HCV IRES Captures an Actively Translating 80S Ribosome.

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.

The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA.

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.

Homology recognition without double-stranded DNA-strand separation in D-loop formation by RecA.

RecA protein and RecA/Rad51 orthologues are required for homologous recombination and DNA repair in all living creatures. RecA/Rad51 catalyzes formation of the D-loop, an obligatory recombination intermediate, through an ATP-dependent reaction consisting of two phases: homology recognition between double-stranded (ds)DNA and single-stranded (ss)DNA to form a hybrid-duplex core of 6-8 base pairs and subsequent hybrid-duplex/D-loop processing. How dsDNA recognizes homologous ssDNA is controversial. The aromatic residue at the tip of the ß-hairpin loop (L2) was shown to stabilize dsDNA-strand separation. We tested a model in which dsDNA strands were separated by the aromatic residue before homology recognition and found that the aromatic residue was not essential to homology recognition, but was required for D-loop processing. Contrary to the model, we found that the double helix was not unwound even a single turn during search for sequence homology, but rather was unwound only after the homologous sequence was recognized. These results suggest that dsDNA recognizes its homologous ssDNA before strand separation. The search for homologous sequence with homologous ssDNA without dsDNA-strand separation does not generate stress within the dsDNA; this would be an advantage for dsDNA to express homology-dependent functions in vivo and also in vitro.

A diet supplemented with cholic acid elevates blood pressure accompanied by albuminuria in rats.

A diet supplemented with cholic acid (CA), the primary 12α-hydroxylated bile acid, can induce hepatic lipid accumulation in rats without obesity. This study examined the effects of a CA-supplemented diet on blood pressure (BP). After acclimation, WKAH/HkmSlc rats (3 weeks old) were divided into two groups and fed with a control AIN-93-based diet or a CA-supplemented diet (0.5 g CA/kg) for 13 weeks. The CA diet increased systolic and diastolic BP as well as hepatic lipid concentrations in the rats. No changes were found in the blood sodium concentration. Urinary albumin concentration increased in CA-fed rats. An increase was observed in the hepatic expression of ATP-binding cassette subfamily B member 1B that correlated BPs and urinary albumin concentration accompanied by an increase in portal taurocholic acid concentration. These results suggest that 12α-hydroxylated bile acids are involved in increased BP and albuminuria via alteration of hepatic function.

Synergistic regulation of hepatic Fsp27b expression by HNF4α and CREBH.

The CIDE (cell death-inducing DFF45-like effector) family composed of CIDEA, CIDEB, CIDEC/FSP27 (fat-specific protein 27), has a critical role in growth of lipid droplets. Of these, CIDEB and CIDEC2/FSP27B are abundant in the liver, and the steatotic livers, respectively. Hepatocyte nuclear factor 4α (HNF4α) has an important role in lipid homeostasis because liver-specific HNF4α-null mice (Hnf4aΔHep mice) exhibit hepatosteatosis. We investigated whether HNF4α directly regulates expression of CIDE family genes. Expression of Cideb and Fsp27b was largely decreased in Hnf4aΔHep mice, while expression of Cidea was increased. Similar results were observed only in CIDEC2, the human orthologue of the Fsp27b, in human hepatoma cell lines in which HNF4α expression was knocked down. Conversely, overexpression of HNF4α strongly induced CIDEC2 expression in hepatoma cell lines. Furthermore, HNF4α transactivated Fsp27b by direct binding to an HNF4α response element in the Fsp27b promoter. In addition, Fsp27b is known to be transactivated by CREBH that is regulated by HNF4α, and expression of CREBH was induced by HNF4α in human hepatoma cells. Co-transfection of HNF4α and CREBH resulted in synergistic transactivation and induction of Fsp27b compared to that of HNF4α or CREBH alone. These results suggest that HNF4α, in conjunction with CREBH, plays an important role in regulation of Fsp27b expression.

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions.

RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair.

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.

Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2.

The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

PPARα activation partially drives NAFLD development in liver-specific Hnf4a-null mice.

HNF4α regulates various genes to maintain liver function. There have been reports linking HNF4α expression to the development of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis. In this study, liver-specific Hnf4a-deficient mice (Hnf4aΔHep mice) developed hepatosteatosis and liver fibrosis, and they were found to have difficulty utilizing glucose. In Hnf4aΔHep mice, the expression of fatty acid oxidation-related genes, which are PPARα target genes, was increased in contrast to the decreased expression of PPARα, suggesting that Hnf4aΔHep mice take up more lipids in the liver instead of glucose. Furthermore, Hnf4aΔHep/Ppara-/- mice, which are simultaneously deficient in HNF4α and PPARα, showed improved hepatosteatosis and fibrosis. Increased C18:1 and C18:1/C18:0 ratio was observed in the livers of Hnf4aΔHep mice, and the transactivation of PPARα target gene was induced by C18:1. When the C18:1/C18:0 ratio was close to that of Hnf4aΔHep mouse liver, a significant increase in transactivation was observed. In addition, the expression of Pgc1a, a coactivator of PPARs, was increased, suggesting that elevated C18:1 and Pgc1a expression could contribute to PPARα activation in Hnf4aΔHep mice. These insights may contribute to the development of new diagnostic and therapeutic approaches for NAFLD by focusing on the HNF4α and PPARα signaling cascade.

The ratio of 12α to non-12-hydroxylated bile acids reflects hepatic triacylglycerol accumulation in high-fat diet-fed C57BL/6J mice.

In our previous study, enterohepatic 12α-hydroxylated (12α) bile acid (BA) levels were found to be correlated with hepatic triacylglycerol concentration in rats fed high-fat (HF) diet. Since BA composition is diverse depending on animal species, we evaluated whether such a relationship is applicable in mice in response to an HF diet. C57BL/6JJmsSLC (B6) male mice were fed HF diet for 13 weeks and analyzed for triacylglycerol, cholesterol, oxysterols, and other metabolites in the liver. The BA composition was determined in the liver, small intestinal contents, portal plasma, aortic plasma, and feces. Neutral sterols were also measured in the feces. The ratio of 12α BA/non-12 BA increased in the liver, portal plasma, small intestinal contents, and feces of HF-fed B6 mice. Moreover, a positive correlation was observed between the ratio of fecal 12α BAs/non-12 BAs and hepatic triacylglycerol concentration. The concentration of 7α-hydroxycholesterol was increased in the liver of HF-fed B6 mice, whereas no increase was observed in the hepatic expression of cytochrome P450 family 7 subfamily A member 1. The present study showed that the ratio of 12α BA/non-12 BA in feces is closely associated with hepatic triacylglycerol accumulation in B6 mice fed HF diet.

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains.

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 Å resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.

The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation.

Double-stranded (ds)DNA, not dsRNA, has an ability to form a homologous complex with single-stranded (ss)DNA or ssRNA of homologous sequence. D-loops and homologous triplexes are homologous complexes formed with ssDNA by RecA/Rad51-family homologous-pairing proteins, and are a key intermediate of homologous (genetic/DNA) recombination. R-loop formation independent of transcription (R-loop formation in trans) was recently found to play roles in gene regulation and development of mammals and plants. In addition, the crRNA-Cas effector complex in CRISPR-Cas systems also relies on R-loop formation to recognize specific target. In homologous complex formation, ssDNA/ssRNA finds a homologous sequence in dsDNA by Watson-Crick base-pairing. crRNA-Cas effector complexes appear to actively melt dsDNA to make its bases available for annealing to crRNA. On the other hand, in D-loop formation and homologous-triplex formation, it is likely that dsDNA recognizes the homologous sequence before the melting of its double helix by using its intrinsic molecular function depending on CH2 at the 2'-position of the deoxyribose, and that the major role of RecA is the extension of ssDNA and the holding dsDNA at a position suitable for homology search. This intrinsic dsDNA function would also play a role in R-loop formation. The dependency of homologous-complex formation on 2'-CH2 of the deoxyribose would explain the absence of homologous complex formation by dsRNA, and dsDNA as sole genome molecule in all cellular organisms.

Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity.

The two-component enzyme, 4-hydroxyphenylacetate 3-monooxygenase, catalyzes the conversion of 4-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. In the overall reaction, the oxygenase component (HpaB) introduces a hydroxyl group into the benzene ring of 4-hydroxyphenylacetate using molecular oxygen and reduced flavin, while the reductase component (HpaC) provides free reduced flavins for HpaB. The crystal structures of HpaC from Thermus thermophilus HB8 in the ligand-free form, the FAD-containing form, and the ternary complex with FAD and NAD(+) were determined. In the ligand-free form, two large grooves are present at the dimer interface, and are occupied by water molecules. A structural analysis of HpaC containing FAD revealed that FAD has a low occupancy, indicating that it is not tightly bound to HpaC. This was further confirmed in flavin dissociation experiments, showing that FAD can be released from HpaC. The structure of the ternary complex revealed that FAD and NAD(+) are bound in the groove in the extended and folded conformation, respectively. The nicotinamide ring of NAD(+) is sandwiched between the adenine ring of NAD(+) and the isoalloxazine ring of FAD. The distance between N5 of the isoalloxazine ring and C4 of the nicotinamide ring is about 3.3 A, sufficient to permit hydride transfer. The structures of these three states are essentially identical, however, the side chains of several residues show small conformational changes, indicating an induced fit upon binding of NADH. Inactivity with respect to NADPH can be explained as instability of the binding of NADPH with the negatively charged 2'-phosphate group buried inside the complex, as well as a possible repulsive effect by the dipole of helix alpha1. A comparison of the binding mode of FAD with that in PheA2 from Bacillus thermoglucosidasius A7, which contains FAD as a prosthetic group, reveals remarkable conformational differences in a less conserved loop region (Gly83-Gly94) involved in the binding of the AMP moiety of FAD. These data suggest that variations in the affinities for FAD in the reductases of the two-component flavin-diffusible monooxygenase family may be attributed to difference in the interaction between the AMP moiety of FAD and the less conserved loop region which possibly shows structural divergence.

Crystal structure of the stationary phase survival protein SurE with metal ion and AMP.

The stationary phase survival protein SurE is a metal ion-dependent phosphatase distributed among eubacteria, archaea, and eukaryotes. In Escherichia coli, SurE has activities as nucleotidase and exopolyphosphatase, and is thought to be involved in stress response. However, its physiological role and reaction mechanism are unclear. We report here the crystal structures of the tetramer of SurE from Thermus thermophilus HB8 (TtSurE) both alone and crystallized with Mn(2+) and substrate AMP. In the presence of Mn(2+) and AMP, differences between the protomers were observed in the active site and in the loop located near the active site; AMP-bound active sites with the loops in a novel open conformation were found in the two protomers, and AMP-free active sites with the loops in a conventional closed conformation were found in the other two protomers. The two loops in the open conformation are entwined with each other, and this entwining is suggested to be required for enzymatic activity by site-directed mutagenesis. TtSurE exists as an equilibrium mixture of dimer and tetramer in solution. The loop-entwined structure indicates that SurE acts as a tetramer. The structural features and the absence of negative cooperativity imply the half-of-the-sites reactivity mechanism resulting from a pre-existing tendency toward structural asymmetry.

Corrigendum to: Contribution of histone N-terminal tails to the structure and stability of nucleosomes.

[This corrects the article DOI: 10.1016/j.fob.2013.08.007.].

Crystallization and preliminary X-ray analysis of the oxygenase component (HpaB) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8.

The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase enzyme catalyzes the hydroxylation of 4HPA to 3,4-dihydroxyphenylacetate in the initial step of the degradation pathway of 4HPA. This enzyme consists of two components: an oxygenase (HpaB) and a reductase (HpaC). HpaB hydroxylates 4HPA using an oxygen molecule and a reduced flavin, which is supplied by HpaC. HpaB from Thermus thermophilus HB8 was overexpressed in Escherichia coli and crystallized. Crystals of HpaB were grown in 0.4 M 1,6-hexanediol, 0.1 M sodium acetate pH 5.0 and 25% (v/v) glycerol and diffracted X-rays to a resolution of 1.60 A. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 91.8, b = 99.6, c = 131.1 A. The asymmetric unit volume provides space for only one subunit of the tetrameric HpaB molecule, giving a Matthews coefficient V(M) of 2.8 A3 Da(-1) and a solvent content of 55.1%. Platinum-derivatized crystals of HpaB were prepared by soaking native crystals in a solution containing 1 mM ammonium tetrachloroplatinate(II) for 1 d and diffracted X-rays to a resolution of 2.50 A. MAD data were successfully collected for structural determination using these crystals.

Metal-free and Ca2+-bound structures of a multidomain EF-hand protein, CBP40, from the lower eukaryote Physarum polycephalum.

Acellular slime mold, Physarum polycephalum, has a unique wound-healing system. When cytoplasm of plasmodia is exposed to extracellular fluid, calcium binding protein 40 (CBP40) seals damaged areas, forming large aggregates Ca(2+) dependently. Part of the CBP40 is truncated at the N terminus by a proteinase in plasmodia (CBP40delta), which does not aggregate in the Ca(2+)-bound form. Here we report the crystal structures of CBP40delta in both the metal-free and the Ca(2+)-bound states. Both structures consist of three domains: coiled-coil, intervening, and EF-hand. The topology of the EF-hand domain is similar to that of calpain. The N-terminal half of CBP40Delta interacts with the C-terminal EF-hands through a large hydrophobic interface, necessary for high Ca(2+) affinity. Conformational change upon Ca(2+) binding is small; however, the structure of CBP40delta provides novel insights into the mechanism of Ca(2+)-dependent oligomerization.

Contribution of histone N-terminal tails to the structure and stability of nucleosomes.

Histones are the protein components of the nucleosome, which forms the basic architecture of eukaryotic chromatin. Histones H2A, H2B, H3, and H4 are composed of two common regions, the "histone fold" and the "histone tail". Many efforts have been focused on the mechanisms by which the post-translational modifications of histone tails regulate the higher-order chromatin architecture. On the other hand, previous biochemical studies have suggested that histone tails also affect the structure and stability of the nucleosome core particle itself. However, the precise contributions of each histone tail are unclear. In the present study, we determined the crystal structures of four mutant nucleosomes, in which one of the four histones, H2A, H2B, H3, or H4, lacked the N-terminal tail. We found that the deletion of the H2B or H3 N-terminal tail affected histone-DNA interactions and substantially decreased nucleosome stability. These findings provide important information for understanding the complex roles of histone tails in regulating chromatin structure.