Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1 protein

Abstract

After the pathogenic bacterium Legionella pneumophila is phagocytosed, it injects more than 250 different proteins into the cytoplasm of host cells to evade lysosomal digestion and to replicate inside the host cell. Among these secreted proteins is the protein DrrA/SidM, which has been shown to modify Rab1b, a main regulator of vesicular trafficking in eukaryotic cells, by transfer of adenosine monophosphate (AMP) to Tyr(77). In addition, Legionella provides the protein SidD that hydrolytically reverses the covalent modification, suggesting a tight spatial and temporal control of Rab1 function by Legionella during infection. Small angle x-ray scattering experiments of DrrA allowed us to validate a tentative complex model built by combining available crystallographic data. We have established the effects of adenylylation on Rab1 interactions and properties in a quantitative way. In addition, we have characterized the kinetics of DrrA-catalyzed adenylylation as well as SidD-catalyzed deadenylylation toward Rab1 and have determined the nucleotide specificities of both enzymes. This study enhances our knowledge of proteins subverting Rab1 function at the Legionella-containing vacuole.

FIGURE 1.

Adenylylation-induced effects on Rab1. All data for Rab1 are shown in blue curves, and data for AMP-Rab1 are shown in green curves. GAP-catalyzed GTP hydrolysis for Rab1 and AMP-Rab1 was measured for different concentrations of LepB_325–618 and TBC1D20_1–362. A and B, resulting curves were fitted using a single exponential equation, and resulting k_obs values were plotted against concentrations of LepB_325–618 (A) and TBC1D20_1–362 (B) used in the experiments. C, GEF-catalyzed nucleotide exchange using DrrA_340–533 was measured similarly for AMP-Rab1. For comparison, previously published data of DrrA_340–533-catalyzed nucleotide exchange on Rab1 are also shown (blue dotted curve (15)). Data fitting for A–C (see “Experimental Procedures”) led to the k_cat, K_m, and k_cat/K_m values shown in Table 1. D, ITC measurements for interaction of Mical-3_1841–1990 with Rab1_fl·GppNHp (left, n = 0.880 ± 0.011, ΔH = −7772 ± 162 cal/mol, ΔS = −0.536 ± cal/mol/K), and AMP-Rab1_fl·GppNHp (right). E and F, in contrast to interaction with different proteins, intrinsic GTP hydrolysis (k_obs = 4.3 × 10⁻⁵ s⁻¹ for Rab1; k_obs = 5.0 × 10⁻⁵ s⁻¹ for AMP-Rab1) (E) and intrinsic nucleotide exchange rates (k_obs = 2.9 × 10⁻⁵ s⁻¹ for Rab1 and AMP-Rab1) (F) were not affected by the covalent modification.

FIGURE 2.

A model of full-length DrrA in complex with Rab1 bound to the GEF domain. A, indicated is the domain architecture of DrrA (red, P4M domain green, GEF domain; blue, adenylyltransferase domain (ATase)) in complex with Rab1 (yellow). The high structural homology of glutamine synthetase adenylyltransferase (GSAtase, orange) from E. coli and the adenylyltransferase domain of DrrA allowed modeling of the relative orientation of crystallographic models of fragments 9–218 (PDB ID 3NKU) and 193–550 (PDB ID 3LOI) of DrrA. B, superposition of the x-ray-based model of full-length DrrA in complex with Rab1 on a reference GASBOR ab initio model, shown in transparent gray beads. The view below has been rotated by 90° about the horizontal axis. The red arrow indicates the active site of the DrrA adenylyltransferase domain. C, the distance distribution function P(r) used for ab initio modeling.

FIGURE 3.

Kinetics of DrrA-catalyzed adenylylation. A–C, values for v/[E₀](s⁻¹) were determined from initial slopes measured using constant concentrations of 15 nm DrrA, 2 mm ATP and varying concentrations of Rab1_3–174·GppNHp (A), constant concentrations of 5 μm DrrA, 2 mm ATP and varying concentrations of Rab1_3–174·GDP (B), and constant concentrations of 15 nm DrrA, 10 μm Rab1_3–174·GppNHp and varying concentrations of ATP (C). The data were fitted to obtain k_cat and K_m or s_0.5 as described under “Experimental Procedures.”

FIGURE 4.

Kinetics of SidD-catalyzed deadenylylation. A and B, values for v/[E₀](s⁻¹) were determined from initial slopes measured using constant concentrations of 4 nm SidD and varying concentrations of AMP-Rab1_fl·GDP (A) or AMP-Rab1_fl·GppNHp (B). Plots of rate constants and substrate concentrations were fitted by Michaelis-Menten kinetics to obtain k_cat and K_m as described under “Experimental Procedures.”

FIGURE 5.

Nucleotide specificities of DrrA and SidD. A, Progress curves of adenylylation of 10 μm Rab1_3–174·GppNHp by 15 nm DrrA with 2 mm ATP (black curve), 2 mm GTP (red curve), 2 mm ADP (blue curve), or 2 mm GDP (green curve). The green star indicates the addition of 1.5 μm DrrA. B, progress curves of adenylylation of 10 μm Rab1_3–174·GppNHp by 15 nm DrrA with 2 mm ATP (black curve), 2 mm CTP (red curve), or 2 mm UTP (blue curve). The blue star indicates the addition of 75 nm DrrA. C, relative activities of nucleotidylation of Rab1_3–174·GppNHp were calculated using initial velocities of the progress curves from A and B. Error bars indicate S.D. D, the structural formulae of nucleobases are shown in the order of substrate specificity of DrrA for nucleoside triphosphates. E, progress curves of denucleotidylylation of 20 μm AMP-Rab1_fl·GppNHp (black), 20 μm GMP-Rab1_fl·GppNHp (red), 20 μm CMP-Rab1_fl·GppNHp (green), or 20 μm UMP-Rab1_fl·GppNHp (blue) by 4 nm SidD were fitted using KinTek Explorer to obtain catalytic efficiencies (Table 4). F, the structural formulae of nucleobases are shown in the order of substrate specificity of SidD for Rab1 modified with nucleoside monophosphates.

FIGURE 6.

Timeline of Legionella infection. The timeline of Legionella infection is shown as a black bar with different proteins and approximate time points of their localization at the LCV reported in literature (human Rab1, red bar; Legionella DrrA, LepB, SidD, and LidA, blue bars). For further information, see under “Discussion” (figure partly adapted from Ref. 10). ER-derived, endoplasmic reticulum-derived.