NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way.     

NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.

In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small N-terminal domain. The solution structure of one of the N-terminal domains from Saccharomyces cerevisiae RNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel beta-sheet and two short alpha-helices which pack onto opposite faces of the beta-sheet. Conserved residues involved in packing the alpha-helices onto the beta-sheet form the hydrophobic core of the domain. Three highly conserved and solvent exposed residues are implicated in RNA binding, W22, K38 and K39. The beta-beta-alpha-beta-alpha topology of the domain differs from the structures of known RNA binding domains such as the double-stranded RNA binding domain (dsRBD), the hnRNP K homology (KH) domain and the RNP motif. However, structural similarities exist between this domain and the N-terminal domain of ribosomal protein L9 which binds to 23 S ribosomal RNA.     

Poxvirus K7 protein adopts a Bcl-2 fold: biochemical mapping of its interactions with human DEAD box RNA helicase DDX3

Poxviruses have evolved numerous strategies to evade host innate immunity. Vaccinia virus K7 is a 149-residue protein with previously unknown structure that is highly conserved in the orthopoxvirus family. K7 bears sequence and functional similarities to A52, which interacts with interleukin receptor-associated kinase 2 and tumor necrosis factor receptor-associated factor 6 to suppress nuclear factor κB activation and to stimulate the secretion of the anti-inflammatory cytokine interleukin-10. In contrast to A52, K7 forms a complex with DEAD box RNA helicase DDX3, thereby suppressing DDX3-mediated ifnb promoter induction. We determined the NMR solution structure of K7 to provide insight into the structural basis for poxvirus antagonism of innate immune signaling. The structure reveals an α-helical fold belonging to the Bcl-2 family despite an unrelated primary sequence. NMR chemical-shift mapping studies have localized the binding surface for DDX3 on a negatively charged face of K7. Furthermore, thermodynamic studies have mapped the K7-binding region to a 30-residue N-terminal fragment of DDX3, ahead of the core RNA helicase domains.     

Modeling by homology of RNA binding domain in A1 hnRNP protein

Eukaryotic nuclear RNA binding proteins share a common sequence motif thought to be implicated in RNA binding. One of the two domains present in A1 hnRNP protein, has been modelled by homology in order to make a prediction of the main features of the RNA binding site. Acylphosphatase (EC 3.6.1.7) was selected as template for the modeling experiment. The predicted RNA binding site is a beta-sheet containing the two RNP consensus sequences as well as lysines and arginines conserved among the family.     

The yeast homolog of the U1 snRNP protein 70K is encoded by the SNP1 gene.

The product of the yeast SNP1 gene has high homology to two domains of the metazoan U1 snRNP protein 70K, which binds to stem/loop I of the U1 RNA. However, the absence of other domains conserved in metazoan 70K and the minimal effect of yeast U1 RNA stem/loop I deletion make the assignment of SNP1 as yeast 70K less clear. To address this question, we have expressed the SNP1 gene as a fusion protein in E. coli and developed a gel shift assay for U1 RNA binding. We show here that the product of the yeast SNP1 gene binds directly and specifically to the first 47 nucleotides of yeast U1 RNA, which include the stem/loop 1 structure. We therefore conclude that the SNP1 gene product is the yeast 70K homolog. This is the first yeast protein to be identified as a homolog of a metazoan snRNP protein.     

Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen "closed" conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.     

Structural and biochemical characterization of the yeast exosome component Rrp40

The exosome is a protein complex that is important in both degradation and 3'-processing of eukaryotic RNAs. We present the crystal structure of the Rrp40 exosome subunit from Saccharomyces cerevisiae at a resolution of 2.2 A. The structure comprises an S1 domain and an unusual KH (K homology) domain. Close packing of the S1 and KH domains is stabilized by a GxNG sequence, which is uniquely conserved in exosome KH domains. Nuclear magnetic resonance data reveal the presence of a manganese-binding site at the interface of the two domains. Isothermal titration calorimetry shows that Rrp40 and archaeal Rrp4 alone have very low intrinsic affinity for RNA. The affinity of an archaeal core exosome for RNA is significantly increased in the presence of the S1-KH subunit Rrp4, indicating that multiple subunits might contribute to cooperative binding of RNA substrates by the exosome.     

Structural insights into RNA recognition by RIG-I

Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA (dsRNA). The dsRNA is sheathed within a network of protein domains that include a conserved "helicase" domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of the superfamily 2 helicases, and this structure reveals complex interplay between motor domains, accessory mechanical domains, and RNA that has implications for understanding the nanomechanical function of this protein family and other ATPases more broadly.     

Crystal structure of E. coli YhbY: a representative of a novel class of RNA binding proteins

E. coli YhbY belongs to a conserved family of hypothetical proteins represented in eubacteria, archaea, and plants (Pfam code UPF0044). Three maize proteins harboring UPF0044-like domains are required for chloroplast group II intron splicing, and bioinformatic data suggest a role for prokaryotic UPF0044 members in translation. The crystal structure of YhbY has been determined. YhbY has a fold similar to that of the C-terminal domain of translation initiation factor 3 (IF3C), which binds to 16S rRNA in the 30S ribosome. Modeling studies indicate that the same surface is highly basic in all members of UPF0044, suggesting a conserved RNA binding surface. Taken together, the evidence suggests that members of UPF0044 constitute a previously unrecognized class of RNA binding domain.     

The cytoadherence linked asexual gene family of Plasmodium falciparum: are there roles other than cytoadherence?

The binding of erythrocytes infected with Plasmodium falciparum to the endothelium lining the small blood vessels of the brain and other organs can mediate severe pathology. A region at the right end of chromosome 9 has been implicated in the binding of parasitised erythrocytes to the endothelial receptor CD36. A gene expressed in asexual erythrocytic stage parasites has been identified in this region and termed the cytoadherence linked asexual gene (clag). Antisense RNA production and targeted gene disruption of clag resulted in greatly reduced binding to CD36. Hybridisation to 3D7 chromosomes showed clag to be a part of a gene family of at least nine members. All members analysed so far have a conserved gene structure of at least nine exons, as well as putative transmembrane domains. The possible functions of the gene family are discussed.     

Cooperative binding of the hnRNP K three KH domains to mRNA targets

The heterogeneous nuclear ribonucleoprotein (hnRNP) K homology (KH) domain is an evolutionarily conserved module that binds short ribonucleotide sequences. KH domains most often are present in multiple copies per protein. In vitro studies of hnRNP K and other KH domain bearing proteins have yielded conflicting results regarding the relative contribution of each KH domain to the binding of target RNAs. To assess this RNA-binding we used full-length hnRNP K, its fragments and the yeast ortholog as baits in the yeast three-hybrid system. The results demonstrate that in this heterologous in vivo system, the three KH domains bind RNA synergistically and that a single KH domain, in comparison, binds RNA weakly.     

The Xenopus laevis poly(A) binding protein is composed of multiple functionally independent RNA binding domains.

A family of eukaryotic RNA binding proteins is defined by the conserved RNP motif. The poly(A) binding protein has four such motifs. We report on the isolation and structural characterization of several variant cDNA clones, as well as of a gene encoding this protein in Xenopus laevis embryos. Wild-type protein as well as truncated versions carrying isolated single motifs or artificial combinations of two and more such elements were characterized for their ability to bind specifically to RNA homopolymers. Three of the isolated repeats were functional in specific RNA binding, whereas the N-terminal RNP motif was non-functional. Combinatorial effects in RNA binding were measured with constructs carrying multiple repeats, which were not predictable from the activity of isolated domains.     

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.     

RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis

The mammalian translation initiation factor 4A (eIF4A) is a prototype member of the DEAD-box RNA helicase family that couples ATPase activity to RNA binding and unwinding. In the crystal form, eIF4A has a distended "dumbbell" structure consisting of two domains, which probably undergo a conformational change, on binding ATP, to form a compact, functional structure via the juxtaposition of the two domains. Moreover, additional conformational changes between two domains may be involved in the ATPase and helicase activity of eIF4A. The molecular basis of these conformational changes, however, is not understood. Here, we generated RNA aptamers with high affinity for eIF4A by in vitro RNA selection-amplification. On binding, the RNAs inhibit ATP hydrolysis. One class of RNAs contains members that exhibit dissociation constant of 27 nM for eIF4A and severely inhibit cap-dependent in vitro translation. The binding affinity was increased on Arg substitution in the conserved motif Ia of eIF4A, which probably improves a predicted arginine network to bind RNA substrates. Selected RNAs, however, failed to bind either domain of eIF4A that had been split at the linker site. These findings suggest that the selected RNAs interact cooperatively with both domains of eIF4A, either in the dumbbell or the compact form, and entrap it into a dead-end conformation, probably by blocking the conformational change of eIF4A. The selected RNAs, therefore, represent a new class of specific inhibitors that are suitable for the analysis of eukaryotic initiation, and which pose a potential therapeutic against malignancies that are caused by aberrant translational control.     

The Bacillus subtilis RNA Helicase YxiN is Distended in Solution

The Bacillus subtilis YxiN protein is a modular three-domain RNA helicase of the DEx(D/H)-box protein family. The first two domains form the highly conserved helicase core, and the third domain confers RNA target binding specificity. Small angle x-ray scattering on YxiN and two-domain fragments thereof shows that the protein has a distended structure in solution, in contrast to helicases involved in replication processes. These data are consistent with a chaperone activity in which the carboxy-terminal domain of YxiN tethers the protein to the vicinity of its targets and the helicase core is free to transiently interact with RNA duplexes, possibly to melt out misfolded elements of secondary structure.     

Functional implications for a prototypical K-turn binding protein from structural and dynamical studies of 15.5K

The kink-turn (K-turn) motif is recognized and bound by a family of proteins that act as nucleation factors for ribonucleoparticle assembly. The binding of various proteins to a conserved RNA structural motif known as the K-turn has been shown to be an important component of regulation in the ribosome, in the spliceosome, and in RNA modification. 15.5K is a prototypical example of a K-turn binding protein, which has been shown to bind the 5'-U4 stem-loop of the spliceosome and the box C/D motif. We describe the solution NMR structure of free 15.5K, as well as studies of conformational flexibility from 15N NMR relaxation and H/D exchange experiments. The protein appears well-structured aside from conformational fluctuation in alpha3. Flexibility in fast time scale motions and the observation of limited intermediate and slow motions further characterize the free protein and may suggest local contributions to recognition and binding.     

Solution NMR structure of VF0530 from Vibrio fischeri reveals a nucleic acid-binding function

Protein domain family PF09905 (DUF2132) is a family of small domains of unknown function that are conserved in a wide range of bacteria. Here we describe the solution NMR structure of the 80-residue VF0530 protein from Vibrio fischeri, the first structural representative from this protein domain family. We demonstrate that the structure of VF0530 adopts a unique four-helix motif that shows some similarity to the C-terminal double-stranded DNA (dsDNA) binding domain of RecA, as well as other nucleic acid binding domains. Moreover, gel shift binding data indicate a potential dsDNA binding role for VF0530.     

Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain

We have determined the crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 at 1.9 A resolution. This protein is a member of the Escherichia coli ygcH sequence family, which contains approximately 15 sequence homologs of bacterial origin. These homologs have a high isoelectric point. The crystal structure reveals that TTHB192 consists of two independently folded domains, and that each domain exhibits a ferredoxin-like fold with a four-stranded antiparallel beta-sheet packed on one side by alpha-helices. These two tandem domains face each other to generate a beta-sheet platform. TTHB192 displays overall structural similarity to Sex-lethal protein and poly(A)-binding protein fragments. These proteins have RNA binding activity which is supported by a beta-sheet platform formed by two tandem repeats of an RNA recognition motif domain with signature sequence motifs on the beta-sheet surface. Although TTHB192 does not have the same signature sequence motif as the RNA recognition motif domain, the presence of an evolutionarily conserved basic patch on the beta-sheet platform could be functionally relevant for nucleic acid-binding. This report shows that TTHB192 and its sequence homologs adopt an RNA recognition motif-like domain and provides the first testable functional hypothesis for this protein family.