Structural basis for LeishIF4E-1 modulation by an interacting protein in the human parasite Leishmania major

Abstract

Leishmania parasites are unicellular pathogens that are transmitted to humans through the bite of infected sandflies. Most of the regulation of their gene expression occurs post-transcriptionally, and the different patterns of gene expression required throughout the parasites' life cycle are regulated at the level of translation. Here, we report the X-ray crystal structure of the Leishmania cap-binding isoform 1, LeishIF4E-1, bound to a protein fragment of previously unknown function, Leish4E-IP1, that binds tightly to LeishIF4E-1. The molecular structure, coupled to NMR spectroscopy experiments and in vitro cap-binding assays, reveal that Leish4E-IP1 allosterically destabilizes the binding of LeishIF4E-1 to the 5' mRNA cap. We propose mechanisms through which Leish4E-IP1-mediated LeishIF4E-1 inhibition could regulate translation initiation in the human parasite.

Figure 1.

NMR spectroscopy and crystal structure of LeishIF4E-1 bound to a fragment of Leish4E-IP1. (A) R1, (B) R2, (C) Heteronuclear NOE data are shown for a sample of ∼0.2 mM ¹⁵N¹³C-Leish4E-IP1_1-52 bound to LeishIF4E-1 in 95:5 H₂O/D₂O at pH 6.5. Data points corresponding to unassigned residues or to residues for which measurements could not be obtained are shown in black and were given an arbitrary negative value. Dashed lines indicate two-stable stretches of residues in solution compared to other regions in the Leish4E-IP1_1–52 fragment (See also Supplementary Figures S1 and S2). (D) Cartoon representation of the Leish(IF4E-1/4E-IP1_1–52) complex (PDB ID: 5WB5), where residues 13 to 210 of LeishIF4E-1 and residues 4–43 of Leish4E-IP1_1–52 are shown (See also Supplementary Figures S1 and S2). LeishIF4E-1 and Leish4E-IP1 are shown in cyan and purple, respectively. The N and C termini of each protein and secondary structure elements are indicated. Disordered segments in loops (L1 and L2) from LeishIF4E-1 are shown in dashed lines. LeishIF4E-1 UniProt accession number is E9ADE1 and Leish4E-IP1 accession number is E9AFM3. (E) Dorsal view of the complex described in (D). Disordered segments in loops (L2 and L3) from LeishIF4E-1 are shown in dashed lines.

Figure 2.

Comparison of key interaction motifs between Leish(IF4E-1/4E-IP1) complex and human eIF4E/4E-BP1 or Drosophila eIF4E/Cup complexes. (A) Cartoon representation of the structural alignment of the Leish(IF4E-1/4E-IP1_1-52) and the mammalian eIF4E/m⁷GTP/4E-BP1 complex [PDB ID: 5BXV, (31)]. The Leish(IF4E-1/4E-IP1_1–52) complex is represented and colored as described in Figure 1. eIF4E and 4E-BP1 are shown in orange and yellow, respectively. The m⁷GTP cap in the eIF4E/4E-BP1 complex is omitted for clarity. (B) As described in (A) but showing key interactions centered on the consensus binding motifs [Y(X)₄LΦ] of Leish4E-IP1. Key residues are indicated. (C) As described in (A) but showing interactions centered on the linker region of Leish4E-IP1 (See also Figure 1A–C). (D) As described in (A) but showing interactions over the C-terminal tail of Leish4E-IP1. (E) Structural alignment of Leish(IF4E-1/4E-IP1_1–52) and the eIF4E/Cup complex from Drosophila melanogaster [PDB ID: 4AXG, (46)]. eIF4E and Cup are shown in pink and green, respectively. The linker segment of Cup is disordered in the complex and is shown as dashed lines (See also Supplementary Figure S1B and S4).

Figure 3.

Effect of Leish4E-IP1 on the m⁷GTP cap-binding affinity of LeishIF4E-1. (A) Structural alignment of the Leish(IF4E-1/4E-IP1_1-52) and the mammalian eIF4E/m⁷GTP/4E-BP1 complex [PDB ID: 5BXV, (31)] centered on the cap-binding pocket. LeishIF4E-1 and eIF4E are colored in cyan and orange, respectively. For clarity, Leish4E-IP1_1–52 and 4E-BP1 are omitted. The side chains of W56 and W102 from eIF4E, when bound to 4E-BP1, are in a ‘flipped-in’ conformation, while the side chains of W37 and W83 from LeishIF4E-1, when bound to Leish4E-IP1, are in a ‘flipped-out’ conformation. (B) ¹⁵N-¹H-TROSY-HSQC spectra overlay between ¹⁵N¹³C-LeishIF4E-1 (cyan) acquired on 600 MHz spectrometer and ¹⁵N¹³C-LeishIF4E-1 bound to unlabeled Leish4E-IP1_1-52 in an equimolar ratio (purple). An enlarged portion of the spectra shows the tryptophan side chain area (See also Supplementary Figure S2). (C) Upper panel: Pull-down experiments with Streptavidin-binding peptide (SBP)-tagged LeishIF4E-1 (SBP-LeishIF4E-1) from L. amazonensis. Cell extracts were affinity purified over an adipic-agarose m⁷GDP resin in the absence or presence of an increasing amount of recombinant His-GB1-TEV-Leish4E-IP1_1–52 or His-GB1 protein. Proteins that remained bound to the resin after several washes were separated on SDS-PAGE and analyzed by Western blot using an anti-SBP antibody, detecting LeishIF4E-1. Lower panel: Densitometry analysis of the blot shown in the upper panel. The effect of His-GB1-Tev-Leish4E-IP1_1–52 or His-GB1 on the binding of LeishIF4E-1 to the m⁷GDP resin is presented as a percentage value when compared to SBP-LeishIF4E-1 in the absence of Leish4E-IP1_1–52 (SBP-LeishIF4E-1, 0 μg, set as 100% signal). The data are represented as the mean of at least three independent experiments. Error bars indicate the standard error of the mean.

Figure 4.

Alternative models of Leish(IF4E-1/4E-IP1) function. (A) LeishIF4E-1 (IF4E-1, cyan) is a functional cap-binding protein and activates translation initiation. The binding of Leish4E-IP1 (4E-IP1, purple) inhibits translation initiation by preventing the binding of LeishIF4E-1 to the cap. (B) LeishIF4E-1 maintains mRNAs in a cap-dependent translational inactive state. Upon binding of Leish4E-IP1, LeishIF4E-1 dissociates from the cap, which frees the mRNAs and allows them to interact with a functional cap-binding protein (yellow) and to be actively translated. For clarity of the figure, proteins are not represented to scale.