Essential amino acids of the hantaan virus N protein in its interaction with RNA

The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.     

Molecular dissection of influenza virus nucleoprotein: deletion mapping of the RNA binding domain.

Influenza virus nucleoprotein (NP) is associated with the genome RNA, forming ribonucleoprotein cores. To identify the amino acid sequence involved in RNA binding, we performed Northwestern blot analysis with a set of N- and C-terminal deletion mutants of NP produced in Escherichia coli. The RNA binding region has been mapped between amino acid residues 91 and 188, a stretch of residues that contains a sequence that is not only highly conserved among NPs from A-, B-, and C-type influenza viruses but also similar to the RNA binding domain of a plant virus movement protein.     

Purification and Molecular Characterization of the Brain Synaptic Membrane Glutamate-Binding Protein

A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.     

Two distinct segments of the hepatitis B virus surface antigen contribute synergistically to its association with the viral core particles

The long surface antigen polypeptide (L-HBsAg) of hepatitis B virus (HBV) is believed to mediate contact between the virus envelope and nucleocapsid protein (HBcAg). The N and C termini of L-HBsAg were shortened progressively in order to define the minimum contiguous sequence of amino acids that contains the residues necessary for association with HBcAg. The resulting mutants were expressed in rabbit reticulocyte lysates and their interaction with HBcAg was examined with an immunoprecipitation assay and an equilibrium binding assay in solution to give relative dissociation constants. Binding of HBcAg particles by L-HBsAg displayed two widely differing dissociation constants, indicating two distinct binding sites between the molecules. The two distinct sites, one located between residues 24 and 191 and the other between residues 191 and 322 of L-HBsAg, contribute synergistically to high-affinity binding to HBcAg, but disruption of either of these segments resulted in a much weaker interaction showing only one dissociation constant. Inhibition of the interaction by peptides that bind to the tips of the nucleocapsid spikes differentiated contacts in HBcAg for the two binding domains in L-HBsAg and implied that the amino-terminal binding domain contacts the tips of the HBcAg spikes. Analysis of specific single amino acid mutants of L-HBsAg showed that Arg92 played an important role in the interaction.     

Surface lysine and tyrosine residues are required for interaction of the major herpes simplex virus type 1 DNA-binding protein with single-stranded DNA.

Modification of the herpes simplex virus type 1 major DNA-binding protein (ICP8) with reagents and conditions specific for arginine, lysine, and tyrosine residues indicates that surface lysine and tyrosine residues are required for the interaction of this protein with single-stranded DNA. Modification of either of these two amino acids resulted in a loss and/or modification of binding activity as judged by nitrocellulose filter assays and gel shift. Modification specific for arginine residues did not affect binding within the limits of the assays used. Finally, quenching of the intrinsic tryptophan fluorescence of ICP8 in the presence of single-stranded DNA either suggests involvement of this amino acid in the binding reaction or reflects a conformational change in the protein upon binding.     

Studies on cytochrome c oxidase, VI. Polypeptide IV. the complete primary structure.

The complete primary structure of the cytoplasmically synthesized polypeptide IV from beef heart cytochrome oxidase was determined via isolation and sequencing of overlapping methionine, tryptophan, and arginine fragments. The protein consists of 147 amino acids (Mr 17153). It is characterized as a part of a membrane protein complex by a hydrophobic segment consisting of 19 residues. It is suggested that this segment contacts the lipids of the inner mitochondiral membrane. Additional specific contacts may result from pairwise formation of salt bridges between ionic groups of the protein and the phospholipids. The function of this component of the terminal oxidase is yet unknown.     

A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA.

To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein-RNA complexes on DEAE-Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136-144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113-120; they are suggested to be involved in interaction with signal recognition particle RNA.     

Identification of two novel arginine binding DNAs.

RNA tertiary structure is known to play critical roles in RNA-protein recognition and RNA function. To examine how DNA tertiary structure might relate to RNA structure, we performed in vitro selection experiments to identify single-stranded DNAs that specifically bind arginine, and compared the results with analogous experiments performed with RNA. In the case of RNA, a motif related to the arginine binding site in human immunodeficiency virus TAR RNA was commonly found, whereas in the case of DNA, two novel motifs and no TAR-like structures were found. One DNA motif, found in approximately 40% of the cloned sequences, forms of hairpin structure with a highly conserved 10 nucleotide loop, whereas the second motif is especially rich in G residues. Chemical interference and mutagenesis experiments identified nucleotides in both motifs that form specific arginine binding sites, and dimethylsulfate footprinting experiments identified single guanine residues in both that are protected from methylation in the presence of arginine, suggesting possible sites of arginine contact or conformational changes in the DNAs. Circular dichroism experiments indicated that both DNAs undergo conformational changes upon arginine binding and that the arginine guanidinium group alone is responsible for binding. A model for the G-rich motif is proposed in which mixed guanine and adenine quartets may form a novel DNA structure. Arginine binding DNAs and RNAs should provide useful model systems for studying nucleic acid tertiary structure.     

Structure-based analysis of protein-RNA interactions using the program ENTANGLE

Until recently, drawing general conclusions about RNA recognition by proteins has been hindered by the paucity of high-resolution structures. We have analyzed 45 PDB entries of protein-RNA complexes to explore the underlying chemical principles governing both specific and non-sequence specific binding. To facilitate the analysis, we have constructed a database of interactions using ENTANGLE, a JAVA-based program that uses available structural models in their PDB format and searches for appropriate hydrogen bonding, stacking, electrostatic, hydrophobic and van der Waals interactions. The resulting database of interactions reveals correlations that suggest the basis for the discrimination of RNA from DNA and for base-specific recognition. The data illustrate both major and minor interaction strategies employed by families of proteins such as tRNA synthetases, ribosomal proteins, or RNA recognition motifs with their RNA targets. Perhaps most surprisingly, specific RNA recognition appears to be mediated largely by interactions of amide and carbonyl groups in the protein backbone with the edge of the RNA base. In cases where a base accepts a proton, the dominant amino acid donor is arginine, whereas in cases where the base donates a proton, the predominant acceptor is the backbone carbonyl group, not a side-chain group. This is in marked contrast to DNA-protein interactions, which are governed predominantly by amino acid side-chain interactions with functional groups that are presented in the accessible major groove. RNA recognition often proceeds through loops, bulges, kinks and other irregular structures that permit use of all the RNA functional groups and this is seen throughout the protein-RNA interaction database.     

The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein

The P30 protein of tobacco mosaic virus (TMV) is required for cell to cell movement of viral RNA, which presumably occurs through plant intercellular connections, the plasmodesmata. The mechanism by which P30 mediates transfer of TMV RNA molecules through plasmodesmata channels is unknown. We have identified P30 as an RNA and single-stranded (ss) DNA binding protein. Binding of purified P30 to ss nucleic acids is strong, highly cooperative, and sequence nonspecific with a minimal binding site of 4-7 nucleotides per P30 monomer. In-frame deletions across P30 were used to localize the ss nucleic acid binding domain to within amino acid residues 65-86 of the protein. We propose that binding of P30 to TMV RNA creates an unfolded protein-RNA complex that functions as an intermediate in virus cell to cell movement through plasmodesmata.     

Fluorescence studies on the role of tryptophan in heterogeneous nuclear ribonucleoprotein particles of HeLa cells.

The 40 S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from HeLa cells reveal tryptophan fluorescence with a bi-exponential decay, indicating that only a few of the 'core' proteins contain tryptophan residues. The presence of tryptophan residues distinguishes hnRNP particles from nucleosomes, with which they otherwise share a number of properties. This difference, however, is not essential for protein-RNA binding, as the fluorescence decay remains unchanged when hnRNP particles are dissociated into protein and RNA. However, the Stern-Volmer quenching constant is doubled upon salt dissociation, i.e. tryptophan residues become more accessible to solvent. Thus tryptophan quenching is a useful parameter for monitoring protein-protein interactions in hnRNP particles.     

Effects of mutations in the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.     

Effects of Physical and Chemical Treatments on the Biological Activity of Ricin D

Effects of physical and chemical treatments on the cytoagglutinating activity, toxicity and inhibitory activity of cell-free protein synthesis of ricin D or its constituent polypeptide chains were investigated. The results indicated that the isolated polypeptide chains were much less stable than intact ricin D in acidic pH, heating as well as chemicals, and the Ala chain was more unstable than the lie chain.

Chemical modifications of ricin D with specific reagents revealed that the tryptophan and tyrosine residues as well as the carboxyl groups participated in the phenomena of cyto- agglutination and toxic action of ricin D, whereas arginine residues were considered not to be directly involved. Trinitrophenylation of free amino groups did not result in a loss of cytoagglutinating activity, whereas caused a loss of toxicity, suggesting that free amino groups in the lie chain were involved in the toxic action of ricin D.     

Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.

The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of beta-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.     

Analysis of the murine ecotropic leukemia virus receptor reveals a common biochemical determinant on diverse cell surface receptors that is essential to retrovirus entry.

Two residues, tyrosine 235 and glutamic acid 237, of the ecotropic murine leukemia virus receptor (ATRC1) have been shown to be essential for receptor-mediated virus envelope binding and entry. We performed genetic analyses to examine the biochemical contribution of these residues in a productive virus-receptor interaction. Altered ATRC1 receptors bearing either a phenylalanine, a tryptophan, a histidine, or a methionine at position 235 mediated ecotropic virus entry comparable to that mediated by ATRC1. In contrast, altered ATRC1 receptors bearing alanine, threonine, serine, or proline at position 235 exhibited a 300- to 10,000-fold decrease in receptor capability. Furthermore, substitution of tyrosine or phenylalanine into the corresponding position (242) of the homologous human protein that lacks ecotropic virus receptor capability resulted in acquisition of ecotropic virus receptor function comparable to that of ATRC1. Substitution of a tryptophan or a histidine at that position of the human protein, however, resulted in a much-reduced receptor capability, suggesting a preference for a benzene ring in the hydrophobic side chain. A similar analysis of proteins substituted at position 237 revealed that aspartic acid, but not arginine or lysine, can functionally substitute for glutamic acid 237 in ATRC1 or at the corresponding position in the human protein. These results suggest a requirement for an acidic and a nearby hydrophobic amino acid for efficient ecotropic virus entry. Similar motifs have been identified in the virus binding sites of other retrovirus receptors, suggesting that the initial step of retrovirus entry may be governed by a common mechanism.     

Protein-RNA contacts at crystal packing surfaces

The crystal packing surfaces comprising protein-RNA interactions were analyzed for 50 RNA-protein crystal structures in the Protein Data Bank database. Protein-RNA crystal contacts, which represent nonspecific protein-RNA interfaces, were investigated for their amino acid propensities, hydrogen bond patterns, and backbone and side chain interactions. When compared to biologically relevant interactions, the protein-RNA crystal contacts exhibit similarities as well as differences with respect to the principles of protein-RNA interactions. Similar to what was observed at cognate protein-RNA interfaces, positively charged amino acids have high propensities at noncognate protein-RNA interfaces and preferentially form hydrogen bonds with RNA phosphate groups. In contrast, nonpolar residues are less frequently associated with noncognate interactions. These results highlight the important roles of both electrostatic and hydrogen bonding interactions, facilitated by positively charged amino acids, in mediating both specific and nonspecific protein-RNA interactions.     

Protein-RNA interactions: structural analysis and functional classes

A data set of 89 protein-RNA complexes has been extracted from the Protein Data Bank, and the nucleic acid recognition sites characterized through direct contacts, accessible surface area, and secondary structure motifs. The differences between RNA recognition sites that bind to RNAs in functional classes has also been analyzed. Analysis of the complete data set revealed that van der Waals interactions are more numerous than hydrogen bonds and the contacts made to the nucleic acid backbone occur more frequently than specific contacts to nucleotide bases. Of the base-specific contacts that were observed, contacts to guanine and adenine occurred most frequently. The most favored amino acid-nucleotide pairings observed were lysine-phosphate, tyrosine-uracil, arginine-phosphate, phenylalanine-adenine and tryptophan-guanine. The amino acid propensities showed that positively charged and polar residues were favored as expected, but also so were tryptophan and glycine. The propensities calculated for the functional classes showed trends similar to those observed for the complete data set. However, the analysis of hydrogen bond and van der Waal contacts showed that in general proteins complexed with messenger RNA, transfer RNA and viral RNA have more base specific contacts and less backbone contacts than expected, while proteins complexed with ribosomal RNA have less base-specific contacts than the expected. Hence, whilst the types of amino acids involved in the interfaces are similar, the distribution of specific contacts is dependent upon the functional class of the RNA bound.     

Binding of U1A protein changes RNA dynamics as observed by 13C NMR relaxation studies

Recognition of RNA by proteins and small molecules often involves large changes in RNA structure and dynamics, yet very few studies have so far characterized these motional changes. Here we extend to the protein-bound RNA recent 13C relaxation studies of motions in the RNA recognized by human U1A protein, a well-known model for protein-RNA recognition. Changes in relaxation observed upon complex formation demonstrate that the protein-binding site becomes rigid in the complex, but the upper stem-loop that defines the secondary structure of this RNA experiences unexpected motional freedom. By using a helix elongation strategy, we observe that the upper stem-loop moves independently of the remainder of the structure also in the absence of U1A. Surprisingly, RNA residues making important intermolecular contacts in the structure of the complex exhibit increased flexibility in the presence of the protein. Both of these results support the hypothesis that RNA-binding proteins select a structure that optimizes intermolecular contacts in the manifold of conformations sampled by the free RNA and that protein binding quenches these motions. Together with previous studies of the RNA-bound protein, they also demonstrate that protein-RNA interfaces experience complex motions that modulate the strength of individual interactions.     

Multiple targets for suppression of RNA interference by tomato aspermy virus protein 2B

Viral suppressors of RNA interference (RNAi) appear to have evolved as a response to this innate genomic defense. We report the nucleic acid binding properties of the Cucumovirus RNAi suppressor tomato aspermy virus protein 2B (TAV 2B). Using total internal reflection fluorescence spectroscopy (TIRFS), we show that TAV 2B binds double-stranded RNA corresponding to siRNAs and miRNAs, as well as single-stranded RNA oligonucleotides. A number of positively charged residues between amino acids 20 and 30 are critical for RNA binding. Binding to RNA oligomerizes and induces a conformational change in TAV 2B, causing it to form a primarily helical structure and a 4:2 protein-RNA complex.