Biomolecular NMR Assignments

, Volume 12, Issue 1, pp 189–194 | Cite as

1H, 13C and 15N resonance assignment of domain 1 of trans-activation response element (TAR) RNA binding protein isoform 1 (TRBP2) and its comparison with that of isoform 2 (TRBP1)

  • Harshad Paithankar
  • Pankaj V. Jadhav
  • Amit S. Naglekar
  • Shilpy Sharma
  • Jeetender Chugh


TAR RNA binding protein (TRBP) is a double-stranded RNA binding protein involved in various biological processes like cell growth, development, death, etc. The protein exists as two isoforms TRBP2 and TRBP1. TRBP2 contains additional 21 amino acids at its N-terminus, which are proposed to be involved in its membrane localization, when compared to TRBP1. The resonance assignment (19–228) of the double-stranded RNA binding domains (dsRBD 1 and 2) of TRBP2 has been reported earlier. Here, we report 1H, 13C and 15N resonance assignment for dsRBD1 of TRBP2 (1–105) containing the additional N-terminal residues. This assignment will provide deeper insights to understand the effect of these residues on the structure and dynamics of TRBP2 and would therefore help in further elucidating the differences in the role of these isoforms.


TRBP1 TRBP2 Backbone assignment RNA binding modes NMR spectroscopy 


Trans-activation response element (TAR) RNA binding protein (TRBP)—first acknowledged as the protein involved in interaction with HIV TAR element (Gatignol et al. 1991)—belongs to a family of double stranded RNA binding proteins (dsRBPs). Recent studies have confirmed the involvement of TRBP in essential biological pathways that regulate cell growth and metabolism, including biogenesis of small non-coding RNAs (microRNAs and small interfering RNAs), expression of viral RNAs, etc. via interaction with dsRNAs and/or proteins. (Daniels and Gatignol 2012).

MicroRNAs (miRNAs), small 20–22 nucleotide-long endogenous RNAs, are regulatory RNAs that are involved in various phases of cellular growth and development (Kim et al. 2009). The tightly-regulated miRNA biogenesis pathway involves multi-protein complexes that process precursor (pre-) and primary (pri-) miRNAs into mature miRNAs (Bhatia et al. 2015; Kim et al. 2009). These complexes primarily include an RNase III enzyme and a double stranded RNA binding protein (dsRBP). In these pathways, TRBP acts as a part of RNA induced silencing complex (RISC)-loading complex (RLC), where it first assists the RNase III enzyme—Dicer to position the site where long pre-miRNA is cleaved to shorter miRNA:miRNA* duplex; and then assists Ago and Dicer for selection of mature miRNA strand (Noland et al. 2011).

TRBP is known to exist in humans in two isoforms: TRBP2 (isoform 1) and TRBP1 (isoform 2) (Daniels and Gatignol 2012). The major difference between the two isoforms lies in the fact that TRBP1 (22–366) lacks the initial 21 amino acids present at N-terminal of the TRBP2 (1–366). Although the exact role of these additional 21 residues in TRBP2 is still unknown, it has been reported that it might be involved in the membrane localization of the TRBP2 (Daniels et al. 2009). Both the isoforms of TRBP contain two double stranded RNA binding domains (dsRBDs) that are involved in its interaction with dsRNAs, and a third dsRBD that is involved in protein–protein interactions. The two dsRBDs are known to directly interact with a variety of dsRNAs with helical imperfections (A-form helix of RNA interrupted by loops and bulges at varying locations) by a relatively unknown mechanism.

To gain further insights into the same, we are in the process of performing detailed dynamics study of different domains of TRBP2 at multiple time-scales (data not shown). Furthermore, because the exact role of the additional 21 residues at the N-terminal is not entirely known, it is important to include these residues in the detailed dynamics studies to understand their significance in RNA recognition. Previous studies have reported the crystal structures of TRBP–dsRBD1 (28–54 and 60–96 aa) (PDB id:3LLH) and TRBP–dsRBD2 (161–227 aa) with RNA duplex (PDB id: 3ADL) (Yamashita et al. 2011). In addition to this, the solution structure of TRBP–dsRBD2 (150–225 aa) (PDB id:2CPN) has also been reported (Yamashita et al. 2011). Here, we report 1H, 13C and 15N resonance assignment and structure (CS-Rosetta (Shen et al. 2008, 2009)) calculated using backbone chemical shifts of dsRBD1 of TRBP2 and its comparison with crystal structure of TRBP1-dsRBD1 (28–96 aa).

Materials and methods

Protein expression and purification

The plasmid encoding TRBP2–dsRBD1 was provided as a kind gift by Dr. Jennifer Doudna (University of California, Berkeley, CA, USA). The clone was received in pSV272 vector—which is a modified form of pET27 vector—producing fusion protein containing His6-MBP tag at N-terminus followed by Tobacco Etch Virus (TEV) protease cleavage site before TRBP2–RBD1. The clone was then transformed into Escherichia coli NTC cells for large-scale plasmid purification (for further use and storage) and into BL21(DE3) cells for over-expression of recombinant protein. For preparation of 15N labeled protein, cells were grown in M9 media containing 15NH4Cl (Sigma–Aldrich) as sole source of nitrogen. For preparation of 15N–13C labeled protein sample, 13C-labeled glucose (Cambridge Isotope laboratories) was used as source of carbon in addition to 15NH4Cl (Sigma-Aldrich) as sole source of nitrogen. A single colony was picked and grown at 37 °C overnight in 10 ml M9 media containing 50 µg/ml kanamycin (Sigma-Aldrich). The primary culture was then transferred to 1 L of M9 medium and grown at 37 °C with shaking at 225 rpm. The cells were induced with 1 mM IPTG when cell density reached OD600 ~ 0.8–1.0 and were further incubated at 28 °C for about 8 h. Post-incubation, cells were harvested by centrifugation at 8000×g at 4 °C for 20 min and resuspended in lysis buffer (20 mM Tris at pH 7.5, 500 mM NaCl, 10% glycerol and 10 mM Imidazole). Cell lysis was performed by using 1% Triton X-100 and lysozyme (250 µg/ml of cell suspension). Complete lysis of the cells was ensured by sonication using an ultrasonic homogenizer at 70% amplitude, using a microprobe with 10 s ON and 10 s OFF cycle on ice. The crude cell extract was centrifuged at 32,000×g at 4 °C for 30 min. From the cleared supernatant thus obtained, the soluble nucleic acids were removed by polyethylene imine (PEI) precipitation. Further, protein was precipitated using a saturated solution of ammonium sulfate, resuspended in lysis buffer and further subjected to dialysis in lysis buffer to remove ammonium sulfate. The His6-tagged fusion protein was then purified using a pre-equilibrated Ni–NTA column (HisTrap, 5 ml, GE healthcare). Elution of the bound protein was performed with elution buffer (20 mM Tris at pH 7.5, 500 mM NaCl, 10% glycerol and 300 mM Imidazole). Imidazole was removed from the eluted protein by buffer exchange against cleavage buffer (20 mM Tris at pH 7.5, 25 mM NaCl). Cleavage of the purified fusion protein was carried out by TEV protease for 12–16 h at 4 °C with continuous mixing using cleavage buffer containing 10% glycerol. Post-cleavage, His6-tag and impurities were removed by second round of Ni–NTA purification. The cleaved protein, thus obtained, was collected in column washings with gel filtration buffer (20 mM Tris at pH 7.5, 500 mM NaCl, 10% glycerol). The purity of the protein was further assured by running it through sephacryl S-200HR gel filtration column (GE healthcare). Purified protein was concentrated to ~ 1 mM concentration in Amicon 15 ml filters with 3 kDa cutoff (Merck), was exchanged with NMR buffer (10 mM Sodium phosphate at pH 6.4, 100 mM NaCl, 1 mM EDTA and 1 mM DTT) and was stored at 4 °C until further use.

NMR spectroscopy

All the experiments were recorded on Bruker AVANCE III NMR spectrometer operating at 14.1 T equipped with quad resonance (1H, 13C, 15N, 31P, 2H) cryogenic probe and pulsed field gradients in x, y and z directions. 800 µM sample of 13C–15N–TRBP2–RBD1 in NMR buffer containing 5% 2H2O at 25 °C was used for the data collection to perform resonance assignments. To get sequential connectivity, a battery of triple-resonance experiments, namely HNCA, HN(CO)CA, HNN, CBCANH, CBCA(CO)NH, HNCO and HN(CA)CO, were recorded (Cavanagh 2007; Panchal et al. 2001). 1H–15N HSQC spectrum was recorded on 15N–13C-labeled sample of protein after every triple-resonance experiment to make sure that the protein is stable while the complete set of triple-resonance experiments was being recorded. 15N–TRBP2–dsRBD1 sample in NMR buffer was used to record 15N-edited TOCSY–HSQC spectrum (TOCSY mixing time = 80 ms) for amino acid identification. The data collected was processed in Topspin 3.2 software (Bruker) and analyzed in CARA v1.9.1 ( The 1H chemical shifts were directly referenced to 2,2-dimethyl-2-silapentane-5-sulphonic acid (DSS) (δ = 0 ppm) at 25 °C. 13C and 15N chemical shifts were indirectly referenced as mentioned elsewhere (Wishart et al. 1995).

Structure calculation

The backbone assignment (HN, N, C′, Cα, Cβ) for TRBP2–dsRBD1 in NMR-STAR2.1 format was uploaded in the CS-Rosetta server available at ( (Shen et al. 2008, 2009). Three thousand structures were generated and optimizations were carried out to get best model for the chemical shift data. While calculating the structure, any modification in primary sequence to exclude the flexible residues was disallowed.

Results and discussion

Extent of resonance assignment

The assigned 1H–15N HSQC spectrum of TRBP2–dsRBD1 has been depicted in Fig. 1. The backbone assignment for 97.9% (96 out of 98) of amide groups, 74.5% (73 out of 98) for C′, 93.9% (92 out of 98) for Cα and 67.1% (57 out of 98) of Cβ was achieved for non-proline residues. Also, 92.9% (91 out of 98) Hα and 47.2% (67 out of 143) Hβ side chain protons were successfully assigned. The assignment for the non-native residues—GG, the remnants at the N-terminal after cleavage from the fusion tag (not counted in 1–105 sequence length)—was not obtained. In addition to these, the resonance assignments for I19 and E20 could not be obtained. Chemical shift assignments for TRBP2–dsRBD1 (1–105) have been deposited in BMRB database with accession no. 27262.

Fig. 1

a Backbone resonance assignment marked on 1H–15N HSQC spectrum of TRBP2–dsRBD1 (1–105). Insets are used to zoom crowded peak regions. Pair of side-chain NH2 peaks have been marked with horizontal lines. b Amino acid sequence of TRBP2–dsRBD1 with underlined residues (I19, E20 and all proline residues) for which assignment could not be obtained

The backbone resonance assignment for TRBP2–dsRBD-1-2 (19–228 aa) has earlier been submitted in BMRB (accession number 18324) by Plevin and coworkers (Benoit and Plevin 2013). The results from this communication summarized that the deletion of dsRBD1 or dsRBD2 did not affect the backbone chemical shifts of the TRBP2–dsRBD-1-2 (19–228 aa), thereby depicting absence of inter-domain cooperativity (Wilson et al. 2015). However, upon comparison of the 1H–15N HSQC spectrum of the common residues (19–105 aa) of TRBP2–dsRBD1 (1–105 aa) and TRBP2–dsRBD-1-2 (19–228 aa) (previously recorded) (Fig. 2), large differences were observed. In fact, it was difficult to transfer resonance assignments from TRBP2–dsRBD-1-2 spectrum (19–228 aa) to the TRBP2–dsRBD1 spectrum (1–105 aa) reported here. We expected to have one blue peak for every red peak in the spectrum (Fig. 2), but we could only find correlation in the peripheral peaks. In the crowded region of the spectrum, one-to-one correlation between red and blue peak was not visible highlighting structural differences in the loop and helical regions.

Fig. 2

Overlay of 1H–15N TROSY–HSQC of TRBP–dsRBD-1-2 (19–228 aa) onto 1H–15N HSQC of TRBP–dsRBD1 (1–105 aa). To highlight the chemical shift perturbations, common residues in the two constructs have been colored in red (19–105, TRBP–dsRBD1) and blue (19–105, TRBP–dsRBD-1-2). The absence of one-to-one correlation between red and blue peaks suggested structural perturbations originating due to presence of additional N-terminal residues in TRBP2–dsRBD1. The remaining residues have been marked in gold (1–18, TRBP2–dsRBD1) and grey (106–228, TRBP2–dsRBD-1-2)

Chemical shift perturbations

Combined chemical shift perturbations for backbone 1H and 15N were calculated between TRBP2–dsRBD1 and TRBP2–dsRBD-1-2 and plotted against residue number (Fig. 3). It is clearly evident from the Fig. 3 that in addition to a basal chemical shift perturbation (of the order of 0.1 ppm) all along the protein chain, there are significant deviations in the amino acids M22, L23, A24, A25, K29, H58, H85 and L105. While a shift in L105 is expected as it is a terminal residue, chemical shift perturbations in N-terminal stretch of residues (M22, L23, A24, A25 and K29) indicated structural differences possibly arising due to interaction with additional N-terminal residues (1–18 aa). Further, the residues H58 and H85 belong to the loop and α-helix parts of the protein, respectively, which are involved in RNA recognition. These results hinted towards the possible role of the additional N-terminal residues in TRBP2 over TRBP1 in RNA recognition. However, these results need to be experimentally verified further.

Fig. 3

Backbone 1H and 15N chemical shift perturbations between TRBP2–dsRBD1 and TRBP2–dsRBD-1-2 for the common amino acids (19–105)

The study by Plevin and co-workers had reported the resonance assignments using a buffer system (23.5 mM Phosphate buffer, pH 6.5, 100 mM KCl, 10 mM MgCl2 and 5 mM β-mercaptoethanol) different from the one used in this study (10 mM Phosphate buffer, pH 6.4, 100 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol). To eliminate the possibility of the differences in the buffer leading to the sharp perturbations observed in the spectra reported in Figs. 2 and 3, the 1H–15N HSQC spectra of TRBP2–dsRBD1 were recorded and compared in the two buffer conditions (Supplementary Figs. 1 and 2). Upon comparison, a maximum chemical shift perturbation of < 0.025 ppm was observed (Supplementary Fig. 2) thereby eliminating the role of the buffer in inducing the perturbations. This in turn hints towards the possible role of the additional 21 N-terminal residues in TRBP2 affecting the chemical shifts of residues present in the loop and helical regions of the spectrum.

Structure comparisons with crystal structure of domain 1

Using CS-Rosetta program (Shen et al. 2008, 2009), the tertiary structure of the TRBP2–dsRBD1 was calculated with the help of the backbone chemical shifts (HN, N, C′, Cα and Cβ). The backbone assignment for 97.9% (96 out of 98) of amide groups, 74.5% (73 out of 98) for C′, 93.9% (92 out of 98) for Cα and 67.1% (57 out of 98) of Cβ, 92.9% (91 out of 98) Hα and 47.2% (67 out of 143) Hβ side chain protons were used for structure calculation using CS-Rosetta. The structure calculated was validated using MolProbity (Davis et al. 2007) and ProCheck (Laskowski et al. 1993) (Supplementary Figs. 3 and 4, respectively). MolProbity (for all 10 models) calculated 97.6% (1035/1060) residues in favored (98%) regions; 100.0% (1060/1060) residues in allowed (> 99.8%) regions; and no outliers were predicted. ProCheck calculated 92.1% residues in most favored regions while 7.1% in additional allowed regions; no residues were found in generously allowed or disallowed regions.

The structure optimized by CS-Rosetta shows presence of α1–β1–β2–β3–α2 fold which is common to dsRBDs (Fig. 4) and matches well (RMSD = 1.44 Å) with the already reported crystal structure of dsRBD1 of TRBP for the common residues (28–96 aa). Furthermore, the presence of an additional helix was observed in a stretch of residues 18–24 and was found in close-proximity to middle part of α1 helix, which is one of the RNA recognition motifs of the protein. Interestingly, this newly found helix in TRBP2–dsRBD1, predicted by CS-Rosetta, also showed relatively higher (of the order of 8–10 Hz; data not shown) backbone transverse relaxation rates (R2) as compared to that measured for additional N-terminal residues (of the order of 3 Hz; data not shown) suggesting presence of conformational plasticity or chemical exchange in the residues 18–24.

Fig. 4

Overlay of crystal structure (PDB id 3LLH, green) with that of one of the 10 low energy structures as calculated by CS-Rosetta (red and gold) using backbone chemical shifts. While the common residues in the two structures (28–96) have an RMSD of 1.44 Å, an additional short helix in the stretch of 18–24 aa was also observed


The backbone resonance assignment for dsRBD1 of Isoform 1 (containing the additional N-terminal residues in comparison to isoform 2) of TRBP was obtained. The chemical shift perturbations of backbone amide protein and nitrogen chemical shift between isoform 1 and 2 suggested that additional N-terminal residues perturb the structure of dsRBD1 keeping the overall fold similar. This suggested that the additional 21 residues in TRBP2–dsRBD1 are possibly involved in some interaction with rest of the protein chain (at least in case of dsRBD1). This also suggested that TRBP2 and TRBP1 may have different mode of binding to dsRNAs owing to structural differences in dsRBD1. Our study highlights the importance of these N-terminal residues and it may be worthwhile to include these residue (in other words use isoform 1 instead of isoform 2) while performing the dynamics and structural investigations on TRBP protein in absence and presence of RNA molecules.



Authors thank Prof Jennifer Doudna for the TRBP clones. JC thanks IISER Pune for the start-up grant. JC acknowledges extra mural grant from Department of Science & Technology, India (EMR/2015/001966). SS acknowledges Ramalingaswami fellowship (BT/RLF/Re-entry/11/2012) and SPPU Research and Development grant to Department of Biotechnology. HP thanks IISER Pune for the fellowship; PVJ and ASN acknowledge Department of Biotechnology, India for their Masters in Biotechnology fellowship. Authors thank High-field NMR facility at IISER Pune funded by DST-FIST and IISER Pune.

Supplementary material

12104_2018_9807_MOESM1_ESM.docx (3 mb)
Supplementary material 1 (DOCX 3062 KB)


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Authors and Affiliations

  1. 1.Department of ChemistryIndian Institute of Science Education & ResearchPuneIndia
  2. 2.Department of BiologyIndian Institute of Science Education & ResearchPuneIndia
  3. 3.Department of BiotechnologySavitrabai Phule Pune University (Formerly University of Pune)PuneIndia

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