Z DUMMY: K165 0.9994906 residue_name_number cleaner0 2023-09-18T09:04:03Z DUMMY: T166 protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.39663774 species cleaner0 2023-09-18T09:04:50Z MESH: 26695 0.99588466 experimental_method cleaner0 2023-09-18T09:04:56Z MESH: structural analysis 0.9985169 experimental_method cleaner0 2023-09-18T08:45:06Z MESH: sequence alignment 0.8075491 experimental_method cleaner0 2023-09-18T09:07:03Z MESH: WebLogo chemical CHEBI: cleaner0 2023-09-18T08:56:49Z Methyl 0.998982 protein_state cleaner0 2023-09-18T08:49:41Z DUMMY: wt protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI mutant MESH: cleaner0 2023-09-18T09:05:35Z M1.HpyAVI-P41S mutant MESH: cleaner0 2023-09-18T09:05:53Z M1.HpyAVI-N111T mutant MESH: cleaner0 2023-09-18T09:06:47Z M1.HpyAVI-K165R T166V 0.9894895 chemical cleaner0 2023-09-18T09:38:38Z CHEBI: DNA chemical CHEBI: cleaner0 2023-09-18T09:06:14Z 5′-GAGG-3′ 0.7406915 chemical cleaner0 2023-09-18T09:06:17Z CHEBI: 5′-GAAG-3′ 0.8041685 chemical cleaner0 2023-09-18T09:06:19Z CHEBI: 5′-GGAG-3′ RESULTS paragraph 27668 Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI. This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above. Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695. 0.9995059 residue_name_number cleaner0 2023-09-15T14:50:16Z DUMMY: P41 0.9565232 structure_element cleaner0 2023-09-18T09:07:25Z SO: GGAG protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.998413 protein_state cleaner0 2023-09-18T09:00:19Z DUMMY: highly flexible structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.95510674 residue_range cleaner0 2023-09-18T10:06:42Z DUMMY: 33 and 58 chemical CHEBI: cleaner0 2023-09-18T09:07:49Z DNA 0.99823356 experimental_method cleaner0 2023-09-18T09:08:14Z MESH: Replacement 0.99676514 residue_name cleaner0 2023-09-18T09:08:08Z SO: serine structure_element SO: cleaner0 2023-09-18T08:28:01Z loop 0.5137013 species cleaner0 2023-09-18T09:08:04Z MESH: 26695 DISCUSS title_1 28193 DISCUSSION DISCUSS paragraph 28204 Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear. Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes. Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes. 0.99879885 protein_state cleaner0 2023-09-18T09:08:21Z DUMMY: DNA-bound 0.9980544 evidence cleaner0 2023-09-18T09:30:25Z DUMMY: structure 0.9987933 protein_type cleaner0 2023-09-18T09:08:39Z MESH: γ-class N6-adenine MTase 0.69096255 residue_name cleaner0 2023-09-18T09:38:49Z SO: adenine 0.9610856 chemical cleaner0 2023-09-18T09:38:53Z CHEBI: DNA chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.9975112 ptm cleaner0 2023-09-15T14:55:25Z MESH: N6-methyladenine 0.99863404 taxonomy_domain cleaner0 2023-09-18T09:27:44Z DUMMY: eukaryotic 0.9988544 protein_type cleaner0 2023-09-15T15:05:55Z MESH: N6-adenine MTases 0.99871874 taxonomy_domain cleaner0 2023-09-15T14:51:05Z DUMMY: eukaryotes 0.99865556 experimental_method cleaner0 2023-09-18T09:08:43Z MESH: Biochemical and structural characterization 0.9822526 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI chemical CHEBI: cleaner0 2023-09-18T08:56:49Z methyl 0.99716395 ptm cleaner0 2023-09-15T14:55:25Z MESH: N6-methyladenine 0.9985537 taxonomy_domain cleaner0 2023-09-15T14:51:05Z DUMMY: eukaryotes DISCUSS paragraph 28788 Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution. Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases. 0.998248 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases 0.9987644 oligomeric_state cleaner0 2023-09-15T15:49:20Z DUMMY: monomer protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.998727 oligomeric_state cleaner0 2023-09-15T15:49:54Z DUMMY: dimer 0.9870143 evidence cleaner0 2023-09-18T09:09:24Z DUMMY: crystal 0.99645835 protein_type cleaner0 2023-09-18T09:09:20Z MESH: β-class DNA exocyclic MTases 0.9813897 evidence cleaner0 2023-09-18T09:09:10Z DUMMY: crystal 0.9987569 oligomeric_state cleaner0 2023-09-15T15:49:54Z DUMMY: dimer 0.9984802 protein_type cleaner0 2023-09-15T14:51:23Z MESH: DNA MTases DISCUSS paragraph 29145 The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively. These two structural characteristics may account for the substrate promiscuity of this enzyme. And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI. 0.9987935 protein_state cleaner0 2023-09-18T09:00:19Z DUMMY: highly flexible 0.99759173 residue_range cleaner0 2023-09-18T09:09:34Z DUMMY: 33-58 structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD 0.9977372 residue_range cleaner0 2023-09-18T09:09:37Z DUMMY: 133-163 0.76505375 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9985341 chemical cleaner0 2023-09-18T09:38:58Z CHEBI: DNA structure_element SO: cleaner0 2023-09-18T09:09:53Z minor and major grooves 0.9994406 residue_name_number cleaner0 2023-09-15T14:50:16Z DUMMY: P41 protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI DISCUSS paragraph 29498 The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme. 0.66722006 protein_state cleaner0 2023-09-18T09:34:09Z DUMMY: missing structure_element SO: cleaner0 2023-09-18T08:28:02Z loop 0.71347064 residue_range cleaner0 2023-09-18T10:06:48Z DUMMY: 33 and 58 0.9982004 chemical cleaner0 2023-09-18T09:39:02Z CHEBI: DNA 0.9979321 protein_state cleaner0 2023-09-18T09:34:12Z DUMMY: stable 0.99098516 protein cleaner0 2023-09-15T15:01:22Z PR: M.TaqI 0.9987587 experimental_method cleaner0 2023-09-18T09:10:23Z MESH: Crystallization 0.96516407 complex_assembly cleaner0 2023-09-18T09:10:16Z GO: M1.HpyAVI-DNA DISCUSS paragraph 29817 DNA methylation plays an important role in bacterial pathogenicity. DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori. Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase. As an important biological modification, DNA methylation directly influences bacterial survival. Knockout of M1.HpyAVI largely prevents the growth of H. pylori. Importantly, H. pylori is involved in 90% of all gastric malignancies. Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection. However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics. The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection. Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival. Small molecules targeting these highly conserved residues are likely to emerge less drug resistance. ptm MESH: cleaner0 2023-09-15T14:50:29Z DNA methylation 0.9985331 taxonomy_domain cleaner0 2023-09-15T14:53:03Z DUMMY: bacterial ptm MESH: cleaner0 2023-09-18T09:11:03Z DNA adenine methylation 0.9984968 taxonomy_domain cleaner0 2023-09-15T14:48:40Z DUMMY: bacteria 0.9971612 species cleaner0 2023-09-18T09:25:37Z MESH: H.pylori ptm MESH: cleaner0 2023-09-18T09:11:02Z DNA adenine methylation 0.99845654 protein_type cleaner0 2023-09-18T08:37:54Z MESH: DNA adenine methyltransferase ptm MESH: cleaner0 2023-09-15T14:50:29Z DNA methylation 0.99820757 taxonomy_domain cleaner0 2023-09-15T14:53:03Z DUMMY: bacterial 0.7532001 experimental_method cleaner0 2023-09-18T10:05:20Z MESH: Knockout of protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.9971716 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.99724466 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.9974534 species cleaner0 2023-09-18T09:25:37Z MESH: H.pylori 0.9974273 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.99812746 protein_type cleaner0 2023-09-15T14:58:13Z MESH: adenine MTases protein PR: cleaner0 2023-09-15T14:49:13Z M1.HpyAVI 0.9971631 species cleaner0 2023-09-15T15:04:01Z MESH: H. pylori 0.9993161 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 0.99932635 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 0.9974186 species cleaner0 2023-09-18T09:25:37Z MESH: H.pylori 0.9989734 protein_state cleaner0 2023-09-18T09:11:57Z DUMMY: highly conserved DISCUSS paragraph 30950 In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity. Residues D29 and E216 were identified to play a crucial role in cofactor binding. As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human. 0.9974125 evidence cleaner0 2023-09-18T09:12:11Z DUMMY: structure 0.8668125 protein cleaner0 2023-09-15T14:49:13Z PR: M1.HpyAVI 0.9987343 protein_state cleaner0 2023-09-18T09:00:07Z DUMMY: disordered structure_element SO: cleaner0 2023-09-18T08:23:59Z TRD residue_name_number DUMMY: cleaner0 2023-09-18T09:36:37Z P41 0.9987941 site cleaner0 2023-09-18T09:35:43Z SO: DNA binding region 0.9995447 residue_name_number cleaner0 2023-09-15T14:50:06Z DUMMY: D29 0.9995259 residue_name_number cleaner0 2023-09-15T14:50:11Z DUMMY: E216 0.9988655 evidence cleaner0 2023-09-18T09:12:14Z DUMMY: crystal structure 0.9980763 protein_type cleaner0 2023-09-18T09:27:09Z MESH: N6-adenine MTase 0.9978216 species cleaner0 2023-09-18T09:25:37Z MESH: H.pylori 0.99823475 species cleaner0 2023-09-18T09:25:37Z MESH: H.pylori 0.99853706 species cleaner0 2023-09-15T14:54:51Z MESH: human METHODS title_1 31413 MATERIALS AND METHODS METHODS title_2 31435 Protein expression and purification METHODS paragraph 31471 The ORF encoding M1.HpyAVI was inserted into the expression plasmid pET22b (Novagen, Massachusetts, USA) to produce a recombinant protein containing a C-terminal His-tag. In order to produce soluble protein, a chaperone plasmid PG-KJE8 (TaKaRa, Dalian, China) was co-expressed with M1.HpyAVI. The recombinant protein was purified with a three-step chromatography protocol using a Ni-NTA affinity column, a HiLoad 16/60 Superdex 200 column and a mono-S HR 5/5 column (1ml) (GE Healthcare, Uppsala, Sweden). Mutants of M1.HpyAVI were generated using the Muta-direct Site-directed Mutagenesis kit (SBS Genetech, Beijing, China) and produced using the same protocol with wide type protein. METHODS title_2 32157 Crystallization and data collection METHODS paragraph 32193 Crystallization trials were carried out for both the AdoMet-free and AdoMet-bound proteins using the hanging drop vapor diffusion technique. Crystals used for diffraction data collection of the apoprotein were grown under the condition of 1.0 M Bis-Tris, pH 9.0, 1.4 M ammonium tartrate, and the optimal crystallization condition for AdoMet-bound protein was 1.0 M Bis-Tris, pH 6.0, 14% PEG2000, 0.2 M lithium sulfate. X-ray diffraction data were collected at 100 K on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) using an ADSC Quantum 315r CCD detector. All data were indexed, integrated and scaled using the XDS program. METHODS title_2 32841 Structure determination and refinement METHODS paragraph 32880 The structure of ligand-free M1.HpyAVI was determined by means of molecular replacement using the M.MboIIA (PDB ID 1G60) as a search model. Automated structure determination using Phaser gave a solution showing four subunits sitting in the asymmetric unit. The model was refined using the COOT graphics package manually and phenix.refine. The AdoMet-bound structure was determined by means of molecular replacement using the refined model of the apoprotein, and refined in the same way. Statistics from the data collection and structure refinement are summarized in Table 1. All figures representing the M1.HpyAVI structures were generated using the molecular visualization program PyMol. METHODS title_2 33569 Detection of protein dimerization METHODS paragraph 33603 The interface information of M1.HpyAVI free form and AdoMet-bound form structures were analyzed using the PDBePISA (Proteins, Interface, Structures and Assemblies) web server. METHODS paragraph 33779 The protein molecular weight was determined by gel filtration using a FPLC system coupled to a Superdex 75 HR 10 / 30 column. The sizing standard was calibrated using the gel filtration calibration kit LMW (GE Healthcare, Uppsala, Sweden). METHODS title_2 34019 Binding affinity quantification via microscale thermophoresis (MST) METHODS paragraph 34087 Microscale thermophoresis was performed using the NT115 nanotemper technologies. M1.HpyAVI-wt and M1.HpyAVI-mutant proteins were fluorescently labeled using the protein label kit according to manufacturer's protocol. Affinity measurements were performed by using MST buffer (0.05% Tween-20 added as final concentration). A solution of unlabeled AdoMet was serially diluted from 1 mM to 15 nM. Equal volume of 0.8 μM labeled protein was mixed with the AdoMet and loaded into the silica capillaries. This binding curve can directly be fitted with the nonlinear solution of the law of mass action, with the dissociation constant (KD) as a result. Measurement was performed at 25°C using 40% LED power and 40%IR-laser power. The dissociation constant was calculated using the Nano-temper Analysis software. METHODS title_2 34892 Radioactive methyltransferase analysis METHODS paragraph 34931 Several different DNA duplexes containing single site of 5′- GAGG-3′, 5′- GAAG-3′ or 5′-GGAG-3′ were used as substrate for methyl transfer reaction (Table S2). 0.1 μM of enzyme and 2 μM of S-[methyl-3H] adenosly methionine (China Isotope and Radiation Corporation, Beijing, China) were incubated at 37°C for 5 min, and then 5 μM of DNA substrate was added to initiate the reaction. Aliquots (20 μl) were taken out at 4-min time intervals and quenched with 2 N HCl. Subsequently, DNA of the mixture was purified using a DNA purification column (TIANGEN, Beijing, China) and the scintillation counting of tritiated DNA was quantified by Beckman LS6500 for 10 min. The background radioactivity was determined by omitting the enzyme from the reaction solution. All the reactions were performed in triplicate. SUPPL title_1 35753 SUPPLEMENTARY FIGURES AND TABLES SUPPL footnote 35786 CONFLICTS OF INTEREST SUPPL footnote 35808 The authors declare that they have no conflicts of interest. SUPPL footnote 35869 ACCESSION CODES SUPPL footnote 35885 Atomic coordinates and structure factors for apo-M1.HpyAVI and cofactor-bound M1.HpyAVI have been deposited in the PDB, with accession codes 5HEK and 5HFJ respectively. 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