Backbone resonance assignments of the C-terminal region of human translation initiation factor eIF4B

Translation initiation in eukaryotes is an early step in protein synthesis, requiring multiple factors to recruit the ribosomal small subunit to the mRNA 5’ untranslated region. One such protein factor is the eukaryotic translation initiation factor 4B (eIF4B), which increases the activity of the eIF4A RNA helicase, and is linked to cell survival and proliferation. We report here the protein backbone chemical shift assignments corresponding to the C-terminal 279 residues of human eIF4B. Analysis of the chemical shift values identifies one main helical region in the area previously linked to RNA binding, and confirms that the overall C-terminal region is intrinsically disordered.

of eIF4A helicase function (García-García et al. 2015).Thus, the function of eIF4B is particularly crucial for translation of mRNAs with structured 5'-UTRs (Shahbazian et al. 2010;Sen et al. 2016).At the molecular level, human eIF4B is a 611 residue RNA-binding protein comprising a structured RNA recognition motif (RRM) domain near the N-terminus, as well as a non-canonical RNA-binding region within the C-terminal region (CTR).Early work had identified amino acids 385-423 as the center of this C-terminal RNA-binding region (Méthot et al. 1994);Naranda et al. 1994), and identified an additional stretch of basic residues from 434 to 444 that might be involved.The minimal region of eIF4B required for interaction with eIF4A (in complex with RNA and AMP-PNP) is also located within the CTR (Rozovsky et al. 2008).In addition, several physiologically significant phosphorylation sites were reported within the CTR of eIF4B, among which Ser406 and Ser422 have been shown to stimulate translation in vivo (Shahbazian et al. 2006;Wang et al. 2016).While the structure of the human eIF4B RRM domain has already been determined (Fleming et al. 2003), the eIF4B CTR is predicted to be intrinsically disordered and currently lacks characterization at the atomic level.
With a future goal to look at residue-level functions of the eIF4B C-terminal region, we report chemical shift resonance assignments of the backbone 1 H, 13 C and 15 N nuclei for the complete second half of human eIF4B (residue 333

Biological context
At its core, translation initiation in eukaryotes involves recruitment of the small ribosomal subunit with associated protein factors (the 43S pre-initiation complex) onto the protein-coding mRNA (Jackson et al. 2010).This process is usually achieved by the help of a mRNA cap-binding complex known as eukaryotic translation initiation factor 4F (eIF4F; comprising the three factors eIF4E, eIF4A and eIF4G), as well as eIF4B (or the homologue eIF4H) (Merrick 2015).In a concerted action, these factors recognize the mRNA 5'-cap and unwind the structured elements at the 5'-end untranslated region (5'-UTR) of the mRNA.This action facilitates the recruitment of the 43S pre-initiation complex, the scanning of the mRNA towards the startcodon recognition, and also ribosome assembly (Jackson et al. 2010).Throughout this process, eIF4B stimulates the helicase activity of eIF4A (Rogers et al. 2001;Jackson et al. 2010;Özeş et al. 2011), and increases the processivity to 611), as well as two shorter constructs that divide this region into two parts.The chemical shift values are consistent with an intrinsically disordered peptide, except for a helical segment in a region with previous links to RNA binding.

Methods and experiments
Using the protein sequence of human eIF4B (Uniprot entry P23588), the corresponding DNA codon-optimized for E. coli was obtained (Integrated DNA Technologies).The C-terminal region (CTR; residues 333 to 611) was inserted into the SspI site of the modified pET vector (Addgene plasmid #29666) using the ligation-independent cloning protocol.The vector allows for expression of the construct with an N-terminal hexahistidine (His 6 ) tag followed by a tobacco etch virus (TEV) protease site.Subsequent PCR amplification using forward (GAAGATTGCTAA-CATTGGAAGTGG) and reverse (CTTCCAATGTTAG-CAATCTTCTTCTTTG) primers were used to subclone the CTR-N fragment (residues 333-457), and similarly a second set of primers (CTTCCAAGGCCATAGCCCGAC-CAGCAAA, CGGGCTATGGCCTTGGAAGTACAG-GTTTTC) were used to subclone the CTR-C fragment (residues 458-611).
Freshly transformed E. coli T7 Express lysY (New England Biolabs) were first grown overnight in 10 mL of lysogeny broth (LB) with 50 µg/ml Ampicillin, then transferred to 500 mL cultures at 37 °C in M9 minimal medium containing 1 g/L [ 15 N]NH 4 Cl and 2 g/L [ 13 C]glucose.At an OD 600nm of 0.6, protein expression was induced with a final concentration of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) followed by overnight growth at 20 °C and harvesting the cells by centrifugation.For CTR and CTR-N, the bacteria were resuspended in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.8), 250 mM KCl, 20 mM imidazole, 1 mg/mL lysozyme, 1 mM PMSF, 1% of Triton X-100, 2 mM β-mercaptoethanol, and one protease inhibitor cocktail tablet (Roche).Following sonication on ice (10 min at 40% power, alternating 50s of sonication and 60s pause), the sample was centrifuged at 40,000 x g for 40 min at 4 °C to remove cellular debris.The supernatant was filtered with a 0.7 μm glass Microfilter GF/F (GE Healthcare Life Sciences Whatman) and added to 1 mL NUVIA Ni 2+ affinity chromatography resin (Bio-Rad) in a plastic column, washed first with five column volumes of binding buffer (20 mM Tris-HCl (pH 7.8), 250 mM KCl, 20 mM imidazole, 2 mM β-mercaptoethanol), then three column volumes with a higher salt concentration (800 mM of KCl), and finally five column volumes with increased imidazole concentration (30 mM).The protein was eluted with a buffer containing 20 mM Tris-HCl (pH 7.8), 250 mM KCl, 500 mM imidazole, 2 mM β-mercaptoethanol.Removal of the N-terminal His 6 tag was performed by the addition of TEV protease (15 µg/ mg protein) during a dialysis step against 20 mM Tris-HCl (pH 7.8), 250 mM KCl, 5 mM β-mercaptoethanol.Dialysis of the CTR-N sample included 0.5 M urea.For CTR-C, the bacteria pellet was resuspended in a buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol, 5 mM imidazole and 1 mg/mL lysozyme prior to a sonication and lysate preparation similar to the other samples.After loading the CTR-C lysate onto the same affinity chromatography resin as above, the wash step this time included 10 column volumes of the resuspension buffer, then five column volumes with an increase to 25 mM imidazole, and finally elution with a further increase to 500 mM imidazole.Removal of the N-terminal His 6 tag was performed by overnight digestion with TEV protease (15 µg/mg protein) at 4 °C, following a return to the low imidazole buffer with the use of a PD10 column (Cytiva).For all samples, the protease, His-tag and remaining uncleaved protein were removed by a second Ni 2+ affinity chromatography step.All protein variants were also further purified with anion exchange chromatography (EnrichQ, Bio-rad; HiTrap CM FF, Cytiva).The CTR sample was further purified with reverse-phase HPLC on a C18 column (ReproSil Gold 200) followed by lyophilization.Protein concentration was measured using absorbance at 280 nm with protein extinction coefficients obtained using the ProtParam tool (http://web.expasy.org/protparam): 19,480 M − 1 cm − 1 (CTR), 5500 M − 1 cm − 1 (CTR-N), and 13,980 M − 1 cm − 1 (CTR-C).
The final samples of uniformly 13 C, 15 N-labeled eIF4B constructs were prepared in a buffer of 20 mM sodium phosphate (pH 7.0), 150 mM NaCl, 2 mM dithithreitol (DTT), with 10% (v/v) D 2 O added for the lock.NMR samples contained 170 µL in a 3 mm NMR tube, and assignment spectra were collected at 293 K on a Bruker Avance 700 MHz spectrometer with a triple-resonance gradient room-temperature probe.NMR data were processed by using NMRPipe/ NMRDraw software (Delaglio et al. 1995) and NMR spectra were visualized and analyzed using Sparky (T.D. Goddard & D. G. Kneller, University of California, San Francisco, USA).
Following the protein backbone chemical shift assignment of CTR-N and CTR-C, we were able to mostly complete the assignment of the full CTR.Due to overlap, these assignments correspond only to the 1 H N , 13 C α , 13 C', and 15 N nuclei.Except for the region bridging the two smaller constructs, the assignments are essentially the same as for CTR-N and CTR-C.As expected, the largest difference is for Cys457, which is the last residue in CTR-N (Figs. 1E  and 7.88 ppm/123.2ppm), but in CTR is found at 8.36 ppm/119.5 ppm.Other differences for this CTR region in the 1 H, 15 N-HSQC include Asn452 (Fig. 1E), Lys453 (Fig. 1F), Glu454 (Fig. 1E), Asp456 (Fig. 1F), Thr461 (Fig. 1C), and  2B).Once again we identified a single helical segment with high confidence, located in the same position indicated by the Δδ 13 C α values.There is also high confidence that the majority of eIF4B-CTR corresponds to a disordered coil.In contrast, the two smaller regions of secondary structure predicted by TALOS-N have very low confidence scores.
The primary helical segment that we have identified from Thr369 to Gln390 is of interest, since it is fully located within the 367-423 amino acid region found to bind RNA in vitro (Méthot et al. 1994).The stretch of basic residues from 434 to 444, also proposed to interact with RNA (Méthot et al. 1994);Naranda et al. 1994), furthermore displays some helical character in the Δδ 13 C α analysis (Fig. 2A).In future experiments, the chemical shift assignments will allow for a precise characterization of the interaction of RNA to specific residues in the eIF4B CTR.It will also be possible to look into residues involved in binding other translation initiation factors, and the residue-specific consequence of post-translational modifications such as phosphorylation on Ser406 and Ser422.