Mth10b, a unique member of the Sac10b family, does not bind nucleic acid

The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity in vitro. Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA) showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.     

Bacillus subtilis YkuK protein is distantly related to RNase H

In addition to one hypothetical viral sequence from Bacteriophage KVP40, the PfamA family of unknown function DUF458 (Pfam Accession No. PF04308) encompasses several uncharacterized bacterial proteins including Bacillus subtilis YkuK protein. Using Meta-BASIC, a highly sensitive method for detection of distant similarity between proteins, we assign DUF458 family members to the ribonuclease H-like (RNase H-like) superfamily. DUF458 sequences maintain all core secondary structure elements of RNase H-like fold and share several conserved, presumably active site residues with RNase HI, including an invariant DDE motif. In addition to providing a model structure for a previously uncharacterized protein family, this finding suggests that DUF458 proteins function as nucleases. The unusual phyletic pattern, together with a presence of DUF458 in several thermophilic organisms, may suggest a potential role of these proteins in DNA repair in stressful conditions such as an extreme heat or other stress that causes spore formation.     

Structure prediction and docking-based molecular insights of human YB-1 and nucleic acid interaction

Y-box-binding protein 1 (YB-1), a cold shock domain protein, is one of the most conserved nucleic acid-binding proteins. The multifunctional human YB-1 is a member of a large family of proteins with an evolutionary ancient cold shock domain. The presence of a cold shock domain is a specific feature of Y-box-binding proteins and allows attributing them to a wider group of proteins containing a cold shock domain. This protein is involved in a number of cellular processes including proliferation, differentiation and stress response. The YB-1 performs its function both in the cytoplasm and in the cell nucleus. In this study, we present the structure of full-length human YB-1 protein along with investigation of their nucleic acid-binding preferential. The study also focuses on biases for particular purine and pyrimidine bases. The overall goal of this study was to model and validate full-length YB-1 protein and to compare its nucleic acid-binding studies with previous reports.     

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.     

AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has a role in flower and seed development

The glycine-rich protein AtGRP2 is one of the four members of the cold-shock domain (CSD) protein family in Arabidopsis. It is characterized by the presence of a nucleic acid-binding CSD domain, two glycine-rich domains and two CCHC zinc-fingers present in nucleic acid-binding proteins. In an attempt to further understand the role of CSD/GRP proteins in plants, we have proceeded to the functional characterization of the AtGRP2 gene. Here, we demonstrate that AtGRP2 is a nucleo-cytoplasmic protein involved in Arabidopsis development with a possible function in cold-response. Expression analysis revealed that the AtGRP2 gene is active in meristematic tissues, being modulated during flower development. Down-regulation of AtGRP2 gene, using gene-silencing techniques resulted in early flowering, altered stamen number and affected seed development. A possible role of AtGRP2 as an RNA chaperone is discussed.     

Crystal structure of a heterodimer of editosome interaction proteins in complex with two copies of a cross-reacting nanobody

The parasite Trypanosoma brucei, the causative agent of sleeping sickness across sub-Saharan Africa, depends on a remarkable U-insertion/deletion RNA editing process in its mitochondrion. A approximately 20 S multi-protein complex, called the editosome, is an essential machinery for editing pre-mRNA molecules encoding the majority of mitochondrial proteins. Editosomes contain a common core of twelve proteins where six OB-fold interaction proteins, called A1-A6, play a crucial role. Here, we report the structure of two single-strand nucleic acid-binding OB-folds from interaction proteins A3 and A6 that surprisingly, form a heterodimer. Crystal growth required the assistance of an anti-A3 nanobody as a crystallization chaperone. Unexpectedly, this anti-A3 nanobody binds to both A3(OB) and A6, despite only ~40% amino acid sequence identity between the OB-folds of A3 and A6. The A3(OB)-A6 heterodimer buries 35% more surface area than the A6 homodimer. This is attributed mainly to the presence of a conserved Pro-rich loop in A3(OB). The implications of the A3(OB)-A6 heterodimer, and of a dimer of heterodimers observed in the crystals, for the architecture of the editosome are profound, resulting in a proposal of a 'five OB-fold center' in the core of the editosome.     

The trypanosome leucine repeat gene in the variant surface glycoprotein expression site encodes a putative metal-binding domain and a region resembling protein-binding domains of yeast, Drosophila, and mammalian proteins.

We have identified a new variant surface glycoprotein expression site-associated gene (ESAG) in Trypanosoma brucei, the trypanosome leucine repeat (T-LR) gene. Like most other ESAGs, it is expressed in a life cycle stage-specific manner. The N-terminal 20% of the predicted T-LR protein resembles the metal-binding domains of nucleic acid-binding proteins. The remainder is composed of leucine-rich repeats that are characteristic of protein-binding domains found in a variety of other eucaryote proteins. This is the first report of leucine-rich repeats and potential nucleic acid-binding domains on the same protein. The T-LR gene is adjacent to ESAG 4, which has homology to the catalytic domain of adenylate cyclase. This is intriguing, since yeast adenylate cyclase has a leucine-rich repeat regulatory domain. The leucine-rich repeat and putative metal-binding domains suggest a possible regulatory role that may involve adenylate cyclase activity or nucleic acid binding.     

Crystal structure of TbAlba1 from Trypanosoma brucei

Alba (Acetylation lowers binding affinity) domain is a small, dimeric nucleic acid-binding domain, which is widely distributed in archaea and numbers of eukaryotes. Alba domain containing proteins have been reported to be involved in many cellular processes, such as regulation of translation, maintaining genome stability, regulation of RNA processing and so on. In Trypanosoma brucei (T. brucei), there are four Alba proteins identified, which are named TbAlba1 to TbAlba4. However, the structure and function of TbAlba proteins are still unknown. Here, we solved the crystal structure of TbAlba1 to a resolution of 2.46 Å. TbAlba1 adopts a similar Alba-fold, which comprises of four β-strands (β1-β4) and three long α-helices (α1-α3). Furthermore, TbAlba1 displays some structural features quite different from other Alba proteins. These differences may imply the diverse biological roles of Alba family members.     

Structure and function of argonaute proteins

Argonaute (Ago) family proteins are multidomain proteins expressed in prokaryotic and eukaryotic organisms. In eukaryotes, Ago proteins are most well known for their roles in RNA silencing. In prokaryotes, the functions of Ago proteins are unknown, but based on their similarity to eukaryotic Ago proteins, they could be involved in nucleic acid-directed regulatory pathways related to RNA silencing. Recent structural and biochemical studies have shed new light on the function of this family of proteins. These studies reveal how these proteins recognize and cleave RNA and suggest a function for prokaryotic family members.     

Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis.

Rat cellular retinol-binding protein II (CRBP II) is an abundant 134-residue intestinal protein that binds all-trans-retinol and all-trans-retinal. It belongs to a family of homologous, 15-kDa cytoplasmic proteins that bind hydrophobic ligands in a noncovalent fashion. These binding proteins include a number of proteins that bind long chain fatty acids. X-ray analyses of the structure of two family members, rat intestinal fatty acid-binding protein and bovine myelin P2 protein, indicate that they have a high degree of conformational similarity and that the carboxylate group of their bound fatty acid interacts with a delta-guanidium group of at least 1 of 2 "buried" arginine residues. These 2 Arg residues are conserved in other family members that bind long chain fatty acids and in cellular retinoic acid-binding protein, but are replaced by Gln109 and Gln129 in CRBP II. We have genetically engineered two amino acid substitutions in CRBP II: 1) Gln109 to Arg and 2) Gln129 to Arg. The purified Escherichia coli-derived CRBP II mutant proteins were analyzed by fluorescence and nuclear magnetic resonance spectroscopy. Both mutants exhibit markedly decreased binding of all-trans-retinol and all-trans-retinaldehyde, but no increased binding of all-trans-retinoic acid. Arg substitution for Gln109 but not for Gln129 produces a dramatic increase in palmitate binding activity. Analysis of the endogenous fatty acids associated with the purified E. coli-derived proteins revealed that E. coli-derived intestinal fatty acid binding protein and the Arg109 CRBP II mutant are complexed with endogenous fatty acids in a qualitatively and quantitatively similar manner. These results provide evidence that this internal Arg may play an important role in the binding of long chain fatty acids by members of this protein family.     

Structural Features of the Glutamate Transporter Family

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C₄-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C₄-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning α-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids

Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase superfamily, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a potential regulatory role. Through a wider analysis of other poorly characterized DNA-modifying enzymes we also show that the phage Mu Mom-like proteins, which catalyze the N6-carbamoylmethylation of adenines, are also linked to diverse families of bacterial transposases, suggesting that DNA modification by transposable elements might have a more general presence than previously appreciated. Among the other families of 2-oxoglutarate- and iron(II)-dependent dioxygenases identified in this study, one which is found in algae, is predicted to mainly comprise of RNA-modifying enzymes and shows a striking diversity in protein domain architectures suggesting the presence of RNA modifications with possibly unique adaptive roles. The results presented here are likely to provide the means for future investigation of unexpected epigenetic modifications, such as hydroxymethyl cytosine, that could profoundly impact our understanding of gene regulation and processes such as DNA demethylation.     

Novel Predicted RNA-Binding Domains Associated with the Translation Machinery

Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60–65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions. Received: 15 May 1998 / Accepted: 20 July 1998     

Sm-like protein Hfq: location of the ATP-binding site and the effect of ATP on Hfq-- RNA complexes

Sm-like proteins are ubiquitous ring-shaped oligomers that exhibit a variety of nucleic acid-binding activities. They have been linked functionally to various cellular events involving RNA, and it is generally believed that their activity is exerted via the passive binding of nucleic acids. Our earlier studies of the Sm-like Escherichia coli protein Hfq provided the first evidence indicating that Hfq is an ATP-binding protein. Using a combination of biochemical and genetic techniques, we have now determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. The results of RNA footprinting and binding analyses suggest that ATP binding by the Hfq-RNA complex results in its significant destabilization. RNA footprinting indicates deprotection of Hfq-bound RNA tracts in the presence of ATP, suggestive of their release by the protein. The results reported herein broaden the scope of potential in vivo roles for Hfq and other Sm-like proteins.