Crystal structure of the Legionella pneumophila effector SidL (Lpg0437) in complex with its metaeffector LegA11 (Lpg0436)

Abstract

Legionella pneumophila is an opportunistic human pathogen that causes atypical pneumonia called Legionnaires' Disease. To replicate within host cells, L. pneumophila injects up to 330 effector proteins into the host cytosol via a Dot/Icm type IV secretion system. Several effectors, called metaeffectors, regulate the activity of other effectors within infected host cells through direct protein-protein interactions. LegA11 (AnkJ/Lpg0436) has been identified as a putative metaeffector of SidL (Ceg14/Lpg0437), one of eight L. pneumophila effectors that inhibit host mRNA translation. LegA11 binds and suppresses SidL enzymatic activity, but the molecular basis of this regulation and impact on mRNA translation are unknown. Here, we present the crystal structure of SidL in complex with LegA11 to a resolution of 2.4 Å, revealing a high-affinity 1:1 complex with an extensive interaction interface of ~2300 Ų. Using isothermal titration calorimetry, we determined a dissociation constant of 1.8 nM. In vitro translation assays demonstrate that SidL inhibits mRNA translation, and this activity is completely suppressed by LegA11. Mutagenesis of key interface residues in LegA11 disrupts complex formation and abolishes its metaeffector activity, confirming that LegA11 regulates SidL through direct protein-protein interaction. These findings establish LegA11 as a bona fide metaeffector that contributes to suppression of host mRNA translation by L. pneumophila.

Keywords: Legionella pneumophila; effector; mRNA translation; metaeffector.

Figure 1.. Interaction of SidL and LegA11 observed in analytical size exclusion chromatography.

300 μg SidL_1–666 and/or 125 μg LegA11 in 100 μL buffer were applied to an S200 10/300 increase size exclusion column. The absorption curve of SidL_1–666 is shown in black, the absorption curve of LegA11 is shown in blue, the absorption curve of the mixed proteins is shown in red.

Figure 2.. Overall structure of SidL/LegA11.

Cartoon representation of the three-dimensional structure of the complex formed by SidL and LegA11. The model in the upper left corner represents the complex. The two subdomains of SidL are shown in blue and green, LegA11 is shown in orange. For better visibility, the complex components have been rotated individually, the respective degrees of rotation relative to the orientation of the complex are shown next to the arrows. Secondary structure elements as well as the N- and C-termini of the individual components are labeled.

Figure 3.. Binding interfaces of SidL and LegA11.

Cartoon representation of the binding interfaces between SidL and LegA11. The color code corresponds to that in Figure 2. Side chains of amino acid residues responsible for mediating the interactions are depicted as stick models and are labeled accordingly. Hydrogen bonds and polar interaction are shown as grey dotted lines. A. Interface between SidL_N and LegA11. B. Interface between SidL_C and LegA11.

Figure 4.. Affinity of SidL and LegA11 determined by ITC.

A. Baseline-corrected heat rate (μW) versus time (s) for titration of LegA11 (ligand) into SidL (receptor) at 25 °C, showing exothermic binding events (negative peaks) that diminish after saturation. B. Integrated binding isotherms from four independent experiments plotted as molar heat (kJ/mol) versus molar ratio of LegA11 to SidL. The best-fit one-site model yielded a dissociation constant K_d of 1.8 ^+4.5 _−1.3 nM, with a stoichiometry of 0.92 ± 0.16. Binding was strongly exothermic (average ΔH ≈ −200 kJ/mol). Receptor and ligand concentrations were: 1.45/32.77 μM, 0.72/14.97 μM, 0.63/10.61 μM, and 0.61/10.72 μM (SidL/LegA11). C. Thermodynamic signature of binding showing large enthalpic contribution (ΔH = −194 ± 17 kJ/mol) opposed by an unfavorable entropic term (−TΔS = +144 ± 16 kJ/mol), resulting in a free energy change of ΔG = −50 ± 3 kJ/mol.

Figure 5.. Influence of mutations in LegA11 on binding to SidL_76–645.

1 mg SidL_76–645 and/or 400 μg LegA11 in 100 μL buffer were applied to an S200 10/300 increase size exclusion column. The colors of the respective chromatograms are as follows: SidL_76–645 – black; LegA11-WT – blue; LegA11–4M – dotted blue; SidL_76–645/LegA11-WT – orange; SidL_76–645/LegA11–4M – dotted orange.

Figure 6.. LegA11 restores SidL-mediated translation inhibition via a direct protein-protein interaction.

Rabbit reticulocyte lysates were incubated with Firefly luciferase (Luc) mRNA alone (Positive) or with (A) 5 ng SidI, 5ng SidL, 50 ng SidL, or 100 ng SidL; (B) 65 nM of SidL, SidL and LegA11, or LegA11 alone; or (C) 65 nM of SidL alone, SidL and LegA11 variants, or LegA11 variants alone. Data shown are mean ± standard deviation (s.d.) of samples in triplicates (N=3) and are representative of three independent experiments. AU, arbitrary units. Asterisks (*) denote statistical significance (***P<0.001, *P<0.05, ns, not significant) by one-way ANOVA with Tukey’s post-hoc test.