RNA binding of T-cell intracellular antigen-1 (TIA-1) C-terminal RNA recognition motif is modified by pH conditions

pH-dependent RNA binding of TIA-1 RRM3 1 RNA binding of T-cell intracellular antigen-1 (TIA-1) C-terminal RNA recognition motif is modified by pH conditions* Isabel Cruz-Gallardo1, Ángeles Aroca1, Cecilia Persson2, B. Göran Karlsson2 and Irene Díaz-Moreno1* 1From the Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), cicCartuja, Universidad de Sevilla-CSIC, Avda. Américo Vespucio 49, Sevilla 41092, Spain. 2Swedish NMR Centre, University of Gothenburg, PO Box 465, SE-40530 Gothenburg, Sweden. *Running title: pH-dependent RNA binding of TIA-1 RRM3 To whom the correspondence should be addressed: Dr. Irene Díaz Moreno. Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), cicCartuja, Universidad de Sevilla-CSIC, Avda. Américo Vespucio 49, Sevilla 41092, Spain. Tel.: 34-954489513; Fax: 34-954460065; E-mail: [email protected]. Keywords: DNA/RNA binding protein (D/RBP); pH-dependence; NMR; RNA recognition motif (RRM); T-cell intracellular antigen-1 (TIA-1). Background: T-cell intracellular antigen- 1(TIA-1) is a key DNA/RNA binding protein in RNA metabolism. Results: The binding of TIA-1 RRM3 domain (isolated or fused to RRM2) to RNA is affected by pH changes. Conclusion: The pH-dependence of RRM3/RNA interaction may have a significant effect in the overall TIA-1 function. Significance: Unveiling the auxiliary role of RRM3 in RNA recognition sheds light on the TIA-1 function between cell compartments. SUMMARY T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is constituted by three RNA recognition motifs (RRMs) and a glutamine rich domain and binds to uridine-rich RNA sequences through its C-terminal domains RRM2 and RRM3. Here we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation / deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C- terminal RRM3 domain in TIA-1 RNA recognition is poorly understood and this work provides an insight into its binding mechanisms. Human T-cell intracellular antigen-1 (TIA-1)3 is a 46-kDa DNA/RNA binding protein (D/RBP) with a dual regulatory role at the level of transcriptional and posttranscriptional processes shuttling between the nucleus and the cytoplasm (1,2). In the nucleus, TIA-1 regulates the alternative splicing of pre-mRNA (3-5) and modulates gene transcription by binding to DNA (6). In the cytoplasm, TIA-1 regulates the turnover of several mRNAs (7-9) by repressing their translation. To fulfill this function, TIA-1 binds to 3’ untranslated regions (UTR’s) that are frequently adenine/uridine rich elements (AREs). In addition, recent studies (10) have identified 5’ terminal oligopyrimidine tracts (5’TOP) mRNAs – defined as 4-to-15- nucleotide CU-rich elements in 5’ UTR’s (11,12) – as novel targets for TIA-1/TIAR (TIA-1 related) proteins. Under stress conditions, eIF2α is phosphorylated so the pH-dependent RNA binding of TIA-1 RRM3 2 initiation of translation is inhibited and TIA-1 recruits and escorts noncanonical preinitiation complexes to stress granules (1,13,14). TIA-1 consists of three RNA recognition motifs (RRM1-3) along with a C- terminal glutamine-rich prion-related domain (PRD) (Figure 1A). Both PRD and the N terminal RRM (RRM1) are involved in U1snRNP recruitment to regulated splice sites (4). Isolated RRM1 binds to singlestranded DNA molecules (ssDNA) (6) but not to RNA (15). However, recent reports show that RRM1 presence enhances TIA-1 affinity for certain RNA sequences (16). RRM2 is necessary for TIA-1 binding to RNAs containing U-rich motifs, whereas RRM3 binds weakly but contributes to improve such binding (15,16). All TIA-1 RRMs display the canonical αβ fold with the consensus ribonucleoprotein (RNP1 and RNP2) residues for RNA binding (17,18). RRM3 exhibits a non-canonical N- terminal α-helix in a homology model (19) that is conserved in TIAR (PDB ID 1X4G) and Pub1p – the analogous protein in Sacharomyces cerevisiae – (20). Moreover, the C-terminal motif Trp-Gly-[Arg/Lys] is also conserved and the β-sheet is slightly distorted (Figure 1B and 1C). Due to their common features, TIA-1/TIAR/Pub1p RRM3 domains are considered a new type of RRM family called TIA-1 C-terminal domain like RRM (TRRM) (19,20). The contribution of RRM3 to RNA recognition is still unclear, although it has been reported its contribution in binding poli-U sequences (16), as well as its role in specifically selecting transcripts (20). Given the pleiotropic role of human TIA-1 in the control of critical cellular events, understanding how RNA recognition occurs, along with the biophysical features associated, has a great relevance. This work suggests that the auxiliary role in RNA recognition of isolated RRM3 or RRM3 in the RRM23 context is modulated by slight environmental changes in terms of pH, as small changes in protonation / deprotonation of conserved RRM3 histidine residues induce drastic effects over TIA-1 binding to RNA. EXPERIMENTAL PROCEDURES Protein and RNA Oligonucleotides Preparation – RRM2 domain (residues 80- 173), RRM3 domain (residues 190-288) and RRM23 construct (residues 80-288) were obtained by PCR from the plasmid containing full-length TIA-1 kindly provided by Prof. P. Anderson (Harvard Medical School, USA) and cloned into pETM-11 vector (Invitrogen) as previously described (19). Isotopically 15N and 13C-labeled proteins were expressed in Escherichia coli BL21 (DE3) cells in minimal medium (M9) supplemented either with 15NH4Cl or 15NH4Cl and 13C-glucose following the same protocol as in Aroca et al.(19) for LB medium. His-tagged TIA-1 constructs were purified by nickel affinity chromatography (Ni Sepharose 6 Fast Flow, GE Healthcare). The His-tag, along the TEV cleavage site, was kept at the constructs since no differences on the secondary structure with or without His-tag were observed (19). Samples were concentrated up to 1 mM in 20 mM potassium phosphate buffer, 50 mM KCl (pH 7.0). Protein concentration was determined by spectrophotometry with predicted extinction coefficients. RNA 5’AUUUA 3’ and 5’AUUUAUUUAUUUAUUUAUUUAUUU 3’ oligos were chemically synthesized (IDT, Integrated DNA Technologies). Nuclear Magnetic Resonance Spectroscopy (NMR) – NMR samples of TIA- 1 RRM3 were prepared in 90% H2O / 10% D2O solutions of 20 mM potassium phosphate buffer, 50 mM KCl (pH 7.0) at concentrations in a range 0.05-1 mM. NMR spectra were recorded at 25 ºC on 800MHz Varian INOVA and 500MHz Bruker Avance III spectrometers. The Bruker spectrometer was equipped with cryoprobe technology. The spectra recorded in Varian spectrometer were processed with nmrPipe software (21) to be further analyzed with the CcpNmr program suite (22). Those spectra recorded in Bruker spectrometer were processed using TopSpin 3.0 and analyzed with SPARKY (23). Standard 3D NMR experiments (HNCACB, HNCA and HNCO) were used to assign backbone resonances of RRM3 domain. The coverage of the backbone assignment was 98%. The assigned chemical shifts were deposited at the BMRB with the accession number 18829 and were used to generate the protein 3D structure at CS23D2.0 web server (24). The pdb file generated with NMR chemical-shifts assignments for the 3D structure was used for pH-dependent RNA binding of TIA-1 RRM3 3 data discussion and to build Figures 1, 4, 6 and 7. The pH effects on TIA1 RRM2 and RRM3 domains were followed by recording 15N-HSQC spectra on 700MHz Bruker Avance III and 800MHz Varian INOVA during titrations of a 0.01 mM sample of 15N- labeled RRM2 domain and a 0.05 mM sample of 15N-labeled RRM3 domain, respectively, in 20 mM potassium phosphate buffer, 50 mM KCl (pH 3.8 for RRM2 and 5.0 for RRM3) with small aliquots of 0.1 M NaOH to gradually increase the pH. RNA binding of TIA-1 RRM3 was monitored by acquiring 15N-HSQC spectra along titrations of the 5´-AUUUA-3´ RNA oligonucleotide into a sample of 25 µM 15N- labeled RRM3 in 20 mM potassium phosphate buffer, 50 mM KCl, at both pH 5.5 and 7.5 values. The pH value of the sample was verified after each titration step. Weighted average values of 15N and 1H chemical-shift perturbations (∆δavg) of each resonance was calculated as follows: ∆δavg = (([∆δH]2 + [∆δN/5]2)/ 2)1/2, being ∆δH and ∆δN chemical-shift increments of 1H and 15N, respectively. The threshold value, used to consider significant chemical-shift perturbations from the titration data, was defined as the average of ∆δavg values plus 2 standard deviations (2Sn-1). The pH effect on RNA structure was controlled by registering 1D NMR spectra on a 700MHz Bruker Avance III spectrometer at the pH values of 5.5 and 7.5. Samples contained 50 µM of the 24-mer AU oligo 5’AUUUAUUUAUUUAUUUAUUUAUUU 3’ and were resuspended in the buffer described before using either 90% H2O:10% D20 or 100% D2O as solvents. The comparison of spectra at different pH values (data not shown) revealed no significant changes in linewidth or chemical-shift perturbations. pH Titration Curves – Titration curves were obtained by plotting the change in chemical shift as a function of pH for all of the detectable backbone amide protons (δ 1H) and nitrogens (δ 15N) of TIA-1 RRM3. Data from 12 out of 18 affected residues were fitted to a sigmoidal curve model describing a single protonation event with an associated pKa value: δ = (Ka δH + [H+] δL)/ (Ka + [H+]) where δH and δL are the chemical-shift values at high and low pH. Fits were obtained in Origin (OriginLab, Northampton, MA) 8.0 by non-linear least squares fitting on the proposed model. The pKa value was the average of two independent pH titrations with estimated errors of ±10%. The threshold value, used to identify significant perturbations when data from the titrations were analyzed together, was defined as the averaged chemical-shift perturbations (∆δavg) for the system plus 2 standard deviations (2Sn- 1). The reported pKa values of TIA-1 RRM2 histidines were calculated as in the case of RRM3. 15N-HSQC spectra were assigned according to the work of Kuwasako and collaborators (17) (PDB code 2RNE and BMRB accession number 11376). Circular Dichroism Spectroscopy (CD) – All CD spectra were recorded on a Jasco J- 815 spectropolarimeter equipped with a peltier temperature-control system. RRM3, RRM23 and the RNA oligo samples were in 20 mM potassium phosphate buffer, 50 mM KCl, at both pH 5.5 and 7.5 values. Each spectrum was an average of 20 scans. The secondary structure analysis of RRM3 was performed recording farultraviolet (far-UV) CD spectra (190-250 nm) of 3 µM samples at 25 ºC. RNA binding was monitoring in the UV range (240-330 nm) by adding increasing amounts of TIA-1 RRM23 construct to 3 µM of the 24-mer oligo 5’AUUUAUUUAUUUAUUUAUUUAUUU 3’. A temperature of 10 ºC was chosen to optimize the signal change upon protein binding. The integral of the CD signal between 255 and 265 nm was plotted against the ratio [RRM23]/ [24-mer AU RNA] and fitted to a 1:1 binding site model, as reported before (25). Fluorescence measurements – Emission spectra were recorded in the range 290-500 nm with an excitation wavelength at 282 nm on a Perkin-Elmer LS-5 fluorimeter equipped with a water-thermostat cell holder at 25 ºC. 10 µM RRM3 samples were prepared in 20 mM potassium phosphate buffer, 50 mM KCl, at the pH values of 5.5 and 7.5. The slits wavelength was 5 nm for both excitation and emission and the path length of the cell was 1 cm. The background signal from the buffer was subtracted. RESULTS AND DISCUSSION The pH effect on TIA-1 RRM3 domain – pH NMR titration of TIA-1 RRM3, pH-dependent RNA binding of TIA-1 RRM3 4 over the range of pH 5 – pH 8, showed remarkable chemical-shift differences in superimposed 15N HSQC spectra at high and low pH values (Figure 2). A set of signals corresponding to 18 residues were changing upon increasing pH. Other residues underwent negligible chemical-shift changes since they were insensitive to pH effects. A single resonance was observed for each amide over the tested pH range suggesting that the protonation and deprotonation of side chains is fast on the NMR time scale. Different sigmoidal curves were obtained for perturbed residues with titration data (Figure 3) but most of them could be fitted (see Experimental Procedures) to single pKa values for both 1H and 15N dimensions. As shown in Table 1, pKa values were in the range from 6.3 to 6.9. The averaged chemical-shift perturbations (∆δavg) at high (pH 7.5) and low (pH 5) pH values (see Experimental Procedures) gives an evidence of sensitivity of a particular nucleus to a protonation event. The largest values for backbone amides were observed for the His266 (numbering according to the full length TIA-1 protein, Figure 4A). 18 perturbed amides showed significant perturbations (∆δavg > 0.05 ppm) mainly distributed along four regions of RRM3. Two sequential stretches at N- terminus involved, on one side, Asp192, Glu193 and, on the other, the Val210-Thr215 stretch except for the Leu214. The C- terminus residue slot included Ser250, His253, Val256 and those from Thr262 to Lys269. Residues that overcome the threshold of 0.05 ppm in ∆δavg have been mapped onto the TIA-1 RRM3 backbone (Figure 4B and 4C). As it was expected, strongest effects were found for the three solvent-exposed histidines 248, 253 and 266. In fact, the His248 chemical-shift perturbation could not be determined due to signal broadening beyond the detection limit at pH 7, which is a clear evidence of a pH-dependent changes. Other residues surrounding the three histidines were also perturbed as in the case of Thr211 that is in close proximity to His266. To check that the structure of RRM3 remains unaltered upon pH, far-UV CD spectra and fluorescence emission measurements of the single tryptophan of this domain (Trp272) have been registered (Figure 5). No substantial changes appear in the CD spectra, indicating that the secondary structure of RRM3 is not affected by the pH. On the other hand, the fluorescence maximum appears at ca. 330 nm at both pH values, which corresponds to a partially buried Trp272 residue. The absence of redshifted fluorescence (ca. 350 nm) shows no substantial conformational changes or protein unfolding. The pH effect on RNA binding of TIA-1 RRM3 domain – To assess the effect of pH on RNA recognition by TIA-1 RRM3 domain, its interaction with a short 5-mer AU-rich RNA oligonucleotide, namely 5´- AUUUA-3´, was studied. Since the pKa values for histidine-protonation events were ca. 7 (Table 1), NMR experiments were carried out at low (5.5) and high (7.5) pH values. Averaged chemical-shift perturbations inferred from the titration of 15N-labeled RRM3 with 5´-AUUUA-3´ revealed not only that RRM3 binds RNA using the canonical platform, which comprises aromatic residues mainly localized at three out of four strands from β-sheet, but also that it occurs at both pH values. Due to the small calculated ∆δavg, suggesting a moderate affinity of RRM3 for the tested oligo, the dissociation constant could not be obtained. However striking differences arise from data comparison at tested pH values (Figure 6). Upper panels of Figures 6A and 6B show a detail of the superimposed 15N HSQC spectra along titrations for the same residue revealing chemical-shift perturbations that suggest stronger binding at pH 5.5 than at pH 7.5. These differences were observed for all perturbed residues. Middle and lower panels show the mapping of significantly RRM3 shifted residues (∆δavg ≥ 0.025 ppm) upon RNA binding over the backbone and surface representations. At pH 5.5 titration (Figure 6A), residues undergoing the largest shifts were located at the stretches Tyr206–Gly208 and Lys238–Tyr240, along with Val243, as part of the central β strands (β1 and β3). In addition, the set of residues Lys269–Lys274 from β4 was also involved in RNA recognition. Note that Phe242 resonance overlapped with that corresponding to Gln221, so the ∆δavg values could not be accurately calculated although significant line broadening was observed. Moreover, the pH-dependent RNA binding of TIA-1 RRM3 5 easily-assigned Nε of the indole ring from Trp272 experienced large chemical-shift perturbations, even though its amide signal could not be identified. The titration at pH 7.5 (Figure 6B), in its turn, showed almost the same perturbed residues of RRM3 domain but with significant differences: Tyr206 and Phe242 amide resonances were neither shifted nor broadened. Indeed, the perturbation level of highly affected residues at pH 5.5 (Figure 6A, ∆δavg > 0.050) is lower at pH 7.5 (Figure 6B, 0.025 ≤ ∆δavg ≤ 0.050) – with the exception of Tyr271 –, as a result of weaker binding affinity between TIA-1 and AU-rich RNA at the last pH value. A deep analysis of RNA-TIA-1 RRM3 binding event (Figure 6) revealed that aromatic residues from β1 and β3 strands – namely Tyr206, Tyr240 and Phe242 – constituted the usual RNA docking platform in most RRMs. In addition, Tyr271 and Trp272 (at least its indole amide) from the end of β4, along with the motif formed by Gly273 and Lys274 at the C-terminal region, were also involved in RNA interaction. Indeed, the C-terminal stretch Gly273- Lys274, which is well-conserved among TIA- 1 analogues, has been previously described to be involved in RNA recognition for Pub1p C- terminal domain (20, Figure 1C). Moreover, it is also taking part in forming hydrogen bonds with RNA in other RRMs typelike PTB family, even though the C-terminal stretch differs from the above (26,27). Trp272 is part of a pocket where His248 from α3 and Gln197 from α1 are in close proximity (Figure 4C). Hence, the His248-Trp272 interaction may pack the extra N-terminal α1-helix on the RRM3 domain (19,20). Within this interaction network, protonation / deprotonation events of His248 could hinder the anchorage of Trp272 by disrupting cation-pi contacts, thus affecting the RNA recognition platform of TIA-RRM3, as suggested by the intrinsic tryptophan residue fluorescence (Figure 5). In fact, the chemical-shift perturbation experienced by Trp272 Nε nucleus of the indole ring is substantially larger at acidic pH (Figure 6). As a consequence of protonation / deprotonation of TIA-1 RRM3 histidine residues, electrostatic potential (EP) surfaces change at both pH values (Figure 7), especially those protein regions surrounding histidines. EP changes around His248 environment can explain differences on RNA binding affinity of TIA-1 RRM3 due to pH variations. Thus, the disruption of the RNA recognition platform can explain the lower binding of the TIA-1 RRM3 motif at pH 7.5. The pH effect on the TIA-1 binding to RNA – The assays performed by NMR (Figure 6) with the RRM3 domain to evaluate its interaction with RNA showed too small chemical-shift perturbations to be used for quantitative assessment of binding affinities. To overcome this issue, an additional binding experiment was performed to quantify the pH dependence effect and evaluate whether the overall TIA-1 binding to RNA is significantly affected. A construct that comprises RRM2 and RRM3 domains (TIA-1 RRM23) was used, which is, actually, the responsible unit in TIA-1 / RNA recognition (15). As RRM2 domain contains two histidines (His94 and His96) (17) the pKa values of their –NH groups values were first checked by NMR pH titrations (pKa-His94 ca. 5.3 and pKa-His96 ca. 5.4, Figure 8). This is in agreement with the pKa prediction of the imidazole group for the RNA binding His96 in the context of RRM23 (pKa-His96 ≈ 5.1) using the PROPKA3 server (28). Similar pKa values (pKa < 5.3) have been measured for –NH resonances of those RRM2 residues surrounding both His94 and His96. Altogether indicates that only the three histidines from RRM3 in RRM23 experience the protonation/deprotonation events at the two pH values (pH 5.5 and pH 7.5) used in RNA binding studies. Secondly, we have assessed the affinity of TIA-1 RRM23 for the 24-mer AU RNA by CD (Figure 9). The protein binds to RNA stronger at pH 5.5 than at pH 7.5 (KD ≈ 4.4 nM and KD ≈ 58.5 nM, respectively). Such observations demonstrate that RRM23 binding to RNA is pH dependent and this effect is mainly caused by RRM3 domain since the deprotonation of RRM2 in the context of RRM23 is negligible at both pH values used for RNA binding studies. As previous studies have described, the three domains of TIA-1 protein contribute to RNA binding with RRM2 and RRM3 modules as the main recognition platforms (16) as it happens in other RBPs, such as the central-domains units of KH-type splicing regulatory protein (KSRP) (29) and the N- terminal of Human antigen R (HuR) RRM pH-dependent RNA binding of TIA-1 RRM3 6 modules (25). Whereas TIA-1 RRM2 dominates the interaction, RRM3 enhances it playing an auxiliary role. The pH influence over exposed histidine residues of RRM3 – at positions 248, 253 and 266 – in protonation / deprotonation events around pH 7 is clear. Notably, they are highly conserved among TIA-1 homologous and, in the particular case of His248, it has remained unchanged even in species as Arabidopsis thaliana or Saccharomyces cerevisiae along evolution (Figure 1C). These observations are wellcorrelated with RNA binding, which decreases at pH 7.5, leading to shorten the RNA platform by means of His248 deprotonation. It is tempting to speculate that the canonical folding of RRM3 tunes RNA binding capability of TIA-1 according to small pH changes – around the physiological value –. Even though the RRM3 extra α-helix (α1) fails in directly binding to RNA oligos, its relative orientation regarding α3 and β4 could facilitate stable RNA binding (30). Such a similar effect has been recently reported for the transcription factor EGR1 (early growth response protein 1) in its binding to DNA (31). The His382 is well conserved and modulates protein-DNA binding, acting as a pH-dependent molecular switch. Another well-studied example of molecular switches regarding to enviromental pH is the hemagglutinin fusion peptide of the influenza virus (32,33), responsible for the viral penetration into host cells by membrane fusion. 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(2004) UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605-1612. 39. Koradi, R., Billeter, M., and Wüthrich K. (1996) MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graphics 14, 51-55. pH-dependent RNA binding of TIA-1 RRM3 9 Acknowledgments- The authors wish to thank Dr. P. Anderson (Harvard Medical School, Boston, USA) for kindly provide the plasmid containing the TIA-1 full-length protein and Dr. M. Gorospe (National Institutes of Health, Baltimore, USA) for providing the 24-mer AU RNA oligo. Authors also thank the technical assistance at the NMR services in CITIUS, University of Sevilla and are grateful to Prof. Miguel A. De la Rosa and Dr. Antonio Díaz-Quintana for critical reading of the manuscript. FOOTNOTES *This work was supported by the Andalusian Government (P07-CVI-02896 and BIO198). Dr. Aroca was awarded with a FEBS Short-Term fellowship in 2011. The access to the Bio-NMR Research Infrastructure is co-funded under the 7th Framework Programme of the EC (FP7/2007-2013) grant agreement 261863. 3The abbreviations used are: adenine/uridine rich elements (AREs); averaged chemical-shift perturbations (∆δavg); circular dichroism (CD); dissociation constant (KD); DNA/RNA binding protein (D/RBP); electrostatic potential (EP); human antigen R (HuR); KH-type splicing regulatory protein (KSRP); nuclear magnetic resonance (NMR); prion-related domain (PRD); RNA recognition motif (RRM); single-stranded DNA molecules (ssDNA); T-cell intracellular antigen-1 (TIA-1); 5’ terminal oligopyrimidine tracts (5’TOP); TIA-1 C-terminal domain like RRM (TRRM); TIA-1 related protein (TIAR); untranslated regions (UTR’s). FIGURE LEGENDS FIGURE 1. TIA-1 RRM3 domain. A) TIA-1 domain organization and the constructs used in this study. B) Ribbon representation of the RRM3 domain with labeled secondary structure elements. All structure representations in the text were created with Chimera software (38) using the pdb file generated from chemical-shifts assignments (BMRB accession number 18829) at the CS23D2.0 web server (24). C) Sequence alignment of TIA-1 RRM3 domain (Homo sapiens) with its homologous proteins of Mus musculus (Mm), Homo sapiens (Hs), Saccharomyces cerevisiae (Sc) and Arabidopsis thaliana (At). The secondary structure elements are indicated in the protein sequence along with the three histidines mentioned in this work. Sequences are colored by percent of identity, black (90-100%), dark grey (80%), bright grey (60%) and white (< 50 %). FIGURE 2. NMR pH titration of TIA-1 RRM3 domain in the range of pH 5 – 8. Upper - Overlaid HSQC spectra acquired along the titration where most affected residues are marked. According to the pH, HSQC spectra are colored as follows: pH 5.0, red; pH 5.5, orange; pH 6.0, dark green; pH 6.5, light blue; pH 7.0, purple; pH 7.5, light green and pH 8.0, dark blue. Lower - Selected regions from overlaid HSQC spectra showing substantial chemical-shift perturbations of some residues. FIGURE 3. Representative pKa curves indicating the pH dependence of the chemical shifts of backbone amides of TIA-1 RRM3. Data are fitted to a sigmoidal curve model and describes a single protonation event. FIGURE 4. Distribution of averaged chemical-shift perturbations for pH-affected backbone amides of TIA-1 RRM3. A) The ∆δavg in ppm of signals are calculated according to the Experimental Procedures at high (pH 7.5) and low (pH 5.0) pH values. The red asterisk (*) stands for the His248 resonance, which is broadened beyond the detection limit. B) Mapping of the ∆δavg on TIA-1 RRM3 ribbon. Each view is rotated 180º around the vertical axis. Residues with ∆δavg > 0.05 ppm appear in orange. Residues with ∆δavg below the threshold value are blue while prolines or non-assigned residues are in grey. The three exposed histidines are marked with their side-chain traces in yellow. Note that the ∆δavg of His248 has not been calculated due pH-dependent RNA binding of TIA-1 RRM3 16 Figure 6 pH-dependent RNA binding of TIA-1 RRM3 17 Figure 7 pH-dependent RNA binding of TIA-1 RRM3 18 Figure 8 pH-dependent RNA binding of TIA-1 RRM3 19 Figure 9