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Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination

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Science Advances  15 May 2020:
Vol. 6, no. 20, eaaz8041
DOI: 10.1126/sciadv.aaz8041
  • Fig. 1 Strategy for the formation of a covalently linked AnkXCys:PC:Rab1b complex.

    (A) Thiol-reactive CDP-choline analogs were prepared by attaching a chloroacetamide or bromoacetamide functionality via a short carbon spacer to the choline head group of CDP-choline. Natural CDP-choline is colored black; the synthetically installed linker and the haloacetamide group are colored red. (B) Direct approach. An AnkX cysteine mutant (AnkXCys) is reacted with a thiol-reactive CDP-choline analog to form a binary adduct, which is then used to phosphocholinate Rab1b. (C) Indirect approach. Rab1b is first modified with the thiol-reactive PC group using catalytic quantities of wild-type AnkX (AnkXWT). The resulting Rab1b-PC conjugate subsequently forms the AnkXCys:PC:Rab1b complex by the addition of stoichiometric amounts of AnkXCys.

  • Fig. 2 Preparative formation of a covalently linked AnkXCys:PC:Rab1b complex using thiol-reactive CDP-choline analogs.

    (A) Incorporation of thiol-reactive CDP-choline derivatives into Rab1b and compatibility with different reducing agents. The calculated masses for the expected Rab1b-PC conjugates are as follows: 20,014 Da (Rab1b-PC–C3-Cl), 20,028 Da (Rab1b-PC–C4-Cl), and 20,073 Da (Rab1b-PC–C4-Br). The black dashed lines indicate the mass of unmodified Rab1b (19,729 Da). (B) AnkX residues selected for the mutagenesis to cysteine [Protein Data Bank (PDB) ID: 4BET] (19). Distances between the choline head group of CDP-choline and the γ-C atom are shown in angstrom. The α-C atom was used for glycine residues, which is marked with an asterisk (*). (C) Direct approach for the covalent complex formation between AnkXD265C (50 μM), Rab1b (100 μM), and thiol-reactive CDP-choline derivatives (1 mM). Covalent complex formation was assessed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel shift assay. The red rectangles indicate the bands for the specific AnkXD265C:PC:Rab1b complex. (D) Indirect approach for the covalent complex formation between AnkXG108C (200 μM), Rab1b (50 μM), and thiol-reactive CDP-choline derivatives (1 mM). Covalent complex formation was assessed by SDS-PAGE gel shift assay. The red rectangles indicate the bands for the specific AnkXG108C:PC:Rab1b complexes. (E) Analytical size exclusion chromatography of the binary AnkXG108C:PC:Rab1b complex. The SDS-PAGE gel shows the input of the size exclusion chromatography. A280nm, absorbance at 280 nm; AU, arbitrary units.

  • Fig. 3 Structure of the AnkXG108C:PC:Rab1b:GDP complex.

    (A) Cartoon representation of the AnkXG108C:PC:Rab1b:GDP complex. Dark green, AnkX FIC domain and AnkX ARs 1 to 4; brown, AnkX ARs 5 to 9; light green, AnkX ARs 10 to 13; gold, Rab1b switch I (SI); blue, Rab1b switch II (SII); red, Rab1b C terminus; orange spheres, G108CAnkX and S76Rab1b. The dashed orange line indicates the covalent linkage between G108CAnkX and S76Rab1b. GDP is represented as a balls-and-sticks model. (B) Schematic model of the AnkX-Rab1b binding interface. Supposed interaction partners are the AnkX FIC domain and Rab1b switch II, AnkX ARs 5 to 9, and Rab1b switch I, as well as AnkX ARs 10 to 13 and the Rab1b C terminus. (C) Schematic representation of polar interactions between Rab1b and AnkX. Dashed lines, salt bridges. (D) Schematic representation of hydrophobic interactions between Rab1b and AnkX. Red lines indicate contacts between F143AnkX to Rab1b residues. (E) Surface model of the AnkXG108C:PC:Rab1b:GDP complex. The complex structure is superimposed with active Rab1b (PDB ID: 3NKV) (10) and AnkX1–484 (PDB ID: 4BET) (19). (F) Structural evaluation of Rab1b upon binding to AnkX. Gold, Rab1b switch I; blue, Rab1b switch II; gray, α3 of Rab1b; red, Rab1b C terminus (α4 und α5). Active Rab1b bound to the nonhydrolyzable GTP-analog GppNHp (PDB ID: 3NKV) (10) represents the structure of the small GTPase before AnkX binding. Note that major structural rearrangements take place within the switch II region of Rab1b. (G) Activity assay for AnkX AR truncations. Rab1b (5 μM) was modified with CDP-choline (1 mM) by lysates of overexpressing AnkX AR truncations [total lysate (2 mg/ml)] for 2 hours, and a Western blot (WB) using an α-PC antibody and α-His antibody was performed. The signal of Rab1b phosphocholination was normalized against the signal of the AnkX His6-tag. (H) Activity assay for AnkX alanine substitutions. kcat/KM values have been determined from phosphocholination progress curves using the change in Rab1b tryptophane fluorescence. Rab1b (5 μM) was modified with CDP-choline (1 mM) by catalytic amounts of AnkX (50 nM). (I) Catalytic efficiencies (kcat/KM) of Rab1b Ala mutants within the AnkX-Rab1b binding interface determined with a time-resolved tryptophane fluorescence–based assay and were normalized to wild-type Rab1b (Rab1bWT). Rab1b (5 μM) was modified with CDP-choline (1 mM) by catalytic amounts of AnkX (100 or 250 nM for T74ARab1b, S76ARab1b, Y77ARab1b, and Y109ARab1b). Since phosphocholination of Y109ARab1b did not result in a change of tryptophane fluorescence, the catalytic efficiency of this alanine mutant was analyzed with mass spectrometry and estimated to be of similar scale as the catalytic efficiency of T74ARab1b (fig. S8). Golden bars, Rab1b switch I; blue bars, Rab1b switch II; gray bar, α3 of Rab1b; red bars, Rab1b C terminus. Number sign (#) indicates not determined because of inactivity. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 AnkX mediates phosphocholination by displacing switch II of Rab1b.

    (A) In the AnkXG108C:PC:Rab1b:GDP complex, the AnkX thorn inserts between switch II and α3 of Rab1b. AnkX (light blue) is illustrated as a cartoon model. Red, AnkX insertion domain bearing the AnkX thorn. Rab1b (gray) is illustrated as a surface model. Blue, Rab1b switch II; green, α3 of Rab1b. GDP is represented as sticks and spheres. (B) In active Rab1b (PDB ID: 3NKV) (10), the area between switch II and α3 is initially not accessible for the AnkX thorn. Active Rab1b (magenta) is illustrated as a surface model and was structurally superimposed with the AnkXG108C:PC:Rab1b:GDP complex. Labeling is the same as that in (A). (C) In the AnkXG108C:PC:Rab1b:GDP complex, F143AnkX at the tip of the AnkX thorn (red) binds into a hydrophobic pocket of Rab1b (gray). (D) In active Rab1b (PDB ID: 3NKV) (10), the binding of F143AnkX into the hydrophobic pocket of Rab1b is blocked by Y78Rab1b. (E) Scheme for the suggested role of the AnkX thorn during AnkX-mediated phosphocholination. By insertion of F143AnkX at the tip of the AnkX thorn (T) into the hydrophobic pocket between switch II and α3 of Rab1b, AnkX locally unfolds the adjacent region of S76Rab1b and thus enables S76Rab1b to reach into the catalytic pocket of AnkX. (F) Structural superposition of active Rab1b (PDB ID: 3NKV; blue) (10) with crystal structures of GDP-bound Rab proteins: Rab2b:GDP (PDB ID: 2A5J; green), Rab3b:GDP (PDB ID: 3DZ8; gray), Rab4b:GDP (PDB ID: 2O52; orange), and Rab23:GDP (PDB ID: 1Z22; magenta). Residues at the position of Y78Rab1b are highly conserved and thus severely restrict the structural mobility of related amino acids at the site of S76Rab1b and related amino acids. Switch II and α3 of active Rab1b are depicted as cartoon model. (G) Time-resolved tryptophane fluorescence–based assay to monitor the catalytic activity of F143AnkX mutants. Rab1b (5 μM) was incubated with CDP-choline (1 mM), and upon addition of catalytic amounts of F143AnkX variants (250 nM), the tryptophane fluorescence of Rab1b was recorded. Fluorescence curves were fitted to a single exponential (black lines). (H) Catalytic efficiencies (kcat/KM values) of the F143AnkX mutants were calculated from the fluorescence curves of (G) [number sign (#) indicates not determined because of inactivity]. (I) Phosphocholination activity of the ΔF143AnkX variant toward folded and unfolded AnkX substrates. Folded Rab1b protein (50 μM) or the unfolded octapeptide TITSSYYR (50 μM) was incubated with CDP-choline (10 mM) and either ΔF143AnkX (5 μM) or WTAnkX (5 μM) for 4 days. The degree of phosphocholination was assessed with mass spectrometry and plotted as bar chart.

Supplementary Materials

  • Supplementary Materials

    Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination

    Stefan Ernst, Felix Ecker, Marietta S. Kaspers, Philipp Ochtrop, Christian Hedberg, Michael Groll, Aymelt Itzen

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