N backbone chemical shift assignments of m 7 GTP cap-bound Leishmania initiation factor 4 E-1

Abstract Most of the translational control of gene expression in higher eukaryotes occurs during the initiation step of protein synthesis. While this process is well characterized in mammalian cells, it is less defined in parasites, including the ones that cause human Leishmaniasis. The Leishmania cap-binding isoform 1 (LeishIF4E-1) is the only isoform that binds the specific trypanosomatids-specific hypermethylated 5′ cap, called cap-4, in the human stage of the parasite life cycle. We report here the extensive NMR resonance assignment of LeishIF4E-1 bound to a cap analog, m7GTP. The chemical shift data constitute a prerequisite to understanding specific translation initiation mechanisms used in Leishmania parasites and to developing antiparasitic drugs targeting their translation initiation factors.


Biological context
The translation initiation heterotrimeric complex, eIF4F, recruits the 5′ end of mRNAs to the small ribosomal subunit (Shirokikh and Preiss 2018). In human cells, this complex comprises a cap-binding protein, eIF4E, which specifically binds a methylated cap structure (m 7 GTP) at the 5′ end of most cellular mRNAs. A scaffold protein, eIF4G, binds eIF4E, the DEAD-box RNA helicase eIF4A, the poly-A binding protein PABP, and the multisubunit eIF3, which binds to the small ribosomal subunit. eIF4G interacts with eIF4E through a consensus motif, Y(X) 4 LΦ (where X is any amino acid and Φ is a hydrophobic residue). Despite our increasing knowledge of translation mechanisms in human cells, how the equivalent process is carried out in trypanosomatids, including Leishmania major, is not as well understood.
The specific factors involved in translation initiation vary depending on the developmental stage of Leishmania parasites. These parasites encode six highly diverged capbinding protein isoforms (LeishIF4E-1 through −6) that significantly differ among themselves and from their orthologs in mammalian cells (Yoffe et al. 2004(Yoffe et al. , 2006(Yoffe et al. , 2009Zinoviev et al. 2011). Trypanosomatids also contain a specific hypermethylated cap structure at the 5′ end of their mRNAs, called cap-4 (Reolon et al. 2019;Leiter et al. 2020). In the human developmental stage of the parasite's life cycle, LeishIF4E-1 is the only isoform that is highly expressed and maintains cap-binding activity, suggesting that it is the functional capbinding protein in the human infective stage (Zinoviev et al. 2011). LeishIF4E-1 does not, however, interact with any of the predicted LeishIF4G scaffold proteins, suggesting that LeishIF4E-1 is recruited to the LeishIF4F translation initiation complex through novel interactions, or that translation initiation in amastigotes proceeds through a cap-independent mechanism. A crystal structure of LeishIF4E-1 bound to a fragment of a novel IF4E interacting protein (Meleppattu et al. 2018), Leish4E-IP1, showed that the core of LeishIF4E-1 is structurally conserved when compared to eIF4E. Structural analysis, coupled with biochemical assays, revealed that Leish4E-IP1 represses cap-binding. As the cap was absent in the crystal structure, the structure did not allow us to visualize how LeishIF4E-1 interacts with the 5' mRNA cap. Here, we report the extensive NMR resonance assignment of LeishIF4E-1 bound to a m 7 GTP cap analog.

Sample preparation
We grew E. coli strain BL21 (DE3) cells transformed with this plasmid in M9 minimal media containing 95% 2 H 2 O supplemented with 15 NH 4 Cl (1 g/L) and 13 C 6 -D-glucose (2 g/L), when required, as the sole nitrogen and carbon sources, respectively. We used 1 mM of isopropyl β-D-1thiogalactopyranoside (IPTG) to induce protein expression and grew cells at 20 °C for 16 hours. We harvested and re-suspended cells in buffer containing 50 mM NaH 2 PO 4 / Na 2 HPO 4 , pH 7.8, 500 mM NaCl, 10 mM imidazole, 5 mM 2-β-mercaptoethanol, benzonase and EDTA-free protease inhibitor cocktail tablet (Roche). We lysed cells by sonication at 4 °C and centrifuged the resulting lysate. We purified the His 6 -TEV-LeishIF4E-1 protein on a nickel-affinity column (Ni-NTA; Qiagen) as previously described (Meleppattu et al. 2018). We digested the eluted protein with TEV protease to cleave the His 6 tag for 12 h (overnight reaction). We concentrated the protein by ultrafiltration using a 10 kDa-cutoff filter (Millipore Sigma). We further purified LeishIF4E-1 from the cleaved tag using a Superdex 75 HiLoad 16/60 preparative size exclusion column (GE Healthcare Life Sciences). After size exclusion purification, we concentrated the protein, added 5 mM of m 7 GTP (Millipore Sigma) and incubated overnight. We buffer exchanged samples into an NMR buffer (50 mM NaH 2 PO 4 /Na 2 HPO 4 , pH 6.5, 100 mM NaCl, 2 mM DTT, 5 mM m 7 GTP 5% D 2 O, concentrated to 0.1-0.4 mM) and removed excess of m 7 GTP using a desalting PD-10 column (GE Healthcare). NMR spectroscopy experiments were recorded at 298 K either on Bruker or Varian spectrometers, operating at high field strengths of 600, 750, and 900 MHz, all equipped with a cryogenically cooled probe.

NMR experiments
We recorded all NMR spectra at 298 K on a Varian Inova 900 MHz spectrometer or a Bruker 750 MHz spectrometer, both equipped with cryogenic probes. We processed the data using NMRPipe (Delaglio et al. 1995), and analyzed the data with CARA (Keller 2004) and SPARKY (Goddard et al. 2005). We assigned the backbone chemical shifts of 15 N-13 C-LeishIF4E-1 using TROSY versions of the traditional triple-resonance experiments: HNCA, HNCOCA, HNCO, HNCACO, HNCACB, and HNCOCACB ( Figs. 1 and 2). We used selective amino acid labelling to identify and confirm overlapping resonances ( 15 N-Xxx, where Xxx = Ala, Gly, His, Leu, Lys, Thr). We assigned three tryptophan aromatic side chains using NMR spectra collected on point mutants W37A, W83A and W95A (Fig. 3). W37 and W83 are predicted to interact with the m 7 GTP based on analysis of sequence alignments and crystal structure of cap-free or cap-bound IF4Es (Marcotrigiano et al. 1997;Volpon et al. 2006;Peter et al. 2015;Sekiyama et al. 2015;Meleppattu et al. 2018). The purification yield for the W22A, W25A and W133A mutants were not sufficient to carry out NMR experiments. We deuterated all NMR observable samples. We also used Non Uniform Sampling (NUS) in the two indirect dimensions to collect triple resonance data and used Poisson Gap Sampling to sample 12-15% of the indirect grid (Hyberts et al. 2010). We used the hmsIST program to reconstruct and process the data (Hyberts et al. 2012).

Assignments and data deposition
We obtained the nearly complete backbone NMR assignment of LeishIF4E-1 bound to m 7 GTP, for 177 residues of the 202 non-proline residues, as shown in the 15 N TROSY-HSQC spectrum. All chemical shifts were deposited in the BioMagResBank (www.bmrb.wisc.edu) under accession number 50250. This work paves the way to understanding how LeishIF4E-1 binds the trypanosomatid cap-4, now that two studies have reported its efficient chemical synthesis (Reolon et al. 2019;Leiter et al. 2020). 1 H, 13 C, and 15 N backbone chemical shift assignments of m 7 GTP cap-bound Leishmania…