Molecular basis of an atypical dsDNA 5mC/6mA bifunctional dioxygenase CcTet from Coprinopsis cinerea in catalyzing dsDNA 5mC demethylation.

The eukaryotic epigenetic modifications 5-methyldeoxycytosine (5mC) and N6-methyldeoxyadenine (6mA) have indispensable regulatory roles in gene expression and embryonic development. We recently identified an atypical bifunctional dioxygenase CcTet from Coprinopsis cinerea that works on both 5mC and 6mA demethylation. The nonconserved residues Gly331 and Asp337 of CcTet facilitate 6mA accommodation, while D337F unexpectedly abolishes 5mC oxidation activity without interfering 6mA demethylation, indicating a prominent distinct but unclear 5mC oxidation mechanism to the conventional Tet enzymes. Here, we assessed the molecular mechanism of CcTet in catalyzing 5mC oxidation by representing the crystal structure of CcTet-5mC-dsDNA complex. We identified the distinct mechanism by which CcTet recognizes 5mC-dsDNA compared to 6mA-dsDNA substrate. Moreover, Asp337 was found to have a central role in compensating for the loss of a critical 5mC-stablizing H-bond observed in conventional Tet enzymes, and stabilizes 5mC and subsequent intermediates through an H-bond with the N4 atom of the substrates. These findings improve our understanding of Tet enzyme functions in the dsDNA 5mC and 6mA demethylation pathways, and provide useful information for future discovery of small molecular probes targeting Tet enzymes in DNA active demethylation processes.

A fungal dioxygenase CcTet serves as a eukaryotic 6mA demethylase on duplex DNA.

N6-methyladenosine (6mA) is a DNA modification that has recently been found to play regulatory roles during mammalian early embryo development and mitochondrial transcription. We found that a dioxygenase CcTet from the fungus Coprinopsis cinerea is also a dsDNA 6mA demethylase. It oxidizes 6mA to the intermediate N6-hydroxymethyladenosine (6hmA) with robust activity of 6mA-containing duplex DNA (dsDNA) as well as isolated genomics DNA. Structural characterization revealed that CcTet utilizes three flexible loop regions and two key residues-D337 and G331-in the active pocket to preferentially recognize substrates on dsDNA. A CcTet D337F mutant protein retained the catalytic activity on 6mA but lost activity on 5-methylcytosine. Our findings uncovered a 6mA demethylase that works on dsDNA, suggesting potential 6mA demethylation in fungi and elucidating 6mA recognition and the catalytic mechanism of CcTet. The CcTet D337F mutant protein also provides a chemical biology tool for future functional manipulation of DNA 6mA in vivo.

Structural basis of ubiquitin modification by the Legionella effector SdeA.

Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.

High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases.

Epigenetic therapy has significant potential for cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based 'High-Throughput Methyl Reading' (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two natural products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.

The Molecular Basis of Human ALKBH3 Mediated RNA N1 -methyladenosine (m1 A) Demethylation.

N1 -methyladenosine (m1 A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1 A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two ß-hairpins (ß4-loop-ß5 and ß'-loop-ß'') and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m1 A- and 3-methylcytidine (m3 C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m1 A to N6 -methyladenosine (m6 A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1 A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.

The Molecular Basis of Catalysis by SDR Family Members Ketoacyl-ACP Reductase FabG and Enoyl-ACP Reductase FabI in Type-II Fatty Acid Biosynthesis.

The short-chain dehydrogenase/reductase (SDR) superfamily members acyl-ACP reductases FabG and FabI are indispensable core enzymatic modules and catalytic orientation controllers in type-II fatty acid biosynthesis. Herein, we report their distinct substrate allosteric recognition and enantioselective reduction mechanisms. FabG achieves allosteric regulation of ACP and NADPH through ACP binding across two adjacent FabG monomers, while FabI follows an irreversible compulsory order of substrate binding in that NADH binding must precede that of ACP on a discrete FabI monomer. Moreover, FabG and FabI utilize a backdoor residue Phe187 or a "rheostat" α8 helix for acyl chain length selection, and their corresponding triad residues Ser142 or Tyr145 recognize the keto- or enoyl-acyl substrates, respectively, facilitating initiation of nucleophilic attack by NAD(P)H. The other two triad residues (Tyr and Lys) mediate subsequent proton transfer and (R)-3-hydroxyacyl- or saturated acyl-ACP production.

PDV2 has a dosage effect on chloroplast division in Arabidopsis.

KEY MESSAGE: PDV2 has a dosage effect on chloroplast division in Arabidopsis thaliana , but this effect may vary in different plants. Chloroplasts have to be divided as plants grow to maintain an optimized number in the cell. Chloroplasts are divided by protein complexes across the double membranes from the stroma side to the cytosolic side. PDV2 is a chloroplast division protein on the chloroplast outer membrane. It recruits the dynamin-related GTPase ARC5 to the division site. The C-terminus of PDV2 and the C-terminus of ARC6 interact in the intermembrane space, which is important for the localization of PDV2. Previously, it was shown that overexpression of PDV2 can increase the division of chloroplasts in Arabidopsis and moss, so the authors concluded that PDV2 determines the rate of chloroplast division in land plants. PDV2 was also shown to inhibit the GTPase activity of ARC5 by in vitro experiment. These results look to be contradictory. Here, we identified a null allele of PDV2 in Arabidopsis and studied plants with different levels of PDV2. Our results suggested that the chloroplast division phenotype in Arabidopsis is sensitive to the level of PDV2, while this is not the case for ARC6. The level of PDV2 protein is reduced sharply in fast-growing leaves, while the level of ARC6 is not. The levels of PDV2 and ARC6 in several other plant species at different developmental stages were also investigated. The results indicated that their expression pattern varies in different species. Thus, PDV2 is an important positive factor of chloroplast division with an apparent dosage effect in Arabidopsis, but this effect for different chloroplast division proteins in different plants may vary.

Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity.

Specifier proteins are important components of the glucosinolate-myrosinase system, which mediate plant defense against herbivory and pathogen attacks. Upon tissue disruption, glucosinolates are hydrolyzed to instable aglucones by myrosinases, and then aglucones will rearrange to form defensive isothiocyanates. Specifier proteins can redirect this reaction to form other products, such as simple nitriles, epithionitriles and organic thiocyanates instead of isothiocyanates based on the side chain structure of glucosinolate and the type of the specifier proteins. Nevertheless, the molecular mechanism underlying the different product spectrums of various specifier proteins was not fully understood. Here in this study, we solved the crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana (AtESP) at 2.3 Å resolution. Structural comparisons with the previously solved structure of thiocyanate forming protein, TFP from Thlaspi arvense (TaTFP) reveal that AtESP shows a dimerization pattern different from TaTFP. Moreover, AtESP harbors a slightly larger active site pocket than TaTFP and several residues around the active site are different between the two proteins, which might account for the different product spectrums of the two proteins. Together, our structural study provides important insights into the molecular mechanisms of specifier proteins and shed light on the basis of their different product spectrums.

Redox switching mechanism of the adenosine 5'-phosphosulfate kinase domain (APSK2) of human PAPS synthase 2.

Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the rate-limiting biosynthetic step of the universal sulfuryl donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). In higher eukaryotes, the APSK and ATP sulfurylase (ATPS) domains are fused in a single chain. Humans have two bifunctional PAPS synthetase isoforms: PAPSS1 with the APSK1 domain and PAPSS2 containing the APSK2 domain. APSK2 displays a distinct higher activity for PAPSS2-mediated PAPS biosynthesis during tumorigenesis. How APSK2 achieves excess PAPS production has remained unclear. APSK1 and APSK2 lack the conventional redox-regulatory element present in plant PAPSS homologs. Here we elucidate the dynamic substrate recognition mechanism of APSK2. We discover that APSK1 contains a species-specific Cys-Cys redox-regulatory element that APSK2 lacks. The absence of this element in APSK2 enhances its enzymatic activity for excess PAPS production and promotes cancer development. Our results help to understand the roles of human PAPSSs during cell development and may facilitate PAPSS2-specific drug discovery.

A luminescent Cd(ii) coordination polymer as a multi-responsive fluorescent sensor for Zn2+, Fe3+ and Cr2O7 2- in water with fluorescence enhancement or quenching.

A luminescent Cd(ii) coordination polymer, namely {[Cd(btic)(phen)]·0.5H2O} n (CP-1) (H2btic = 5-(2-benzothiazolyl)isophthalic acid, phen = 1,10-phenanthroline), was constructed through the mixed-ligand method under solvothermal conditions. CP-1 manifests a chain structure decorated with uncoordinated Lewis basic N and S donors. CP-1 exhibits high sensing towards Zn2+, Fe3+ and Cr2O7 2- ions with fluorescence enhancement or quenching. CP-1 exhibited a fluorescence enhancement for Zn2+ ions through weak binding to S and N atoms, and a fluorescence quenching for Fe3+ and Cr2O7 2- ions by an energy transfer process. The binding constants were calculated as 1.812 × 104 mol-1 for Zn2+, 4.959 × 104 mol-1 for Fe3+ and 1.793 × 104 mol-1 for Cr2O7 2-. This study shows CP-1 as a rare multi-responsive sensor material for the efficient detection of Zn2+, Fe3+ and Cr2O7 2- ions.

Structural insights into the mechanism and inhibition of transglutaminase-induced ubiquitination by the Legionella effector MavC.

Protein ubiquitination is one of the most prevalent post-translational modifications, controlling virtually every process in eukaryotic cells. Recently, the Legionella effector MavC was found to mediate a unique ubiquitination through transglutamination, linking ubiquitin (Ub) to UBE2N through UbGln40 in a process that can be inhibited by another Legionella effector, Lpg2149. Here, we report the structures of MavC/UBE2N/Ub ternary complex, MavC/UBE2N-Ub (product) binary complex, and MavC/Lpg2149 binary complex. During the ubiquitination, the loop containing the modification site K92 of UBE2N undergoes marked conformational change, and Lpg2149 inhibits this ubiquitination through competing with Ub to bind MavC. Moreover, we found that MavC itself also exhibits weak deubiquitinase activity towards this non-canonical ubiquitination. Together, our study not only provides insights into the mechanism and inhibition of this transglutaminase-induced ubiquitination by MavC, but also sheds light on the future studies into UBE2N inhibition by this modification and deubiquitinases of this unique ubiquitination.

Structural insights into the γ-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans.

(+)-γ-lactamase catalyzes the specific hydrolysis of (+)-γ-lactam out of the racemic γ-lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (-)-γ-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir. However, no structural data has been reported on how the enzymes bind the γ-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase (IHL, Mh33H4-5540) with (+)-γ-lactamase activity, which constitutes a novel family of γ-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (-)-γ-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-γ-lactam bound, and (-)-γ-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (-)-γ-lactam resemble those of IHLs, but the "cover" loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (-)-γ-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of γ-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis.

Reversible single crystal to single crystal transformation with anion exchange-induced weak Cu2+···I⁻ interactions and modification of the structures and properties of MOFs.

{[Cu(6)(TTTMB)(8)(OH)(4)(H(2)O)(6)]·8(NO(3))·34.5H(2)O}(n) can reversibly transform to {[Cu(6)(TTTMB)(8)I(3)]·9I·26H(2)O}(n) upon a single crystal to single crystal process. The transformation is accompanied by the formation of weak Cu(2+)I(-) interactions and changes in the coordination numbers, as well as modulation in their properties.