Refining the overall structure and subdomain orientation of ribosomal protein S4 delta41 with dipolar couplings measured by NMR in uniaxial liquid crystalline phases.

Prokaryotic protein S4 initiates assembly of the small ribosomal subunit by binding to 16 S rRNA. Residues 43-200 of S4 from Bacillus stearothermophilus (S4 Delta41) bind to both 16 S rRNA and to a mRNA pseudoknot. In order to obtain structure-based insights regarding RNA binding, we previously determined the solution structure of S4 Delta41 using NOE, hydrogen bond, and torsion angle restraints. S4 Delta41 is elongated, with two distinct subdomains, one all helical, the other including a beta-sheet. In contrast to the high resolution structures obtained for each individual subdomain, their relative orientation was not precisely defined because only 17 intersubdomain NOE restraints were determined. Compared to the 1.7 A crystal structure, when the sheet-containing subdomains are superimposed, the helical subdomain is twisted by almost 45 degrees about the long axis of the molecule in the solution structure. Because variations in subdomain orientation may explain how the protein recognizes multiple RNA targets, our current goal is to determine the orientation of the subdomains in solution with high precision. To this end, NOE assignments were re-examined. NOESY experiments on a specifically labeled sample revealed that one of the intersubdomain restraints had been misassigned. However, the revised set of NOE restraints produces solution structures that still have imprecisely defined subdomain orientations and that lie between the original NMR structure and the crystal structure. In contrast, augmenting the NOE restraints with N-H dipolar couplings, measured in uniaxial liquid crystalline phases, clearly establishes the relative orientation of the subdomains. Data obtained from two independent liquid crystalline milieux, DMPC/DHPC bicelles and the filamentous bacteriophage Pf1, show that the relative orientation of the subdomains in solution is quite similar to the subdomain orientation in the crystal structure. The solution structure, refined with dipolar data, is presented and its implications for S4's RNA binding activity are discussed.     

Solution structure of the N-terminal zinc fingers of the Xenopus laevis double-stranded RNA-binding protein ZFa

Several zinc finger proteins have been discovered recently that bind specifically to double-stranded RNA. These include the mammalian JAZ and wig proteins, and the seven-zinc finger protein ZFa from Xenopus laevis. We have determined the solution structure of a 127 residue fragment of ZFa, which consists of two zinc finger domains connected by a linker that remains unstructured in the free protein in solution. The first zinc finger consists of a three-stranded beta-sheet and three helices, while the second finger contains only a two-stranded sheet and two helices. The common structures of the core regions of the two fingers are superimposable. Each finger has a highly electropositive surface that maps to a helix-kink-helix motif. There is no evidence for interactions between the two fingers, consistent with the length (24 residues) and unstructured nature of the intervening linker. Comparison with a number of other proteins shows similarities in the topology and arrangement of secondary structure elements with canonical DNA-binding zinc fingers, with protein interaction motifs such as FOG zinc fingers, and with other DNA-binding and RNA-binding proteins that do not contain zinc. However, in none of these cases does the alignment of these structures with the ZFa zinc fingers produce a consistent picture of a plausible RNA-binding interface. We conclude that the ZFa zinc fingers represent a new motif for the binding of double-stranded RNA.     

Solution structure of the Vts1 SAM domain in the presence of RNA

The yeast Vts1 SAM (sterile alpha motif) domain is a member of a new class of SAM domains that specifically bind RNA. To elucidate the structural basis for RNA binding, the solution structure of the Vts1 SAM domain, in the presence of a specific target RNA, has been solved by multidimensional heteronuclear NMR spectroscopy. The Vts1 SAM domain retains the "core" five-helix-bundle architecture of traditional SAM domains, but has additional short helices at N and C termini, comprising a small substructure that caps the core helices. The RNA-binding surface of Vts1, determined by chemical shift perturbation, maps near the ends of three of the core helices, in agreement with mutational data and the electrostatic properties of the molecule. These results provide a structural basis for the versatility of the SAM domain in protein and RNA-recognition.     

The first structure of an RNA m5C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate

The crystal structure of E. coli Fmu, determined at 1.65 A resolution for the apoenzyme and 2.1 A resolution in complex with AdoMet, is the first representative of the 5-methylcytosine RNA methyltransferase family that includes the human nucleolar proliferation-associated protein p120. Fmu contains three subdomains which share structural homology to DNA m(5)C methyltransferases and two RNA binding protein families. In the binary complex, the AdoMet cofactor is positioned within the active site near a novel arrangement of two conserved cysteines that function in cytosine methylation. The site is surrounded by a positively charged cleft large enough to bind its unique target stem loop within 16S rRNA. Docking of this stem loop RNA into the structure followed by molecular mechanics shows that the Fmu structure is consistent with binding to the folded RNA substrate.     

Structural and functional characterization of the TgDRE multidomain protein, a DNA repair enzyme from Toxoplasma gondii

The parasite Toxoplasma gondii expresses a 55 kDa protein or TgDRE that belongs to a novel family of proteins characterized by the presence of three domains, a human splicing factor 45-like motif (SF), a glycine-rich motif (G-patch), and a RNA recognition motif (RRM). The two latter domains are mainly known as RNA-binding domains, and their presence in TgDRE, whose partial DNA repair function was demonstrated, suggests that the protein could also be involved in the RNA metabolism. In this work, we characterized the structure and function of the different domains by using single or multidomain proteins to define their putative role. The SF45-like domain has a helical conformation and is involved in the oligomerization of the protein. The G-patch domain, mainly unstructured on its own as well as in the presence of the SF upstream and RRM downstream domains, is able to bind small RNA oligonucleotides. We also report the structure determination of the RRM domain from the NMR data. It adopts a classical betaalphabetabetaalphabeta topology consisting of a four-stranded beta sheet packed against two alpha helices but does not present the key residues for the RNA interaction. In contrast, our analysis shows that the RRM of TgDRE is not only unable to bind small RNA oligonucleotides but it also shares the protein-protein interaction characteristics with two unusual RRMs of the U2AF heterodimeric splicing factor. The presence of both RNA- and protein-binding domains seems to indicate that TgDRE could also be involved in RNA metabolism.     

Unusual mRNA pseudoknot structure is recognized by a protein translational repressor

Translation of ribosomal proteins in the alpha operon of E. coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon. RNA structure mapping experiments have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin. Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices. The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected. The overall structure is an unusual "double pseudoknot" linking a hairpin upstream of the ribosome binding site with sequences 2-10 codons downstream of the initiation codon. Stabilization of this structure by S4 could account for translational repression.     

Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery

The X-ray structure of the phylogenetically conserved middle portion of human eukaryotic initiation factor (eIF) 4GII has been determined at 2.4 A resolution, revealing a crescent-shaped domain consisting of ten alpha helices arranged as five HEAT repeats. Together with the ATP-dependent RNA helicase eIF4A, this HEAT domain suffices for 48S ribosomal complex formation with a picornaviral RNA internal ribosome entry site (IRES). Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of this essential component of the translation initiation machinery that, respectively, bind eIF4A and a picornaviral IRES. The structural and biochemical results provide mechanistic insights into both cap-dependent and cap-independent translation initiation.     

In vitro RNA selection identifies RNA ligands that specifically bind to eukaryotic translation initiation factor 4B: the role of the RNA remotif.

Translation initiation factor elF-4B is an RNA-binding protein that promotes the association of the mRNA to the 40S ribosomal subunit. One of its better characterized features is the ability to stimulate the activity of the DEAD box RNA hilicase elF-4A. In addition to an RNA recognition motif (RRM) located near its amino-terimus, elF-4B contains an RNA-binding region in its carboxy-terminal half. The elF-4A helicase stimulatory activity resides in the carboxy-terminal half of elF-4B, and the RRM has little impact on this function.To better understand the role of the elF-4B RRM, it was of interest to identify its specific RNA target sequence. To this end, it vitro RNA selection/amplifications were performed using various portions of elF-4B. These experiments were designed to test the RNA recognition specificity of the two elF-4B regions implicated in RNA binding and to assess the influence of elF-4A on the RNA-binding specificity. The RRM was shown to bind with high affinity to an RNA stem-loop structure with conserved primary sequence elements. Discrete point mutations in an in vitro-selected RNA identified residues critical for RNA binding. Neither the carboxy-terminal RNA-interaction region, nor elF-4A, influenced the structure of the high-affinity RNA ligands selected by elF-4B, and elF-4A by itself did not select any specific RNA target. Previous studies have demonstrated an interaction of elF-4B with ribosomes, and it was suggested that this association is mediated through binding to ribosomal RNA. We show that the RRM of elF-4B interacts directly with 18S rRNA and this interaction is inhibited by an excess of the elF-4B in vitro-selected RNA. ElF-4B could bind simultaneously to two different RNA molecules, supporting a model whereby elF-4B promotes ribosome binding to the 5 untranslated region of a mRNA by bridging it to 18S rRNA.     

Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains.

BACKGROUND: Nova-1 and Nova-2 are related neuronal proteins that were initially cloned using antisera obtained from patients with the autoimmune neurological disease paraneoplastic opsoclonus-myoclonus ataxia (POMA). Both of these disease gene products contain three RNA-binding motifs known as K-homology or KH domains, and their RNA ligands have been identified via binding-site selection experiments. The KH motif structure has been determined previously using NMR spectroscopy, but not using X-ray crystallography. Many proteins contain more than one KH domain, yet there is no published structural information regarding the behavior of such multimers. RESULTS: We have obtained the first X-ray crystallographic structures of KH-domain-containing proteins. Structures of the third KH domains (KH3) of Nova-1 and Nova-2 were determined by multiple isomorphous replacement and molecular replacement at 2.6 A and 2.0 A, respectively. These highly similar RNA-binding motifs form a compact protease-resistant domain resembling an open-faced sandwich, consisting of a three-stranded antiparallel beta sheet topped by three alpha helices. In both Nova crystals, the lattice is composed of symmetric tetramers of KH3 domains that are created by two dimer interfaces. CONCLUSIONS: The crystal structures of both Nova KH3 domains are similar to the previously determined NMR structures. The most significant differences among the KH domains involve changes in the positioning of one or more of the alpha helices with respect to the betasheet, particularly in the NMR structure of the KH1 domain of the Fragile X disease protein FMR-1. Loop regions in the KH domains are clearly visible in the crystal structure, unlike the NMR structures, revealing the conformation of the invariant Gly-X-X-Gly segment that is thought to participate in RNA-binding and of the variable region. The tetrameric arrangements of the Nova KH3 domains provide insights into how KH domains may interact with each other in proteins containing multiple KH motifs.     

Structural and biochemical basis for misfolded RNA recognition by the Ro autoantigen

The Ro autoantigen is ring-shaped, binds misfolded noncoding RNAs and is proposed to function in quality control. Here we determine how Ro interacts with misfolded RNAs. Binding of Ro to misfolded precursor (pre)-5S ribosomal RNA requires a single-stranded 3' end and helical elements. As mutating most sequences of the helices and tail results in modest decreases in binding, Ro may be able to associate with a range of RNAs. Ro binds several other RNAs that contain single-stranded tails. A crystal structure of Ro bound to a misfolded pre-5S rRNA fragment reveals that the tail inserts into the cavity, while a helix binds on the surface. Most contacts of Ro with the helix are to the backbone. Mutagenesis reveals that Ro has an extensive RNA-binding surface. We propose that Ro uses this surface to scavenge RNAs that fail to bind their specific RNA-binding proteins.     

Ribosome-associated factor Y adopts a fold resembling a double-stranded RNA binding domain scaffold.

Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112-residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a betaalphabetabetabetaalpha topology and represents a compact two-layered sandwich of two nearly parallel alpha helices packed against the same side of a four-stranded beta sheet. The 23 C-terminal residues of the protein are disordered. Long-range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C-terminal region of helix 1 and the loop linking this helix with strand beta2 show significant conformational exchange in the ms- micro s time scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C-terminal segments of both alpha helices, which most probably constitutes an RNA binding site. The observed betaalphabetabetabetaalpha topology of pY resembles the alphabetabetabetaalpha topology of double-stranded RNA-binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA-binding site in pY does not coincide with the RNA-binding surface of the dsRBDs.     

RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids

The RNA-binding/dimerization domain of the NS1 protein of influenza A virus (73 amino acids in length) exhibits a novel dimeric six-helical fold. It is not known how this domain binds to its specific RNA targets, one of which is double-stranded RNA. To elucidate the mode of RNA binding, we introduced single alanine replacements into the NS1 RNA-binding domain at specific positions in the three-dimensional structure. Our results indicate that the dimer structure is essential for RNA binding, because any alanine replacement that causes disruption of the dimer also leads to the loss of RNA-binding activity. Surprisingly, the arginine side chain at position 38, which is in the second helix of each monomer, is the only amino-acid side chain that is absolutely required only for RNA binding and not for dimerization, indicating that this side chain probably interacts directly with the RNA target. This interaction is primarily electrostatic, because replacement of this arginine with lysine had no effect on RNA binding. A second basic amino acid, the lysine at position 41, which is also in helix 2, makes a strong contribution to the affinity of binding. We conclude that helix 2 and helix 2', which are antiparallel and next to each other in the dimer conformation, constitute the interaction face between the NS1 RNA-binding domain and its RNA targets, and that the arginine side chain at position 38 and possibly the lysine side chain at position 41 in each of these antiparallel helices contact the phosphate backbone of the RNA target.