The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a ‘lure and lock’ model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the ‘lure’ step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on ‘top’ of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein–RNA complexes.     

Protein-RNA sequence covariation in a ribosomal protein-rRNA complex

Comparative sequence analysis has successfully predicted secondary structure and tertiary interactions in ribosomal and other RNAs. Experiments presented here ask whether the scope of comparative sequence-based predictions can be extended to specific interactions between proteins and RNA, using as a system the well-characterized C-terminal RNA binding domain of ribosomal protein L11 (L11-C76) and its 58 nucleotide binding region in 23S rRNA. The surface of L11-C76 alpha-helix 3 is known to contact RNA; position 69 in this helix is conserved as serine in most organisms but varies to asparagine (all plastids) or glutamine (Mycoplasma). RNA sequence substitutions unique to these groups of organisms occur at base pairs 1062/1076 or 1058/1080, respectively. The possibility that rRNA base pair substitutions compensate for variants in L11 alpha-helix 3 has been tested by measuring binding affinities between sets of protein and RNA sequence variants. Stability of the RNA tertiary structure, as measured by UV melting experiments, was unexpectedly affected by a 1062/1076 base pair substitution; additional mutations were required to restore a stably folded structure to this RNA. The results show that the asparagine variant of L11-C76 residue 69 has been compensated by substitution of a 1062/1076 base pair, and plausibly suggest a direct contact between the amino acid and base pair. For some of the protein and RNA mutations studied, changes in binding affinity probably reflect longer-range adjustments of the protein-RNA contact surface.     

Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli

Penicillin-binding proteins (PBPs) and beta-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced gamma-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two gamma-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.     

Salting the charged surface: pH and salt dependence of protein G B1 stability

This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is or=18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems.     

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.     

Simulations of the dynamics at an RNA-protein interface.

Molecular dynamics simulations of the RNA-binding domain of the U1A spliceosomal protein in complex with its cognate RNA hairpin, performed at low and high ionic strength in aqueous solution, suggest a pathway for complex dissociation. First, cations condense around the RNA and compete with the protein for binding sites. Then solvated ions specifically destabilize residues at the RNA-protein interface. For a discrete cluster of residues at the complex interface, the simulations reveal an increased deviation from the crystal structure at high salt concentrations while the remaining protein scaffold is stabilized under these conditions. The microscopic picture of salt influence on the complex suggests guidelines for rational design of interface inhibitors targeted at RNA-protein complexes.     

Interactions of the N-terminal domain of ribosomal protein L11 with thiostrepton and rRNA

Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2'-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives approximately 15-fold enhancement when L11 or L11-C76 is bound. Although the pyrene tag weakens protein binding, unbiased protein-RNA association constants were obtained in competition experiments with untagged RNA. It was found that (i) intact B. stearothermophilus L11 binds rRNA with K approximately 1.2 x 10(9) m(-1) in buffers with 0.2 m KCl, about 100-fold tighter than Escherichia coli L11; (ii) the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity (approximately 8-fold enhancement at 0.2 m KCl), (iii) L11 stimulates thiostrepton binding by 2.3 +/- 0.6 x 10(3)-fold, predicting an overall thiostrepton affinity for the ribosome of approximately 10(9) m(-1), and (iv) the yeast homolog of L11 shows no stimulation of thiostrepton binding. The latter observation resolves the question of why eukaryotes are insensitive to the antibiotic. These measurements also show that it is plausible for thiostrepton to compete directly with EF-G.GDP for binding to the L11-RNA complex, and provide a quantitative basis for further studies of L11 function and thiostrepton mechanism.     

Solution X-ray scattering reveals a novel structure of calmodulin complexed with a binding domain peptide from the HIV-1 matrix protein p17

The solution structures of complexes between calcium-saturated calmodulin (Ca (2+)/CaM) and a CaM-binding domain of the HIV-1 matrix protein p17 have been determined by small-angle X-ray scattering with use of synchrotron radiation as an intense and stable X-ray source. We used three synthetic peptides of residues 11-28, 26-47, and 11-47 of p17 to demonstrate the diversity of CaM-binding conformation. Ca (2+)/CaM complexed with residues 11-28 of p17 adopts a dumbbell-like structure at a molar ratio of 1:2, suggesting that the two peptides bind each lobe of CaM, respectively. Ca (2+)/CaM complexed with residues 26-47 of p17 at a molar ratio of 1:1 adopts a globular structure similar to the NMR structure of Ca (2+)/CaM bound to M13, which adopted a compact globular structure. In contrast to these complexes, Ca (2+)/CaM binds directly with both CaM-binding sites of residues 11-47 of p17 at a molar ratio of 1:1, which induces a novel structure different from known structures previously reported between Ca (2+)/CaM and peptide. A tertiary structural model of the novel structure was constructed using the biopolymer module of Insight II 2000 on the basis of the scattering data. The two domains of CaM remain essentially unchanged upon complexation. The hinge motions, however, occur in a highly flexible linker of CaM, in which the electrostatic residues 74Arg, 78Asp, and 82Glu interact with N-terminal electrostatic residues of the peptide (residues 12Glu, 15Arg, and 18Lys). The acidic residues in the N-terminal domain of CaM interact with basic residues in a central part of the peptide, thereby enabling the central part to change the conformations, while an acidic residue in the C-terminal domain interacts with two basic residues in the two helical sites of the peptide. The overall structure of the complex adopts an extended structure with the radius of gyration of 20.5 A and the interdomain distance of 34.2 A. Thus, the complex is principally stabilized by electrostatic interactions. The hydrophobic patches of Ca (2+)/CaM are not responsible for the binding with the hydrophobic residues in the peptide, suggesting that CaM plays a role to sequester the myristic acid moiety of p17.     

Effect of lysine side chain length on intra-helical glutamate--lysine ion pairing interactions

Ion-pairing interactions are important for protein stabilization. Despite the apparent electrostatic nature of these interactions, natural positively charged amino acids Lys and Arg have multiple methylenes linking the charged functionality to the backbone. Interestingly, the amino acids Lys and Orn have positively charged side chains that differ by only one methylene. However, only Lys is encoded and incorporated into proteins. To investigate the effect of side chain length of Lys on ion-pairing interactions, a series of 12 monomeric alpha-helical peptides containing potential Glu-Xaa (i, i+3), (i, i+4) and (i, i+5) (Xaa = Lys, Orn, Dab, Dap) interactions were studied by circular dichroism (CD) spectroscopy at pH 7 and 2. At pH 7, no Glu-Xaa (i, i+5) interaction was observed, regardless of the Xaa side chain length. Furthermore, only Lys was capable of supporting Glu-Xaa (i, i+3) interactions, whereas any Xaa side chain length supported Glu-Xaa (i, i+4) interactions. Side chain conformational analysis by molecular mechanics calculations showed that the side chain length of Lys enables the Glu-Xaa (i, i+3) interaction with lower energy conformations compared to residues with side chain lengths shorter than that of Lys. Furthermore, these calculated low energy conformers were consistent with conformations of intra-helical Glu-Lys salt bridges in a non-redundant protein structure database. Importantly, the CD spectra for peptides with Glu-Lys interactions did not alter significantly upon changing the pH because of a greater contribution to these interactions by forces other than electrostatics. Incorporating side chains just one methylene shorter (Orn) resulted in significant pH dependence or lack of interaction, suggesting that nature has chosen Lys to form durable interactions with negatively charged functional groups.     

Electrostatic interactions guide the active site face of a structure-specific ribonuclease to its RNA substrate

Restrictocin, a member of the alpha-sarcin family of site-specific endoribonucleases, uses electrostatic interactions to bind to the ribosome and to RNA oligonucleotides, including the minimal specific substrate, the sarcin/ricin loop (SRL) of 23S-28S rRNA. Restrictocin binds to the SRL by forming a ground-state E:S complex that is stabilized predominantly by Coulomb interactions and depends on neither the sequence nor structure of the RNA, suggesting a nonspecific complex. The 22 cationic residues of restrictocin are dispersed throughout this protein surface, complicating a priori identification of a Coulomb interacting surface. Structural studies have identified an enzyme-substrate interface, which is expected to overlap with the electrostatic E:S interface. Here, we identified restrictocin residues that contribute to binding in the E:S complex by determining the salt dependence [partial differential log(k 2/ K 1/2)/ partial differential log[KCl]] of cleavage of the minimal SRL substrate for eight point mutants within the protein designed to disrupt contacts in the crystallographically defined interface. Relative to the wild-type salt dependence of -4.1, a subset of the mutants clustering near the active site shows significant changes in salt dependence, with differences of magnitude being >or=0.4. This same subset was identified using calculated salt dependencies for each mutant derived from solutions to the nonlinear Poisson-Boltzmann equation. Our findings support a mechanism in which specific residues on the active site face of restrictocin (primarily K110, K111, and K113) contribute to formation of the E:S complex, thereby positioning the SRL substrate for site-specific cleavage. The same restrictocin residues are expected to facilitate targeting of the SRL on the surface of the ribosome.     

The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.     

Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells

The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged β-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection.     

NMR determination of pKa values for Asp,Glu, His,and Lys mutants at each variable contiguous enzyme-inhibitor contact position of the turkey ovomucoid third domain

From the larger set of 191 variants at all the variable contact positions in the turkey ovomucoid third domain, we selected a subset that consists of Asp, Glu, His, and Lys residues at eight of the nine contiguous P6-P3' positions (residues 13-21), the exception being P3-Cys16 which is involved in a conserved disulfide bridge. Two-dimensional [1H,1H]-TOCSY data were collected for each variant as a function of sample pH. This allowed for the evaluation of 31 of the 32 pK(a) values for these residues, the exception being that of P5-Lys14, whose signals at high pH could not be resolved from those of other Lys residues in the molecule. Only two of the titrating residues are present in the wild-type protein (P6-Lys13 and P1'-Glu19); hence, these measurements complement earlier measurements by A. D. Robertson and co-workers. This data set was supplemented with results from the pH dependence of NMR spectra of four additional single mutants, P1-Leu18Gly, P1-Leu18Ala, P2-Thr17Val, and P3'-Arg21Ala, and two double mutants, P2-Thr17Val/P3'-Arg21Ala and P8-Tyr11Phe/P6-Lys13Asp. Probably the most striking result was observation of a P2-Thr17...P1'-Glu19 hydrogen bond and a P1'-Glu19-P3'-Arg21 electrostatic interaction within the triad of P2, P1', and P3' (residues 17, 19, and 21, respectively). In several cases, the pK(a) of a particular residue was sensed by resonances not only in that residue but also in residue(s) with which it interacts. Remarkably, in several interacting systems, resonances from different protons within the same residue yielded different pHmid values.     

Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II.

To elucidate the domains on the extrinsic 23 kDa protein involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II, we modified amino or carboxyl groups of the 23 kDa protein to uncharged methyl ester groups with N-succinimidyl propionate or glycine methyl ester in the presence of a water-soluble carbodiimide, respectively. The N-succinimidyl propionate-modified 23 kDa protein did not bind to the 33 kDa protein associated with PSII membranes, whereas the glycine methyl ester-modified 23 kDa protein completely bound. This indicates that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein associated with the PSII membranes. Mapping of the N-succinimidyl propionate-modified sites of the 23 kDa protein was performed using Staphylococcus V8 protease digestion of the modified protein followed by determination of the mass of the resultant peptide fragments with MALDI-TOF MS. The results showed that six domains (Lys11-Lys14, Lys27-Lys38, Lys40, Lys90-Lys96, Lys143-Lys152, Lys166-Lys174) were modified with N-succinimidyl propionate. In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants. These positively charged lysyl residues on the 23 kDa protein may be involved in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M. & Frankel, L.K. (2003) Biochemistry42, 2056-2061].     

Covariance analysis of RNA recognition motifs identifies functionally linked amino acids.

The RNA recognition motif (RRM) is one of the most common eukaryotic protein motifs. RRM sequences form a conserved globular structure known as the RNA-binding domain (RBD) or the ribonucleoprotein domain. Many proteins that contain RRM sequences bind RNA in a sequence-specific manner. To investigate the basis for the RNA-binding specificity of RRMs, we subjected 330 aligned RRM sequences to covariance analysis. The analysis revealed a single network of covariant amino acid pairs comprising the buried core of the RBD and a surface patch. Structural studies have implicated a subset of these residues in RNA binding. The covariance linkages identify a larger set of amino acid residues, including some not directly in contact with bound RNA, that may influence RNA-binding specificity.     

RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein.

An RNA-binding protein of 28 kD (28RNP) has been previously isolated from spinach chloroplasts and was found to be required for 3' end processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic and glycine-rich amino terminal domain. Each domain by itself, as well as in combination with other domains, was expressed in bacterial cells and the polypeptides were purified to homogeneity. We have investigated the RNA-binding properties of the different structural domains using UV-crosslinking, saturation binding and competition between the different domains on RNA-binding. It was found that the acidic domain does not bind RNA, but that each of the RNA-binding domains, expressed either individually or together, do bind RNA, although with differing affinities. When either the first or second RNA-binding domain was coupled to the acidic domain, the affinity for RNA was greatly reduced. However, the acidic domain has a positive effect on the binding of the full-length protein to RNA, because the mature protein binds RNA with a better affinity than the truncated protein which lacks the acidic domain. In addition, it was found that a stretch of two or three G residues is enough to mediate binding of the 28RNP, whereas four U residues were insufficient. The implications of the RNA-binding properties of 28RNP to its possible function in the processing of chloroplast RNA is discussed.     

Roles of catalytic domain residues in interfacial binding and activation of group IV cytosolic phospholipase A2

Group IV cytosolic phospholipase A(2) (cPLA(2)) has been shown to play a critical role in eicosanoid biosynthesis. cPLA(2) is composed of the C2 domain that mediates the Ca(2+)-dependent interfacial binding of protein and the catalytic domain. To elucidate the mechanism of interfacial activation of cPLA(2), we measured the effects of mutations of selected ionic and hydrophobic residues in the catalytic domain on the enzyme activity and the membrane binding of cPLA(2). Mutations of anionic residues located on (Glu(419) and Glu(420)) or near (Asp(436), Asp(438), Asp(439), and Asp(440)) the active site lid enhanced the affinity for cPLA(2) for anionic membranes, implying that the electrostatic repulsion between these residues and the anionic membrane surface might trigger the opening of the active site. This notion is further supported by a biphasic dependence of cPLA(2) activity on the anionic lipid composition of the vesicles. Mutations of a cluster of cationic residues (Lys(541), Lys(543), Lys(544), and Arg(488)), while significantly enhancing the activity of enzyme, abrogated the specific activation effect by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). These data, in conjunction with cell activity of cPLA(2) and mutants transfected into HEK293 cells, suggest that the cationic residues form a specific binding site for PtdIns(4,5)P(2) and that the specific PtdIns(4,5)P(2) binding is involved in cellular activation of cPLA(2). Also, three hydrophobic residues at the rim of the active site (Ile(399), Leu(400), and Leu(552)) were shown to partially penetrate the membrane, thereby promoting membrane binding and activation of cPLA(2). Based on these results, we propose an interfacial activation mechanism for cPLA(2) which involves the removal of the active site lid by nonspecific electrostatic repulsion, the interdomain hinge movement induced by specific PtdIns(4,5)P(2) binding, and the partial membrane penetration by catalytic domain hydrophobic residues.     

Intrinsically disordered electronegative clusters improve stability and binding specificity of RNA-binding proteins

RNA-binding proteins play crucial roles in various cellular functions and contain abundant disordered protein regions. The disordered regions in RNA-binding proteins are rich in repetitive sequences, such as poly-K/R, poly-N/Q, poly-A, and poly-G residues. Our bioinformatic analysis identified a largely neglected repetitive sequence family we define as electronegative clusters (ENCs) that contain acidic residues and/or phosphorylation sites. The abundance and length of ENCs exceed other known repetitive sequences. Despite their abundance, the functions of ENCs in RNA-binding proteins are still elusive. To investigate the impacts of ENCs on protein stability, RNA-binding affinity, and specificity, we selected one RNA-binding protein, the ribosomal biogenesis factor 15 (Nop15), as a model. We found that the Nop15 ENC increases protein stability and inhibits nonspecific RNA binding, but minimally interferes with specific RNA binding. To investigate the effect of ENCs on sequence specificity of RNA binding, we grafted an ENC to another RNA-binding protein, Ser/Arg-rich splicing factor 3. Using RNA Bind-n-Seq, we found that the engineered ENC inhibits disparate RNA motifs differently, instead of weakening all RNA motifs to the same extent. The motif site directly involved in electrostatic interaction is more susceptible to the ENC inhibition. These results suggest that one of functions of ENCs is to regulate RNA binding via electrostatic interaction. This is consistent with our finding that ENCs are also overrepresented in DNA-binding proteins, whereas underrepresented in halophiles, in which nonspecific nucleic acid binding is inhibited by high concentrations of salts.