The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells.

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.

Molecular basis for substrate recognition by lysine methyltransferases and demethylases.

Lysine methylation has emerged as a prominent covalent modification in histones and non-histone proteins. This modification has been implicated in numerous genomic processes, including heterochromatinization, cell cycle progression, DNA damage response, DNA replication, genome stability, and epigenetic gene regulation that underpins developmental programs defining cell identity and fate. The site and degree of lysine methylation is dynamically modulated through the enzymatic activities of protein lysine methyltransferases (KMTs) and protein lysine demethylases (KDMs). These enzymes display distinct substrate specificities that in part define their biological functions. This review explores recent progress in elucidating the molecular basis of these specificities, highlighting structural and functional studies of the methyltransferases SUV4-20H1 (KMT5B), SUV4-20H2 (KMT5C), and ATXR5, and the demethylases UTX (KDM6A), JMJD3 (KDM6B), and JMJD2D (KDM4D). We conclude by examining these findings in the context of related KMTs and KDMs and by exploring unresolved questions regarding the specificities and functions of these enzymes.

Manipulating unconventional CH-based hydrogen bonding in a methyltransferase via noncanonical amino acid mutagenesis.

Recent studies have demonstrated that the active sites of S-adenosylmethionine (AdoMet)-dependent methyltransferases form strong carbon-oxygen (CH···O) hydrogen bonds with the substrate's sulfonium group that are important in AdoMet binding and catalysis. To probe these interactions, we substituted the noncanonical amino acid p-aminophenylalanine (pAF) for the active site tyrosine in the lysine methyltransferase SET7/9, which forms multiple CH···O hydrogen bonds to AdoMet and is invariant in SET domain enzymes. Using quantum chemistry calculations to predict the mutation's effects, coupled with biochemical and structural studies, we observed that pAF forms a strong CH···N hydrogen bond to AdoMet that is offset by an energetically unfavorable amine group rotamer within the SET7/9 active site that hinders AdoMet binding and activity. Together, these results illustrate that the invariant tyrosine in SET domain methyltransferases functions as an essential hydrogen bonding hub and cannot be readily substituted by residues bearing other hydrogen bond acceptors.

Crystal structure and functional analysis of JMJD5 indicate an alternate specificity and function.

JMJD5 is a Jumonji C (JmjC) protein that has been implicated in breast cancer tumorigenesis, circadian rhythm regulation, embryological development, and osteoclastogenesis. Recently, JMJD5 (also called KDM8) has been reported to demethylate dimethylated Lys-36 in histone H3 (H3K36me2), regulating genes that control cell cycle progression. Here, we report high-resolution crystal structures of the human JMJD5 catalytic domain in complex with the substrate 2-oxoglutarate (2-OG) and the inhibitor N-oxalylglycine (NOG). The structures reveal a ß-barrel fold that is conserved in the JmjC family and a long shallow cleft that opens into the enzyme's active site. A comparison with other JmjC enzymes illustrates that JMJD5 shares sequence and structural homology with the asparaginyl and histidinyl hydroxylase FIH-1 (factor inhibiting hypoxia-inducible factor 1 [HIF-1]), the lysyl hydroxylase JMJD6, and the RNA hydroxylase TYW5 but displays limited homology to JmjC lysine demethylases (KDMs). Contrary to previous findings, biochemical assays indicate that JMJD5 does not display demethylase activity toward methylated H3K36 nor toward the other methyllysines in the N-terminal tails of histones H3 and H4. Together, these results imply that JMJD5 participates in roles independent of histone demethylation and may function as a protein hydroxylase given its structural homology with FIH-1 and JMJD6.

Substrate and product specificities of SET domain methyltransferases.

SET domain lysine methyltransferases (KMTs) catalyze the site- and state-specific methylation of lysine residues in histone and non-histone substrates. These modifications play fundamental roles in transcriptional regulation, heterochromatin formation, X chromosome inactivation and DNA damage response, and have been implicated in the epigenetic regulation of cell identity and fate. The substrate and product specificities of SET domain KMTs are pivotal to eliciting these effects due to the distinct functions associated with site and state-specific protein lysine methylation. Here, we review advances in understanding the molecular basis of these specificities gained through structural and biochemical studies of the human methyltransferases Mixed Lineage Leukemia 1 (MLL1, also known as KMT2A) and SET7/9 (KMT7). We conclude by exploring the broader implications of these findings on the biological functions of protein lysine methylation by SET domain KMTs.

SET7/9 catalytic mutants reveal the role of active site water molecules in lysine multiple methylation.

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ε-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ε-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ε-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

ATP synthase b subunit dimerization domain: a right-handed coiled coil with offset helices.

The dimerization domain of Escherichia coli ATP synthase b subunit forms an atypical parallel two-stranded coiled coil. Sequence analysis reveals an 11-residue abcdefghijk repeat characteristic of right-handed coiled coils, but no other naturally occurring parallel dimeric structure of this class has been identified. The arrangement of the helices was studied by their propensity to form interhelix disulfide linkages and analysis of the stability and shape of disulfide-linked dimers. Disulfides formed preferentially between cysteine residues in an a position of one helix and either of the adjacent h positions of the partner. Such heterodimers were far more stable to thermal denaturation than homodimers and, on the basis of gel-filtration chromatography studies, were similar in shape to both non-covalent dimers and dimers linked through flexible Gly(1-3)Cys C-terminal extensions. The results indicate a right-handed coiled-coil structure with intrinsic asymmetry, the two helices being offset rather than in register. A function for the right-handed coiled coil in rotational catalysis is proposed.

The "second stalk" of Escherichia coli ATP synthase: structure of the isolated dimerization domain.

The b subunit of E. coli F(0)F(1)-ATPase links the peripheral F(1) subunits to the membrane-integral F(0) portion and functions as a "stator", preventing rotation of F(1). The b subunit is present as a dimer in ATP synthase, and residues 62-122 are required to mediate dimerization. To understand how the b subunit dimer is formed, we have studied the structure of the isolated dimerization domain, b(62-122). Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) indicate that the b(62-122) dimer is extremely elongated, with a frictional ratio of 1.60, a maximal dimension of 95 A, and a radius of gyration of 27 A, values that are consistent with an alpha-helical coiled-coil structure. The crystal structure of b(62-122) has been solved and refined to 1.55 A. The protein crystallized as an isolated, monomeric alpha helix with a length of 90 A. Combining the crystal structure of monomeric b(62-122) with SAXS data from the dimer in solution, we have constructed a model for the b(62-122) dimer in which the two helices form a coiled coil with a right-handed superhelical twist. Analysis of b sequences from E. coli and other prokaryotes indicates conservation of an undecad repeat, which is characteristic of a right-handed coiled coil and consistent with our structural model. Mutation of residue Arg-83, which interrupts the undecad pattern, to alanine markedly stabilized the dimer, as expected for the proposed two-stranded, right-handed coiled-coil structure.