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. 2003 Sep;12(9):1925–1933. doi: 10.1110/ps.0390103

Solution structure of the C-terminal domain from poly(A)-binding protein in Trypanosoma cruzi: A vegetal PABC domain

Nadeem Siddiqui 1, Guennadi Kozlov 1, Iván D’Orso 2, Jean-François Trempe 1, Kalle Gehring 1
PMCID: PMC2323990  PMID: 12930992

Abstract

PABC is a phylogenetically conserved peptide-binding domain primarily found within the C terminus of poly(A)-binding proteins (PABPs). This domain recruits a series of translation factors including poly(A)-interacting proteins (Paip1 and Paip2) and release factor 3 (RF3/GSPT) to the initiation complex on mRNA. Here, we determine the solution structure of the Trypanosoma cruzi PABC domain (TcPABC), a representative of the vegetal class of PABP proteins. TcPABC is similar to human PABC (hPABC) and consists of five α-helices, in contrast to the four helices observed in PABC domains from yeast (yPABC) and hyper plastic disk proteins (hHYD). A mobile N-terminal helix is observed in TcPABC that does not pack against the core of the protein, as found in hPABC. Characteristic to all PABC domains, the last four helices of TcPABC fold into a right-handed super coil. TcPABC demonstrates high-affinity binding to PABP interacting motif-2 (PAM-2) and reveals a peptide-binding surface homologous to that of hPABC. Our results demonstrate the last four helices in TcPABC are sufficient for peptide recognition and we predict a similar binding mode in PABC domains. Furthermore, these results point to the presence of putative PAM-2 site-containing proteins in trypanosomes.

Keywords: NMR, translation factors, poly(A)-binding proteins, PABC domains, trypanosomes

Poly(A)-binding proteins (PABPs) are ubiquitous RNA binding proteins found in all eukaryotes and are implicated in stabilizing mRNA and promoting translation (Gingras et al. 1999). PABP consists of an N terminus with four highly conserved RNA recognition motifs (RRMs) and a C terminus containing a phylogenetically conserved peptide-binding domain referred to as PABC. The RRMs bind on to the 3′-poly(A) tail (Deo et al. 1999) and serve to protect mRNA from deadenylation, which is the rate-limiting step in mRNA decay (Wang et al. 1999; Grosset et al. 2000). Furthermore, this domain associates with initiation factor 4G (eIF4G) and subsequently stimulates (Haghighat and Sonenberg 1997) eIF4E to interact with the 5′-m7GpppX cap structure on mRNA (Tarun and Sachs 1996; Le et al. 1997; Tarun et al. 1997; Imataka et al. 1998; Gray et al. 2000). Simultaneous binding of eIF4G to PABP and eIF4E loops the mRNA transcript by having its 5′ and 3′ termini joined (Tarun and Sachs 1996; Wells et al. 1998). The circularization of mRNA promotes translation by facilitating terminating ribosomes to cycle on the same mRNA transcript (Fig. 1 ▶). Two PABP interacting proteins, Paip1 and Paip2, were shown to stimulate (Craig et al. 1998) and inhibit (Khaleghpour et al. 2001b) protein translation, respectively. Both proteins contain two independent motifs (Khaleghpour et al. 2001a) that contact PABP and are referred to as PABP interacting motifs PAM-1 and PAM-2 (Roy et al. 2002). The PAM-1 site interacts with the N-terminal RRM region of PABP, whereas the PAM-2 motif interacts with the PABC domain. Release factor 3(RF3)/GSPT also interacts with PABP through a PAM-2 motif within its N terminus (Uchida et al. 2002). In general, a 12-residue PAM-2 motif is necessary and sufficient for binding to PABC (Kozlov et al. 2001, 2002). Although the physiological role of PABC is not entirely clear, it is thought to be responsible for recruiting translation factors, such as Paip1 and 2, and RF3/GSPT to the poly(A)-tail and the 5′–3′ initiation complex on mRNA (Deo et al. 2001; Kozlov et al. 2001).

Figure 1.

Figure 1.

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A model of mRNA circularization via PABP, eIF4G, and eIF4E interaction and proposed role for PABC domain. PABP N-terminal RNA recognition motif interacts with eIF4G, which associates with eIF4E, thereby linking the mRNA termini. Circularization of mRNA promotes translation by facilitating terminating ribosomes to cycle on the same transcript. The PABC domain of PABP recruits a series of translation factors such as Paip1, Paip2, and release factor 3, through the conserved PAM-2 motif, to the initiation complex on mRNA.

A phylogenetic analysis of the C-terminal domain from PABPs (Kozlov et al. 2002) reveals that they can be categorized into three main classes: animal, vegetal, and a "divergent" group. The divergent group includes Saccharomyces cerevisiae, Schizosaccharomyces pombe, and a PABC-like domain found in human hyper plastic disk proteins (hHYDs), which belongs to the family of ubiquitin ligases (Henderson et al. 2002). At present, the role of a PABC domain within ubiquitin ligases remains unclear; however, it is suggested that they bind and ubiquitinate proteins containing PAM-2 like sequences as an alternative mechanism to regulate PABP function and subsequently translation (Deo et al. 2001). Recently, the structure of PABC from human (Kozlov et al. 2001) and two from the divergent class, S. cerevisiae (Kozlov et al. 2002) and HYD (Deo et al. 2001), have been solved. PABCs are completely α-helical in nature and adopt a fold resembling a right-handed super coil. One of the main differences between the three structures is the presence of an extra N-terminal α-helix on human PABC (hPABC). The last four α-helices in hPABC encompass the peptide-binding site and, among PABC domains, sequence conservation is highest among helices α2, α3, and α5, which are required for peptide recognition (Kozlov et al. 2001). Even though the PABC domain in hHYD has four helices, it is structurally more similar to hPABC than to yeast PABC (yPABC). Furthermore, Paip-1 was demonstrated to interact with hHYD, indicating that the last four helices are sufficient for protein binding (Deo et al. 2001). Although the sequence identity between hPABC and yPABC is 40%, the yeast structure shows several distinct features, in particular a strongly bent C-terminal helix that results in altered specificity and affinity for peptide binding (Kozlov et al. 2002). For instance, yPABC binds PAM-2 peptides but with much lower affinity. Studies have shown that the N-terminal region of RF3/SUP35 is required for yeast PABP recognition (Cosson et al. 2002). However, a clear PAM-2 site could not be identified within its N terminus, indicating that different sequence specificity exists in yeast.

The vegetal class of PABPs contains predominantly plant species, although a branch including trypanosomes is also present. This is not surprising because trypanosomes contain several genes encoding homologs of proteins found in either chloroplasts or the cytosol of plants and algae (Hannaert et al. 2003). Trypanosomes are protozoan parasites known to cause disease and infection in humans and other animals. For instance, Trypanosoma cruzi is the causative agent for Chagas’ disease, an endemic illness in Latin American countries. The PABP in this organism (PABP1) is constitutively expressed in all stages of the parasite’s life cycle. The translation system in trypanosomes is unique in that their gene expression is not regulated through transcription initiation but via posttranscriptional regulatory mechanisms including modification of the half-life of mRNA (Clayton 2002). For instance, uridine-rich binding protein-1 (TcUBP-1) inhibits PABP from binding mRNA and consequently contributes to destabilizing mRNA in order to regulate mRNA turnover (D’Orso and Frasch 2001,D’Orso and Frasch 2002).

Here, we report the solution structure of PABC from T. cruzi (TcPABC), a representative of the vegetal class of PABC domains. Similarly to hPABC, TcPABC consists of five α-helices. However, the N-terminal helix is mobile relative to the last four helices that fold into the characteristic right-handed super coil. Nevertheless, the structure maintains high affinity for PAM-2 motifs and contains a peptide-binding surface homologous to hPABC.

Results

Secondary structure determination

The C-terminal domain of PABP1 from T. cruzi, residues 1–85 (see Materials and Methods), was prepared as an isotopically labeled recombinant protein for structural studies by NMR spectroscopy. The secondary structural composition of TcPABC was determined by analysis of chemical shift values from Cα, Cβ, and Hα atoms and 3JHNHα coupling constants obtained from HNCACB, CBCACONH, and HNHA experiments (Wüthrich 1986; Wishart and Sykes 1994). In the case of Hα or Cβ shifts, a negative value from the difference between measured chemical shifts and random coil values indicates the presence of α-helical structure, whereas positive deviations are attributed to β-sheets. The contrary holds for Cα deviations. In the case of 3JHNHα couplings, values <6 Hz indicate α-helical content, whereas values >8 Hz indicate β-sheets. Based on these standards, the consensus (Fig. 2 ▶) indicates 5 α-helices located within residues L11–L15, L18–V36, A42–L50, M53–L59, and D63–L79. Similar to all PABC domains, TcPABC has only α-helical conformations. Furthermore, the secondary structure is most similar to hPABC with the presence of an N-terminal α-helix (Fig. 3 ▶).

Figure 2.

Figure 2.

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JHN-Hα coupling and chemical shift analysis for secondary structure determination in TcPABC (residues 1–85). (A) JHN-Hα coupling values for residues <6 Hz (filled boxes) and >6 Hz (open boxes). Chemical shift deviations from random coil values for (B) Cα, (C) Cβ, and (D) Hα atoms are shown as a function of residue number. Consensus of the results indicates that TcPABC contains 5 α-helices, represented above the graphs as black cylinders.

Figure 3.

Figure 3.

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Sequence alignment of PABC domains and potential ligands. (A) Sequence alignment of TcPABC with PABCs from the vegetal class and with hPABC, yPABC, and hHYD. The 5 α-helices are represented above the sequences as cylinders. The first α-helix, shown in gray, is present in both human and trypanosome PABC. (B) Sequence comparison of human RF3 PAM-2 motif and putative RNA binding proteins derived from A. thaliana (BAB02607), B. napus (AAF00075), and O. sativa (AAK50577) that contain a PAM-2 site (NCBI accession numbers are in parentheses). Highlighted is the human PAM-2 consensus sequence. The figure was created using BOXSHADE (EMBnet). Identical amino acids are highlighted in black and homologous residues in gray.

Heteronuclear NOE analysis

Dynamic properties of TcPABC on the nano- to picosecond timescales were explored by measuring 1H-15N heteronuclear NOE values (hNOEs), which measure the reorientation rate of the amide nitrogen–hydrogen internuclear vector. The spectra were acquired at 500 MHz and thus, in theory, values range from −3.6 to 0.82 for unfolded and folded residues, respectively (Peng and Wagner 1994). A total of 77 of 85 amide-proton signals were obtained (Fig. 4A ▶). No data are presented for prolines (P20, P40, P65), missing amides (G1, S2), or amides with significant overlap (S3, N10, I58, L61, T64, D69) on the 1H-15N heteronuclear single quantum correlation (HSQC) spectra. As expected, negative hNOEs values were obtained for unfolded regions (residues L4–Q9 and N84–V85), indicating high flexibility. For residues A16–R81, covering helices α2–α5, values were between 0.55 and 0.85, indicative of a slow tumbling rigid conformation. Intriguingly, hNOE values between 0.28 and 0.49 were obtained for residues L11–L15, which encompass the first helix. This indicates that the first helix, although structured, is relatively flexible in solution. This flexibility explains why no long-range NOEs are found for this helix within 2D and 3D NOESY experiments. In hPABC, the first helix shows numerous long-range NOEs to helices α2, α4, and α5 (Kozlov et al. 2001).

Figure 4.

Figure 4.

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Plots of 1H-15N heteronuclear NOE value, NOE constraints, and RMSD statistics for TcPABC (residues 1–85). Black cylinders above the graph represent α-helices with the primary sequence of TcPABC shown below. (A) 1H-15N heteronuclear NOE values obtained at a frequency of 500 MHz. (B) A summary of all assigned unambiguous NOEs: intraresidue, sequential, medium, and long-range NOEs are shown in medium gray, black, dark gray, and light gray, respectively. (C) A comparison of the average backbone RMSD per residue of the 20 lowest structures calculated without (diamonds) and with (circles) 15N-1H residual dipolar couplings.

Solution structure of TcPABC

The 3D structure of TcPABC was calculated using standard molecular dynamics protocols as described in Materials and Methods. Figure 4B,C ▶ illustrates the distribution of NOEs and the resulting backbone RMSD as a function of sequence position. The backbone RMSD is approximately inversely proportional to the number of NOEs for each residue. Long-range NOEs could not be identified for residues Q9–K24 and M53–I58, which cover helices α1, the N-terminal of α2, and helix α4. This is reflected by high RMSD values within these regions (Fig. 4C ▶).

A set of 77 1H-15N residual dipolar couplings (RDCs) was measured on 15N-labeled TcPABC in Pf1 phage and added to our calculations for further refinement. Including RDCs for helix α1 did not lead to any meaningful orientation within the structure, verifying that RDCs cannot be used for mobile regions (Meiler et al. 2001). Thus, RDCs were only used for regions with heteronuclear NOE values above 0.55. Hence, for our final round of calculations, 55 1H-15N RDCs for residues N17–R81 (helices α2–α5) were applied. RDC values were not obtained for prolines (P20, P40, P65) or amides with significant overlap (I58, L61, T64, D69) on the 1H-15N HSQC spectra. The same data set without RDCs leads to a distinctly higher backbone RMSD of 0.75 Å versus 0.55 Å with RDCs, improving the convergence of the structures by 36%. In parallel, the RDC Qfactor (Cornilescu et al. 1998) drops from 0.545 to 0.156 using RDCs; thus, the agreement of the structure after refinement with the measured couplings is greatly improved. This is especially true for the loop region following helix α3 and all of helix α4, (residues G48–G66; Fig. 4C ▶), which becomes much better defined with the use of RDCs, balancing the relative lack of NOEs within the region. The last four helices fold into a well-defined bundle, as supported by the residues that form the hydrophobic core and provide the most long-range NOEs (L27, L31, Y32, V36, A43, L50, L59, L62, L68, V72, and L76). As illustrated in the calculated ensemble of structures (Fig. 5 ▶), there is narrow deviation for the last four helices, whereas the first helix shows greater variance from the mean structure.

Figure 5.

Figure 5.

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Stereo drawing of the superposition for backbone atoms of 20 structures with lowest energy and least violations for TcPABC (residues 8–82). The RMSD to the mean structure for the defined region of TcPABC, N17–L79, with RDCs is 0.55 ± 0.14 Å for backbone atoms and 0.91 ± 0.13 Å for all heavy atoms.

Peptide binding site in TcPABC

The peptide-binding site on TcPABC was determined using chemical shift perturbation analysis with a PAM-2 peptide. The PAM-2 consensus sequence for hPABC (Kozlov et al. 2001) was used to search (NCBI-BLAST) the T. cruzi genome for potential PABC ligands. Because T. cruzi and related genomes (Trypanosoma brucei and Leishmania major from the order Kinetoplastida) have been incompletely sequenced, no proteins containing PAM-2 sites could be identified. Proteins with PAM-2 motifs were found in genomes phylogenetically close to T. cruzi. Sequence comparison of putative RNA binding proteins from Arabidopsis thaliana, Brassica napus, and Oryza sativa revealed a PAM-2 motif most similar to human RF3 (Fig. 2B ▶). The PAM-2 motif found in these putative RNA binding proteins indicates that they could play a role in translation regulation by interacting with PABC.

The peptide selected for this study was derived from the N-terminal sequence in human RF3 (residues A47–R74; NCBI accession no. NP060564). Human RF3 (hRF3) peptide was added to a 15N-labeled sample of TcPABC and the residues that show the greatest chemical shift changes on a 15N-1H HSQC spectrum were monitored to identify regions within TcPABC that participate in peptide binding. On titration at low peptide ratios (0.1–0.3 mM peptide: 1 mM protein), amide peaks from many residues broadened and some disappeared with increasing amounts of hRF3. At higher concentrations of hRF3, all missing peaks reappeared. Because intermediate exchange was observed, HNCACB and CBCACONH experiments were carried out on the TcPABC-RF3 complex to reassign the PABC backbone. Comparison of amide chemical shifts (ppm) obtained from a 15N-1H HSQC spectrum with and without peptide shows residues E29 (0.227), Y32 (0.32), K45 (0.364), M49 (0.484), A75 (0.205), E77 (0.232), and V78 (0.391) having the largest change on binding to hRF3. These residues, residing on helices α2, α3, and α5, define a peptide-binding surface (Fig. 6B ▶) analogous to hPABC (Kozlov et al. 2001).

Figure 6.

Figure 6.

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Identification of the peptide-binding site in TcPABC and the family of PABC domains. (A) A plot of chemical shift changes of amide signals as a function of residue number. (B) Cα trace colored according to the size of chemical shift change on RF3 binding. Residues with largest chemical shift change (Y32, K45, M49, and V78) are labeled. (C) Ribbon diagrams of PABC domains from (I) human PABC (PDB 1G9L), (II) human PABC domain in hyper plastic disk proteins (PDB 1I2T), (III) S. cerevisiae PABC (1IFW), and (IV) PABC from T. cruzi (PDB 1NMR). All ribbon diagrams represent a snapshot of each structure. Backbone RMSD and DALI Z scores of TcPABC are 1.66 Å (Z = 5.6), 1.42 Å (Z = 6.0), and 1.97 Å (Z = 3.0) for the PABC domain of human, hyper plastic disks, and yeast, respectively. Distance matrix alignment scores (DALI Z) were calculated at EMBL-European Bioinformatics Institute (EBI). Z scores represent the statistical significance of the best structural alignment.

Discussion

PABC is a highly conserved peptide-binding domain found in the C-terminal region of PABPs and within the HYD family of ubiquitin ligases. It appears that the function of PABC is to recruit translation factors containing PAM-2 to the poly(A)-tail of mRNA. The C-terminal domain of PABP from T. cruzi (TcPABC) represents a vegetal PABC domain. TcPABC shares up to 75% sequence similarity with vegetal PABCs (Fig. 2A ▶). In general, PABC domains are highly similar throughout all species; however, primary sequence conservation is lowest within its N terminus. Structurally, the N-terminal helix is absent within PABCs in HYDs (Deo et al. 2001) and yeast (Kozlov et al. 2002). In hPABC, the N-terminal helix is present and folds into the domain (Fig. 6C, I ▶). Our results show that TcPABC has an intermediate structure with the presence of a mobile N-terminal helix (residues L11–L15). According to secondary structure predictions (MLRC software), an N-terminal helix is most probably present throughout vegetal PABC domains. In addition, this helix is predicted to be longer in plants than in human or trypanosome PABCs. Residues L545–A547 in helix α1 of hPABC are highly conserved throughout animal PABC domains and provide important long-range contacts to the last four helices, which aids the first helix to pack against the protein. Similar residues, conserved as (I/V-G/V-A), are present in plant PABCs, but not in trypanosomes. This suggests that the mobility of the N-terminal helix is a feature unique to trypanosomes.

The remaining helices in TcPABC, α2–α5, fold into a well-defined 4 α-helical core resembling the characteristic arrowhead shape observed in PABC structures (Fig. 6C,IV ▶). A pairwise Cα backbone overlay between the last four helices of TcPABC (residues 17–79) and existing PABC structures exhibits highest structural homology with hPABC (1.66 Å) and hHYD (1.47 Å). Our results suggest that all plant PABCs will adopt a similar fold to TcPABC. However, it remains to be determined whether the mobility of the first helix extends throughout plant PABCs.

A putative protein sequence encoding for RF3 was identified in the trypanosome database (TIGR accession no. 1101628), although the sequence encompassing its N-terminal region was not fully sequenced. Because PAM-2 sequences are located within the N terminus of RF3s, it will be important to clone the full-length protein to detect a probable PAM-2 site within RF3 in trypansomes. Titration experiments of TcPABC with hRF3 PAM-2 peptide revealed a peptide-binding surface highly comparable to hPABC (Kozlov et al. 2001). Intermediate exchange was observed during titration experiments, indicating that TcPABC’s affinity for hRF3 is within a low micromolar range (1–10 μM). A similar exchange regime and affinity was observed for hPABC (data not shown) on binding of hRF3. Our results show that, even though TcPABC possesses a mobile N-terminal helix, the affinity for the hRF3 peptide is not compromised. This is not surprising because the residues that participate in peptide binding are conserved within helices α2, α3, and α5. Removal of the first helix in hPABC gives similar 15N-1H correlation spectra to the full-length domain, indicating that the last four helices remain intact (Kozlov et al. 2002). Furthermore, Paip-1 was demonstrated to bind with hHYD (Deo et al. 2001). From our results, we predict that hPABC without the first helix will still maintain high affinity for PAM-2 peptides, indicating that the N-terminal helix is not required for peptide binding. Because TcPABC and hHYD share high structural homology, we also predict that hHYD will bind to PAM-2 peptides with comparable affinities. The presence of an N-terminal helix throughout animal and vegetal PABCs indicates a function independent of peptide recognition.

The C terminus of a bound PAM-2 peptide is positioned between the hydrophobic groove of helices α2 and α3, whereas the N terminus is stacked between helices α3 and α5 (Kozlov et al. 2001, 2002). In hPABC, residues (E19, F22, K35, M39, and V68) in helices α2, α3, and α5 in hPABC are most affected on binding PAM-2 motifs (Kozlov et al. 2001). Similarly, homologous residues Y32, K45, M49, and V78 in TcPABC (Fig. 6B ▶) also show the largest changes on binding PAM-2. This indicates that peptide recognition by TcPABC occurs by the same mechanism as in hPABC. The residues that participate in binding are highly conserved (Fig. 2A ▶), indicating that peptide recognition is highly conserved across all families of PABC domains.

Altogether, our results indicate that TcPABC recruits proteins with PAM-2 sites such as RF3, Paip homologs, and RNA binding proteins to the poly(A)-tail of mRNA. Determination of TcPABC interacting proteins is important for the study of translational regulatory mechanisms in trypanosomes an other Kinetoplastid parasites. Future work will be directed to using a yeast two-hybrid screen to search for biological partners and to identify protein partners for TcPABC.

Materials and methods

Cloning and expression of the C-terminal domain of PABP from T. cruzi

Primary sequence comparison between the C-terminal region in human PABP (accession no. AD08718) and T. cruzi PABP1 (accession no. AAC46487) revealed that highest homology exists between residues 518 and 636 from human and residues 453 and 550 from T. cruzi. Using secondary structure prediction software and comparison with the structured region of the C-terminal region in human PABP (Kozlov et al. 2001), residues 468–550 from T. cruzi were established as the PABC domain. A 249-bp fragment corresponding to this domain was amplified by PCR with oligonucleotides PABC-1 5′-GGATCCTCTTTGGCTTCACAGGGACAG-3′ and PABC-2 5′-GAATTCCTAAACGTTCATGTGGCGATTC-3′ (restriction sites are underlined), using genomic T. cruzi as the template. The fragment was cloned into the EcoRI and BamHI sites of a pGEX-2T vector (Amersham Biosciences). The construct, TcPABC, was transformed into BL21 Gold-DE3 (Stratagene) and selected on an LB agar plate supplemented with 100 μg/mL ampicillin. For NMR sample preparation, TcPABC was expressed in either 1× Luria broth or M9 media containing 15NH4Cl (Isotech Inc.) or 15NH4Cl and D-(13C6) glucose (Cambridge Isotope Laboratory), all supplemented with 100 μg/mL ampicillin. The cultures were grown at 37°C until OD600 reached ∼0.8. Thereafter, the temperature was reduced to 30°C and 1mM isopropyl-1-thio-β-D-galactopyranoside was added to the culture and shaken for 3 h to induce expression of GST-TcPABC fusion protein.

Purification, characterization of TcPABC, and sample preparation for NMR analysis

The harvested cells were resuspended in lysis buffer (1× phosphate buffered saline supplemented with 100 μg/mL of bovine lysozyme and 1 mM of the protease inhibitor phenyl-methyl-sulfonyl fluoride at pH 7.1) and kept on ice for 20 min. The total extract was centrifuged at 15000g and the supernatant was collected for subsequent purification. The recombinant GST-TcPABC protein was purified by affinity chromatography using a Glutathione-Sepharose 4B resin (Amersham Biosciences). The N-terminal GST tag was cleaved on the resin by treatment with thrombin (2 units/mg of fusion protein) overnight at 4°C. A final purification was completed using an HPLC gel-filtration column (Superdex 75 HR 10/30, Amersham Biosciences) to remove thrombin protease and other impurities. Characterization and sequence composition of TcPABC using SDS-PAGE analysis and ESI mass spectrometry confirmed the presence of 9.09 kD protein comprising 83 residues of TcPABC and a 2-residue (Gly-Ser) N-terminal extension. For NMR analysis, purified TcPABC was exchanged into a buffer containing 50 mM NaHPO3, 150 mM NaCl, 1 mM NaN3, and 10% D2O at pH 6.3. 2D homonuclear NOESY and 13C-NOESY experiments used samples prepared with 100% D2O (Cambridge Isotope Laboratory). The final concentrations of the protein in NMR samples were between 2 and 3 mM.

NMR spectroscopy

All NMR experiments were recorded at 303K using standard double and triple resonance techniques on 15N- or 15N, 13C-labeled samples (Bax and Grzesiek 1993). All of the experiments were done on a Bruker DRX 500 with the exception of the 13C-edited NOESY, which was collected on a Varian Inova 800 spectrometer. The following multidimensional experiments were recorded and evaluated: (1) for backbone assignments: HNCACB and CBCA(CO)NH (Grzesiek et al. 1992; Constantine et al. 1993); (2) for side-chain and NOE assignments: 15N-TOCSY, 15N-edited NOESY, 2D homonuclear NOESY in H2O and D2O, and a 13C-edited NOESY in D2O; (3) for dihedral angle restraints: 3J-HN-Hα coupling constants were obtained from an HNHA (Kuboniwa et al. 1994); (4) for 15N-1H residual dipolar couplings: an IPAP-HSQC experiment on an isotropic sample without phage and on a sample containing 18 mg/mL Pf1 phage (Hansen et al. 1998; Ottiger et al. 1998); and (5) for backbone dynamics: 15N-1H heteronuclear NOE data were measured by taking the ratio of peak intensities from experiments performed with and without 1H presaturation. Hydrogen bond constraints were introduced to secondary structure regions as determined by chemical shift analysis, HNHA experiments, and medium-range NOE patterns. Hydrogen bonds were defined as a restraint from the carbonyl oxygen atom to the amide hydrogen (i, i + 4), using a standard length of 1.5 Å for hydrogen bonds. All NMR spectra were processed using either XWIN-NMR software version 2.5 or 3.1 (Bruker Biospin) or GIFA software (Malliavin et al. 1998). Evaluation of spectra and manual assignments were completed with XEASY software (Bartels et al. 1995).

Peptide preparation, purification, and NMR experiments

The N-terminal region of human RF3 (NCBI accession no. NP060564), residues A47–R74 (Fig. 4B ▶), was synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis and purified by reverse-phase chromatography on a Vydac C18 column. The composition and purity of peptides was verified by ion-spray quadropole mass spectroscopy. Titration experiments on TcPABC with RF3 were carried out by measuring the change in chemical shifts of amide signals {[(Δ1H ppm)2 + (Δ15N ppm × 0.2)2]0.5} from 15N-1H HSQC spectra. All spectra were acquired on a Bruker 600 MHz AVANCE spectrometer at 303K.

Analysis and structure calculation

CNS 1.1 software (Brunger et al. 1998) was used to generate an initial fold of TcPABC with a basic set of NOEs acquired from manual assignments of 3D 1H-15N NOESY and 2D homonuclear NOE spectra including dihedral angle and hydrogen bond constraints (Wüthrich 1989). These calculations generated a fold that was used as a model template for automated assignments by ARIA 1.1 (Nilges et al. 1997). The final structure of TcPABC was calculated using standard protocols in CNS 1.1 with a total set of 1156 unambiguous constraints (Table 1) collected from the experiments described earlier. In the final round of calculations, CNS 1.1 was extended to incorporate RDC restraints for further refinement. The axial and rhombic components of the alignment tensor were defined from a histogram of measured RDCs (Clore et al. 1998a) and optimized by a grid search method (Clore et al. 1998b). Twenty structures were selected based on lowest overall energy and least violations to represent the final structures. PROCHECK-NMR was used to generate Ramachandran plots to check the protein’s stereochemical geometry (Laskowski et al. 1996). The coordinates of TcPABC have been deposited in the RCSB under PDB code 1NMR and the NMR assignments under BMRB accession no. 5698. PABC structures for comparison with TcPABC were taken from PDB entries 1G9L for hPABC, 1I2T for hHYD, and 1IFW for yPABC.

Table 1.

Structural statistics for 20 selected conformers for TcPABC

Constraints used for structure calculation
    Intraresidue NOEs(n = 0) 559
    Sequential NOEs(n = 1) 200
    Medium-range NOEs(n = 2,3,4) 146
    Long-range NOEs(n > 4) 69
    Dihedral angle constraints 78
    Hydrogen bonds 49
    15N-1H residual dipolar couplings 55
    Total number of constraints 1156
Average RMS difference to mean structure (Å) for residues 16–79
    Backbone atoms 0.55 ± 0.14
    All heavy (nonhydrogen) atoms 0.91 ± 0.13
Average energy values (kcal mole−1)
    Etotal 192.14 ± 7.48
    Ebond 6.89 ± 0.52
    Eangle 52.80 ± 2.42
    Eimproper 6.48 ± 0.50
    EVdW 90.01 ± 8.01
    ENOE 15.56 ± 2.32
    Edihedral 1.52 ± 0.26
    Esani 18.87 ± 1.05
Deviation form idealized covalent geometry
    Bonds (Å) 0.0023 ± 0.0001
    Angles (°) 0.387 ± 0.011
    Improper (°) 0.267 ± 0.011
RMS deviation from experimental data
    Distance restraints (Å) 0.014 ± 0.001
    Dihedral angle restraints (°) 0.344 ± 0.029
Average Ramachandran statistics for 20 lowest energy structures
    Residues in most favored region 76.7%
    Residues in additional allowed regions 20.0%
    Residues in generously allowed regions 3.3%
    Residues in disallowed regions 0.0%
Analysis of residual dipolar couplings
    RMSD (Hz) 1.259 ± 0.036
    Q-factor 0.156 ± 0.004
    Correlation coefficient 0.979 ± 0.003
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Acknowledgments

This work was supported by Canadian Institute of Health Research grant #14219 (to K.G.). I.D. is a doctoral fellow from the National Research Council (CONICET), Argentina. We acknowledge the Canadian National High Field NMR Center (NANUC) for their assistance and use of their facilities. Operation of NANUC is funded by the Canadian Institute of Health Research, the Natural Science and Engineering Council of Canada, and the University of Alberta. We also thank T. Sprules, P. Gutierrez, A. Denisov, L. Volpon, M. Osborne, D. Elias, and M. Bachetti for their assistance and helpful discussions.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

Abbreviations

  • HSQC, heteronuclear single quantum coherence

  • HYD, hyper plastic disk protein

  • IF, initiation factor

  • NMR, nuclear magnetic resonance

  • NOE, nuclear overhauser effect

  • PABC, C-terminal domain of poly(A)-binding protein

  • PABP, poly(A)-binding protein

  • PAM, poly(A)-binding protein interacting motif

  • Paip, PABP interacting protein

  • PDB, Protein Data Bank

  • RF3, release factor 3

  • RRM, RNA recognition motif

  • RDCs, residual dipolar couplings

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0390103.

References

  1. Bartels, C., Xia, T., Billeter, M., Güntert, P., and Wüthrich, K. 1995. The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR 6 1–10. [DOI] [PubMed] [Google Scholar]
  2. Bax, A. and Grzesiek, S. 1993. Methodological advances in protein NMR. Acc. Chem. Res. 26 131–138. [Google Scholar]
  3. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54 905–921. [DOI] [PubMed] [Google Scholar]
  4. Clayton, C.E. 2002. Life without transcriptional control? From fly to man and back again. EMBO J. 21 1881–1888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clore, G.M., Gronenborn, A.M., and Bax, A. 1998a. A robust method for determining the magnitude of the fully asymmetric alignment tensor of oriented macromolecules in the absence of structural information. J. Magn. Reson. 133 216–221. [DOI] [PubMed] [Google Scholar]
  6. Clore, G.M., Gronenborn, A.M., and Tjandra, N. 1998b. Direct structure refinement against residual dipolar couplings in the presence of rhombicity of unknown magnitude. J. Magn. Reson. 131 159–162. [DOI] [PubMed] [Google Scholar]
  7. Constantine, K.L., Goldfarb, V., Wittekind, M., Friedrichs, M.S., Anthony, J., Ng, S.C., and Mueller, L. 1993. Aliphatic 1H and 13C resonance assignments for the 26–10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy. J. Biomol. NMR 3 41–54. [DOI] [PubMed] [Google Scholar]
  8. Cornilescu, G., Marquardt, J.L., Ottiger, M., and Bax, A. 1998. Validation of protein structure from anisotropic carbonyl chemical shifts in dilute liquid crystalline phase. J. Am. Chem. Soc. 120 6836–6837. [Google Scholar]
  9. Cosson, B., Couturier, A., Chabelskaya, S., Kiktev, D., Inge-Vechtomov, S., Philippe, M., and Zhouravleva, G. 2002. Poly(A)-binding protein acts in translation termination via eukaryotic release factor 3 interaction and does not influence [PSI(+)] propagation. Mol. Cell. Biol. 22 3301–3315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Craig, A.W., Haghighat, A., Yu, A.T., and Sonenberg, N. 1998. Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation. Nature 392 520–523. [DOI] [PubMed] [Google Scholar]
  11. Deo, R.C., Bonanno, J.B., Sonenberg, N., and Burley, S.K. 1999. Recognition of polyadenylate RNA by the poly(A)-binding protein. Cell 98 835–845. [DOI] [PubMed] [Google Scholar]
  12. Deo, R.C., Sonenberg, N., and Burley, S.K. 2001. X-ray structure of the human hyperplastic discs protein: An ortholog of the C-terminal domain of poly(A)-binding protein. Proc. Natl. Acad. Sci. 98 4414–4419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. D’Orso, I. and Frasch, A.C. 2001. TcUBP-1, a developmentally regulated U-rich RNA-binding protein involved in selective mRNA destabilization in trypanosomes. J. Biol. Chem. 276 34801–34809. [DOI] [PubMed] [Google Scholar]
  14. ———. 2002. TcUBP-1, an mRNA destabilizing factor from trypanosomes, homodimerizes and interacts with novel AU-rich element- and poly(A)-binding proteins forming a ribonucleoprotein complex. J. Biol. Chem. 277 50520–50528. [DOI] [PubMed] [Google Scholar]
  15. Gingras, A.C., Raught, B., and Sonenberg, N. 1999. eIF4 initiation factors: Effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68 913–963. [DOI] [PubMed] [Google Scholar]
  16. Gray, N.K., Coller, J.M., Dickson, K.S., and Wickens, M. 2000. Multiple portions of poly(A)-binding protein stimulate translation in vivo. EMBO J. 19 4723–4733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grosset, C., Chen, C.Y., Xu, N., Sonenberg, N., Jacquemin-Sablon, H., and Shyu, A.B. 2000. A mechanism for translationally coupled mRNA turnover: Interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex. Cell 103 29–40. [DOI] [PubMed] [Google Scholar]
  18. Grzesiek, S., Dobeli, H., Gentz, R., Garotta, G., Labhardt, A.M., and Bax, A. 1992. 1H, 13C, and 15N NMR backbone assignments and secondary structure of human interferon-γ. Biochemistry 31 8180–8190. [DOI] [PubMed] [Google Scholar]
  19. Haghighat, A. and Sonenberg, N. 1997. eIF4G dramatically enhances the binding of eIF4E to the mRNA 5′-cap structure. J. Biol. Chem. 272 21677–21680. [DOI] [PubMed] [Google Scholar]
  20. Hannaert, V., Saavedra, E., Duffieux, F., Szikora, J.P., Rigden, D.J., Michels, P.A., and Opperdoes, F.R. 2003. From the cover: Plant-like traits associated with metabolism of Trypanosoma parasites. Proc. Natl. Acad. Sci. 100 1067–1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hansen, M.R., Mueller, L., and Pardi, A. 1998. Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat. Struct. Biol. 5 1065–1074. [DOI] [PubMed] [Google Scholar]
  22. Henderson, M.J., Russell, A.J., Hird, S., Munoz, M., Clancy, J.L., Lehrbach, G.M., Calanni, S.T., Jans, D.A., Sutherland, R.L., and Watts, C.K. 2002. EDD, the human hyperplastic discs protein, has a role in progesterone receptor coactivation and potential involvement in DNA damage response. J. Biol. Chem. 277 26468–26478. [DOI] [PubMed] [Google Scholar]
  23. Imataka, H., Gradi, A., and Sonenberg, N. 1998. A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation. EMBO J. 17 7480–7489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Khaleghpour, K., Kahvejian, A., De Crescenzo, G., Roy, G., Svitkin, Y.V., Imataka, H., O’Connor-McCourt, M., and Sonenberg, N. 2001a. Dual interactions of the translational repressor Paip2 with poly(A) binding protein. Mol. Cell. Biol. 21 5200–5213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Khaleghpour, K., Svitkin, Y.V., Craig, A.W., DeMaria, C.T., Deo, R.C., Burley, S.K., and Sonenberg, N. 2001b. Translational repression by a novel partner of human poly(A) binding protein, Paip2. Mol. Cell 7 205–216. [DOI] [PubMed] [Google Scholar]
  26. Kozlov, G., Trempe, J.F., Khaleghpour, K., Kahvejian, A., Ekiel, I., and Gehring, K. 2001. Structure and function of the C-terminal PABC domain of human poly(A)-binding protein. Proc. Natl. Acad. Sci. 98 4409–4413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kozlov, G., Siddiqui, N., Coillet-Matillon, S., Trempe, J.F., Ekiel, I., Sprules, T., and Gehring, K. 2002. Solution structure of the orphan PABC domain from Saccharomyces cerevisiae poly(A)-binding protein. J. Biol. Chem. 277 22822–22828. [DOI] [PubMed] [Google Scholar]
  28. Kuboniwa, H., Grzesiek, S., Delaglio, F., and Bax, A. 1994. Measurement of HN-H α J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods. J. Biomol. NMR 4 871–878. [DOI] [PubMed] [Google Scholar]
  29. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R., and Thornton, J.M. 1996. AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8 477–486. [DOI] [PubMed] [Google Scholar]
  30. Le, H., Tanguay, R.L., Balasta, M.L., Wei, C.C., Browning, K.S., Metz, A.M., Goss, D.J., and Gallie, D.R. 1997. Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)-binding protein and increase its RNA binding activity. J. Biol. Chem. 272 16247–16255. [DOI] [PubMed] [Google Scholar]
  31. Malliavin, T.E., Pons, J.L., and Delsuc, M.A. 1998. An NMR assignment module implemented in the Gifa NMR processing program. Bioinformatics 14 624–631. [DOI] [PubMed] [Google Scholar]
  32. Meiler, J., Prompers, J.J., Peti, W., Griesinger, C., and Bruschweiler, R. 2001. Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins. J. Am. Chem. Soc. 123 6098–6107. [DOI] [PubMed] [Google Scholar]
  33. Nilges, M., Macias, M.J., O’Donoghue, S.I., and Oschkinat, H. 1997. Automated NOESY interpretation with ambiguous distance restraints: The refined NMR solution structure of the pleckstrin homology domain from β-spectrin. J. Mol. Biol. 269 408–422. [DOI] [PubMed] [Google Scholar]
  34. Ottiger, M., Delaglio, F., and Bax, A. 1998. Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J. Magn. Reson. 131 373–378. [DOI] [PubMed] [Google Scholar]
  35. Peng, J.W. and Wagner, G. 1994. Investigation of protein motions via relaxation measurements. Methods Enzymol. 239 563–596. [DOI] [PubMed] [Google Scholar]
  36. Roy, G., De Crescenzo, G., Khaleghpour, K., Kahvejian, A., O’Connor-McCourt, M., and Sonenberg, N. 2002. Paip1 interacts with poly(A) binding protein through two independent binding motifs. Mol. Cell. Biol. 22 3769–3782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tarun Jr., S.Z., and Sachs, A.B. 1996. Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J. 15 7168–7177. [PMC free article] [PubMed] [Google Scholar]
  38. Tarun Jr., S.Z., Wells, S.E., Deardorff, J.A., and Sachs, A.B. 1997. Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. Proc. Natl. Acad. Sci. 94 9046–9051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Uchida, N., Hoshino, S., Imataka, H., Sonenberg, N., and Katada, T. 2002. A novel role of the mammalian GSPT/eRF3 associating with poly(A)-binding protein in cap/poly(A)-dependent translation. J. Biol. Chem. 277 50286–50292. [DOI] [PubMed] [Google Scholar]
  40. Wang, Z., Day, N., Trifillis, P., and Kiledjian, M. 1999. An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro. Mol. Cell. Biol. 19 4552–4560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wells, S.E., Hillner, P.E., Vale, R.D., and Sachs, A.B. 1998. Circularization of mRNA by eukaryotic translation initiation factors. Mol. Cell 2 135–140. [DOI] [PubMed] [Google Scholar]
  42. Wishart, D.S. and Sykes, B.D. 1994. Chemical shifts as a tool for structure determination. Methods Enzymol. 239 363–392. [DOI] [PubMed] [Google Scholar]
  43. Wüthrich, K. 1986. NMR of proteins and nucleic acids. John Wiley and Sons, Inc., New York.
  44. ———. 1989. The development of nuclear magnetic resonance spectroscopy as a technique for protein structure determination. Acc. Chem. Res. 22 36–44. [Google Scholar]

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