Raymond C. Trievel

RomA and its highly conserved strain ortholog LegAS4 are SET and ankyrin domain-containing effector proteins of the intracellular bacterial pathogen Legionella pneumophila . These enzymes are secreted into host cells, where they translocate to the nucleus and methylate Lys14 in histone H3 (H3K14), a novel post-translation modification (PTM) that reprograms gene expression and promotes bacterial replication. To elucidate their H3K14 substrate specificity, we determined the crystal structures of LegAS4 and RomA bound to histone H3 peptides and characterized nucleosome binding and methylation by the enzymes. The results reveal a distinctive bipartite engagement of the histone H3 N-terminal tail by the enzymes’ SET and ankyrin domains. Further, the ankyrin domain distinguishes different PTMs at H3R2 and H3K4, which is crucial for nucleosome engagement and H3K14 methylation. Together, these studies yield new insights into histone H3 reading and writing by the L. pneumophila histone lysine methyltransferases during host cell infection.

Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We demonstrate that the NOCT protein is differentially expressed and processed in a cell and tissue type specific manner as a means to control its localization to the cytoplasm or mitochondria. Our studies also show that the N-terminus of NOCT is necessary and sufficient to confer mitochondrial localization. We then measured the impact of cytoplasmic NOCT on the transcriptome and report that it regulates the levels of hundreds of mRNAs that are enriched for components of signaling pathways, neurological functions, and regulators of osteoblast differentiation. Recent biochemical data indicate that NOCT dephosphorylates nicotinamide adenine dinucleotide (NAD) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in manner specified by its intracellular localization.

The N-methyltransferase TylM1 from Streptomyces fradiae catalyzes the final step in the biosynthesis of the deoxyamino sugar mycaminose, a substituent of the antibiotic tylosin. The high-resolution crystal structure of TylM1 bound to the methyl donor S-adenosylmethionine (AdoMet) illustrates a network of carbon-oxygen (CH•••O) hydrogen bonds between the substrate’s sulfonium cation and residues within the active site. These interactions include hydrogen bonds between the methyl and methylene groups of the AdoMet sulfonium cation and the hydroxyl groups of Tyr14 and Ser120 in the enzyme. To examine the functions of these interactions, we generated Tyr14 to phenylalanine (Y14F) and Ser120 to alanine (S120A) mutations to selectively ablate the CH•••O hydrogen bonding to AdoMet. The TylM1 S120A mutant exhibited a modest decrease in the catalytic efficiency relative to wild type (WT) enzyme, whereas the Y14F mutation resulted in an approximately 30-fold decrease in catalytic efficiency. In contrast, site-specific substitution of Tyr14 by the noncanonical amino acid p-aminophenylalanine partially restored activity comparable to the WT enzyme. Correlatively, quantum mechanical calculations of the activation barrier energies of WT TylM1 and the Tyr14 mutants suggest that substitutions which abrogate hydrogen bonding with the AdoMet methyl group impair methyl transfer. Together, these results offer insights into roles of CH•••O hydrogen bonding in modulating the catalytic efficiency of TylM1.

Tetrel bonds represent a category of non-bonding interaction wherein an electronegative atom donates a lone pair of electrons into the sigma antibonding orbital of an atom in the carbon group of the periodic table. Prior computational studies have implicated tetrel bonding in the stabilization of a preliminary state that precedes the transition state in S_N2 reactions, including methyl transfer. Notably, the angles between the tetrel bond donor and acceptor atoms coincide with the prerequisite geometry for the S_N2 reaction. Prompted by these findings, we surveyed crystal structures of methyltransferases in the Protein Data Bank and discovered multiple instances of carbon tetrel bonding between the methyl group of the substrate S-adenosylmethionine (AdoMet) and electronegative atoms of small molecule inhibitors, ions, and solvent molecules. The majority of these interactions involve oxygen atoms as the Lewis base, with the exception of one structure in which a chlorine atom of an inhibitor functions as the electron donor. Quantum mechanical analyses of a representative subset of the methyltransferase structures from the survey revealed that the calculated interaction energies and spectral properties are consistent with the values for bona fide carbon tetrel bonds. The discovery of methyl tetrel bonding offers new insights into the mechanism underlying the S_N2 reaction catalyzed by AdoMet-dependent methyltransferases. These findings highlight the potential of exploiting these interactions in developing new methyltransferase inhibitors.

The circadian protein Nocturnin (NOCT) belongs to the exonuclease, endonuclease and phosphatase superfamily and is most similar to the CCR4-class of deadenylases that degrade the poly-adenosine tails of mRNAs. NOCT-deficient mice are resistant to high-fat diet induced weight gain, and exhibit dysregulation of bone formation. However, the mechanisms by which NOCT regulates these processes remain to be determined. Here, we describe a pair of high-resolution crystal structures of the human NOCT catalytic domain. The active site of NOCT is highly conserved with other exoribonucleases, and when directed to a transcript in cells, NOCT can reduce translation and abundance of that mRNA in a manner dependent on key active site residues. In contrast to the related deadenylase CNOT6L, purified recombinant NOCT lacks in vitro ribonuclease activity, suggesting that unidentified factors are necessary for enzymatic activity. We also find the ability of NOCT to repress reporter mRNAs in cells depends upon the 3′ end of the mRNA, as reporters terminating with a 3′ MALAT1 structure cannot be repressed by NOCT. Together, these data demonstrate that NOCT is an exoribonuclease that can degrade mRNAs to inhibit protein expression, suggesting a molecular mechanism for its regulatory role in lipid metabolism and bone development.

The C-terminal domain of cobalamin-dependent methionine synthase (MetH) has an essential role in catalyzing the reactivation of the enzyme following the oxidation of its cobalamin cofactor. This reactivation occurs through reductive methylation of the cobalamin using S-adenosylmethionine (AdoMet) as the methyl donor. Herein, we examine the molecular recognition of AdoMet by the MetH reactivation domain utilizing structural, biochemical, and computational approaches. Crystal structures of the E. coli MetH reactivation domain in complex with AdoMet, the methyl transfer product S-adenosylhomocysteine (AdoHcy), and the AdoMet analog inhibitor sinefungin illustrate that the ligands exhibit an analogous conformation within the solvent-exposed substrate binding cleft of the enzyme. AdoMet binding is stabilized by an intramolecular sulfur-oxygen chalcogen bond between the sulfonium and carboxylate groups of the substrate and by water-mediated carbon-oxygen hydrogen bonding between the sulfonium cation and the side chains of Glu1097 and Glu1128 that bracket the substrate binding cleft. AdoMet and sinefungin exhibited similar binding affinities for the MetH reactivation domain, whereas AdoHcy displayed an affinity for the enzyme that was an order of magnitude lower. Mutations of Glu1097 and Glu1128 diminished the AdoMet/AdoHcy binding selectivity ratio to approximately twofold, underscoring the role of these residues in enabling the enzyme to discriminate between substrate and product. Together, these findings indicate that Glu1097 and Glu1128 in MetH promote high affinity recognition of AdoMet and that sinefungin and potentially other AdoMet-based methyltransferase inhibitors can abrogate MetH reactivation, which would result in off-target effects associated with alterations in methionine homeostasis and one-carbon metabolism.

Recent studies have demonstrated that carbon–oxygen (CH···O) hydrogen bonds have important roles in S-adenosylmethionine (AdoMet) recognition and catalysis in methyltransferases. Here, we investigate noncovalent interactions that occur between the AdoMet sulfur cation and oxygen atoms in methyltransferase active sites. These interactions represent sulfur–oxygen (S···O) chalcogen bonds in which the oxygen atom donates a lone pair of electrons to the σ antibonding orbital of the AdoMet sulfur atom. Structural, biochemical, and computational analyses of an asparagine mutation in the lysine methyltransferase SET7/9 that abolishes AdoMet S···O chalcogen bonding reveal that this interaction enhances substrate binding affinity relative to the product S-adenosylhomocysteine. Corroborative quantum mechanical calculations demonstrate that sulfonium systems form strong S···O chalcogen bonds relative to their neutral thioether counterparts. An inspection of high-resolution crystal structures reveals the presence of AdoMet S···O chalcogen bonding in different classes of methyltransferases, illustrating that these interactions are not limited to SET domain methyltransferases. Together, these results demonstrate that S···O chalcogen bonds contribute to AdoMet recognition and can enable methyltransferases to distinguish between substrate and product.