5S rRNA-recognition module of CTC family proteins and its evolution

The effects of amino acid replacements in the RNA-binding sites of homologous ribosomal proteins TL5 and L25 (members of the CTC family) on ability of these proteins to form stable complexes with ribosomal 5S RNA were studied. It was shown that even three simultaneous replacements of non-conserved amino acid residues by alanine in the RNA-binding site of TL5 did not result in noticeable decrease in stability of the TL5-5S rRNA complex. However, any replacement among five conserved residues in the RNA-binding site of TL5, as well as of L25 resulted in serious destabilization or complete impossibility of complex formation. These five residues form an RNA-recognition module in TL5 and L25. These residues are strictly conserved in proteins of the CTC family. However, there are several cases of natural replacements of these residues in TL5 and L25 homologs in Bacilli and Cyanobacteria, which are accompanied by certain changes in the CTC-binding site of 5S rRNAs of the corresponding organisms. CTC proteins and specific fragments of 5S rRNA of Enterococcus faecalis and Nostoc sp. were isolated, and their ability to form specific complexes was tested. It was found that these proteins formed specific complexes only with 5S rRNA of the same organism. This is an example of coevolution of the structures of two interacting macromolecules.     

N-terminal domain, residues 1-91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E.

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.     

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.     

General Stress Protein CTC from Bacillus subtilis Specifically Binds to Ribosomal 5S RNA

Two recombinant proteins of the CTC family were prepared: the general stress protein CTC from Bacillus subtilis and its homolog from Aquifex aeolicus. The general stress protein CTC from B. subtilis forms a specific complex with 5S rRNA and its stable fragment of 60 nucleotides, which contains internal loop E. The ribosomal protein TL5 from Thermus thermophilus, which binds with high affinity to 5S rRNA in the loop E region, was also shown to replace the CTC protein from B. subtilis in its complexes with 5S rRNA and its fragment. The findings suggest that the protein CTC from B. subtilis binds to the same site on 5S rRNA as the protein TL5. The protein CTC from A. aeolicus, which is 50 amino acid residues shorter from the N-terminus than the proteins TL5 from T. thermophilus and CTC from B. subtilis, does not interact with 5S rRNA.     

A chemical interference study on the interaction of ribosomal protein L11 from Escherichia coli with RNA molecules containing its binding site from 23S rRNA.

The interaction between ribosomal protein L11 from Escherichia coli and in vitro synthesized RNA containing its binding site from 23S rRNA was characterized by identifying nucleotides that interfered with complex formation when chemically modified by diethylpyrocarbonate or hydrazine. Chemically modified RNA was incubated with L11 under conditions appropriate for specific binding of L11 and the resulting protein-RNA complex was separated from unbound RNA on Mg(2+)-containing polyacrylamide gels. The ability to isolate L11 complexes on such gels was affected by the extent of modification by either reagent. Protein-bound and free RNAs were recovered and treated with aniline to identify their content of modified bases. Exclusion of RNA containing chemically altered bases from L11-associated material occurred for 29 modified nucleotides, located throughout the region corresponding to residues 1055-1105 in 23S rRNA. Ten bases within this region did not reproducibly inhibit binding when modified. Multiple bands of RNA were consistently observed on the nondenaturing gels, suggesting that significant intermolecular RNA-RNA interactions had occurred.     

The Thermus thermophilus5S rRNA–Protein Complex: Identification of Specific Binding Sites for Proteins L5 and L18 in the 5S rRNA

Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilushave earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5–RNA complex crystallized, and its structure determined to 2.3 Å. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment–L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.     

Three helical domains form a protein binding site in the 5S RNA-protein complex from eukaryotic ribosomes.

A ribosomal protein binding site in the eukaryotic 5S rRNA has been delineated by examining the effect of sequence variation and nucleotide modification on the RNA's ability to exchange into the EDTA-released, yeast ribosomal 5S RNA-protein complex. 5S RNAs of divergent sequence from a variety of eukaryotic origins could be readily exchanged into the yeast complex but RNA from bacterial origins was rejected. Nucleotide modifications in any of three analogous helical regions in eukaryotic 5S RNAs of differing origin reduced the ability of this RNA molecule to form homologous or heterologous RNA-protein complexes. Because sequence comparisons did not indicate common nucleotide sequences in the interacting helical regions, a model is suggested in which the eukaryotic 5S RNA binding protein does not simply recognize specific nucleotide sequences but interacts with three strategically oriented helical domains or functional groups within these domains. Two of the domains bear a limited sequence homology with each other and contain an unpaired nucleotide or "bulge" similar to that recently reported for one of the 5S RNA binding proteins in Escherichia coli (Peattie, D.A., Douthwaite, S., Garrett, R.A. and Noller, H.F. (1981) Proc. Natl. Acad. Sci. 78, 7331-7335). The results further indicate that the single ribosomal protein of eukaryotic 5S RNA-protein complexes interacts with the same region of the 5S rRNA molecule as do the multiple protein components in complexes of prokaryotic origin.     

Computational analysis of hydrogen bonds in protein-RNA complexes for interaction patterns

Structural analysis of protein-RNA complexes is labor-intensive, yet provides insight into the interaction patterns between a protein and RNA. As the number of protein-RNA complex structures reported has increased substantially in the last few years, a systematic method is required for automatically identifying interaction patterns. This paper presents a computational analysis of the hydrogen bonds in the most representative set of protein-RNA complexes. The analysis revealed several interesting interaction patterns. (1) While residues in the beta-sheets favored unpaired nucleotides, residues in the helices showed no preference and residues in turns favored paired nucleotides. (2) The backbone hydrogen bonds were more dominant than the base hydrogen bonds in the paired nucleotides, but the reverse was observed in the unpaired nucleotides. (3) The protein-RNA complexes contained more paired nucleotides than unpaired nucleotides, but the unpaired nucleotides were observed more frequently interacting with the proteins. And (4) Arg-U, Thr-A, Lys-A, and Asn-U were the most frequently observed pairs. The interaction patterns discovered from the analysis will provide us with useful information in predicting the structure of the RNA binding protein and the structure of the protein binding RNA.     

Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy.

It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements.     

Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site

BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408. Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair. The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex. CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.     

Xanthate sulfur as a hydrogen bond acceptor: the free xanthate anion and ligand sulfur in nickel tris ethylxanthate

Xanthates, like thiolates, form a variety of complexes with metals in which coordinating sulfur can serve as a hydrogen bond acceptor. Nickel tris xanthate complexes [Ni(xan)₃]⁻, (xan = o-ethylxanthate, N-(carbamoylmethyl)ethylxanthate) have been synthesized and compared by a combination of X-ray crystallographic and spectroscopic measurements. Recent results from our studies of N-H?S hydrogen bonding interactions in metal-xanthate complexes shows N-S distances to be longer than those in related thiolate complexes, indicative of weaker hydrogen bonds for the xanthates. The complex (Et₄N)[N-(carbamoylmethyl)ethylxanthate)] adopts an extended conformation in both the solid state and solution and lacks either intraligand or intermolecular N-H?S hydrogen bonds. The complex (CTA)[Ni(exa)₃] exhibits N-H?S hydrogen bonds between the amide group of the counterion and the ligand sulfur. The amide-sulfur N-H?S distance is 3.567 Å.     

Use of natural-abundance 15N-nmr spectroscopy to investigate the secondary structure of peptides: Gramicidin S

The natural-abundance ¹⁵N-nuclear magnetic resonance (nmr) spectrum of the cyclic decapeptide gramicidin S has been measured and assigned in the solvents dimethyl sulfoxide, methanol, and 2,2,2-trifluoroethanol. Three methods have been investigated to distinguish between peptide groups which are exposed to or shielded from the solvent. The solvent dependence of the ¹⁵N chemical shift is correlated with the two types of peptide group in gramicidin S? those with the carbonyl group exposed or shielded from the solvent. The second method monitors the lability of the N? H proton (via the collapse of the reduced ¹⁵N-¹H coupling) in the presence of added base used to promote intermolecular exchange—peptide protons shielded from the solvent exchange more slowly. The third method looks at the temperature dependence of the ¹⁵N chemical shifts in dimethyl sulfoxide. Here the data are not so distinctive as to allow the differentiation between solvent-exposed or shielded N? H bonds at all peptide groups.     

Acidic groups docked to well defined wetted pockets at the core of the binding interface: a tale of scoring and missing protein interactions in CAPRI

Some challenging targets in CAPRI (T24/25 and T26) involve binding solvent accessible acidic residues at the core of the binding interface, where they are always found immersed in crystal waters. In fact, Asp and Glu residues are more likely to form part of the hydrogen bond network of their surrounding crystal water molecules than to form a buried salt bridge. Interestingly, many of the crystal waters mediating the intermolecular interactions of the acidic groups are already present in the unbound structure, reinforcing the notion that some water molecules behave as an extension of the protein structure. This is in contrast to acidic groups found in the periphery of the binding interface that form ubiquitous salt bridges that cement the high affinity complex, while at the same time they are exposed to rapidly exchanging water molecules. Because of this, dichotomy implicit solvent scoring functions fail to properly rank these complexes by prioritizing salt bridges rather than water mediated contacts. A detailed analysis of Target 24, for which our group predicted two out of the four successful homology model complex structures, and Target 26 reveal how crystal waters shape the binding cavities of acidic groups prior to binding, in agreement with the theory of anchor residues as mediators of protein recognition.     

Mutual induced fit binding of Xenopus ribosomal protein L5 to 5S rRNA

A library of random mutations in Xenopus ribosomal protein L5 was generated by error-prone PCR and used to delineate the binding domain for 5S rRNA. All but one of the amino acid substitutions that affected binding affinity are clustered in the central region of the protein. Several of the mutations are conservative substitutions of non-polar amino acid residues that are unlikely to form energetically significant contacts to the RNA. Thermal denaturation, monitored by circular dichroism (CD), indicates that L5 is not fully structured and association with 5S rRNA increases the t(m) of the protein by 16 degrees C. L5 induces changes in the CD spectrum of 5S rRNA, establishing that the complex forms by a mutual induced fit mechanism. Deuterium exchange reveals that a considerable amount of L5 is unstructured in the absence of 5S rRNA. The fluorescence emission of W266 provides evidence for structural changes in the C-terminal region of L5 upon binding to 5S rRNA; whereas, protection experiments demonstrate that the N terminus remains highly sensitive to protease digestion in the complex. Analysis of the amino acid sequence of L5 by the program PONDR predicts that the N and C-terminal regions of L5 are intrinsically disordered, but that the central region, which contains three essential tyrosine residues and other residues important for binding to 5S rRNA, is likely to be structured. Initial interaction of the protein with 5S rRNA likely occurs through this region, followed by induced folding of the C-terminal region. The persistent disorder in the N-terminal domain is possibly exploited for interactions between the L5-5S rRNA complex and other proteins.     

A ribosomal protein from Thermus thermophilus is homologous to a general shock protein

The gene encoding the ribosomal protein from Thermus thermophilus, TL5, which binds to the 5S rRNA, has been cloned and sequenced. The codon usage shows a clear preference for G/C rich codons that is characteristic for many genes in thermophilic bacteria. The deduced amino acid sequence consists of 206 residues. The sequence of TL5 shows a strong similarity to a general shock protein from Bacillus subtilis, named CTC. The protein CTC is homologous in its N-terminal part to the 5S rRNA binding protein, L25, from E coli. An alignment of the TL5, CTC and L25 sequences displays a number of residues that are totally conserved. No clear sequence similarity was found between TL5 and other proteins which are known to bind to 5S rRNA. The evolutionary relationship of a heat shock protein in mesophiles and a ribosomal protein in thermophilic bacteria as well as a possible role of TL5 in the ribosome are discussed.     

Contribution of hydrogen bonds to protein stability

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.     

Dissecting the protein-RNA interface: the role of protein surface shapes and RNA secondary structures in protein-RNA recognition

Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein-RNA interactions.     

Proton nuclear magnetic resonance studies on glutamine-binding protein from Escherichia coli. Formation of intermolecular and intramolecular hydrogen bonds upon ligand binding

Proton nuclear magnetic resonance studies have revealed several structural and dynamic properties of the glutamine-binding protein of Escherichia coli. When this protein binds L-glutamine, six low-field, exchangeable proton resonances appear in the region from +5.5 to +10 parts per million downfield from water (or +10.2 to +14.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate). This suggests that the binding of L-glutamine induces specific conformational changes in the protein molecule, involving the formation of intermolecular and intramolecular hydrogen bonds between the glutamine-binding protein and L-glutamine, and within the protein molecule. The oxygen atom of the gamma-carbonyl group of L-glutamine is likely to be involved in the formation of an intermolecular hydrogen bond between the ligand and the binding protein. We have shown that at least one phenylalanine and one methyl-containing residue are spatially close to this intermolecular hydrogen-bonded proton. The intermolecular and intramolecular hydrogen-bonded protons of the ligand-protein complex undergo solvent exchange. The local conformations around these intermolecular and intramolecular hydrogen bonds are quite stable when subjected to pH and temperature variations. From these results, the utility of proton nuclear magnetic resonance spectroscopy for investigating such binding proteins has been shown, and a picture of the ligand-binding process can be drawn.     

5S-rRNA-containing ribonucleoproteins from rabbit muscle and liver. Complex and partial primary structures

A 5S-rRNA-containing ribonucleoprotein was purified to homogeneity from a rabbit muscle extract through its affinity to phosphofructokinase-1 and then structurally characterized. This RNP was compared to the 5S-rRNA-containing ribonucleoprotein extracted from rabbit liver ribosomal 60S subunits with EDTA. Analytical gel filtration revealed a molecular mass of 70-80 kDa for both complexes. Gel electrophoresis of the ribosomal complex revealed three protein components, one migrating as a band of 35 kDa and two other small polypeptides of apparently 16.5 kDa and 17.5 kDa. In the sarcoplasmic RNP these small polypeptides were absent. However, besides a major component of 35 kDa, up to five slightly larger and smaller species of 31.5-36.5 kDa were detected. Despite this heterogeneity, only one N-terminal amino acid sequence was obtained for the isolated sarcoplasmic protein, suggesting a C-terminal heterogeneity of one single polypeptide. Within the first 46 amino acid residues no difference between the sequences of the isolated 35-kDa components of sarcoplasmic and ribosomal complexes was found. Homology criteria indicated that this component belongs to the ribosomal protein L5 family. The RNA was identified by complete enzymatic sequencing as 5S rRNA; it was also identical in both complexes and is strongly homologous to 5S rRNA of man. Both L5-5S-RNA complexes could be resolved by hydroxyapatite chromatography into three species still consisting of both protein and RNA. 5'-Terminal dephosphorylation experiments showed that this heterogeneity is exclusively due to the differing number (1-3) of 5'-terminal phosphates. The two additional low-molecular-mass proteins were stably associated to the ribosomal RNP at high salt concentrations in a stoichiometry of about 2:1. They were identified as the acidic phosphoproteins P2/P3 by N-terminal sequencing. High phosphate concentrations facilitated their dissociation from the L5-5S-RNA complex. For the sarcoplasmic L5-5S-RNA complex a hitherto unknown interaction with phosphofructokinase-1, affecting the enzymatic properties, was demonstrated.