Molecular mechanism of elongation factor 1A inhibition by a Legionella pneumophila glycosyltransferase

Abstract

Legionnaires' disease is caused by a lethal colonization of alveolar macrophages with the Gram-negative bacterium Legionella pneumophila. LpGT (L. pneumophila glucosyltransferase; also known as Lgt1) has recently been identified as a virulence factor, shutting down protein synthesis in the human cell by specific glucosylation of EF1A (elongation factor 1A), using an unknown mode of substrate recognition and a retaining mechanism for glycosyl transfer. We have determined the crystal structure of LpGT in complex with substrates, revealing a GT-A fold with two unusual protruding domains. Through structure-guided mutagenesis of LpGT, several residues essential for binding of the UDP-glucose-donor and EF1A-acceptor substrates were identified, which also affected L. pneumophila virulence as demonstrated by microinjection studies. Together, these results suggested that a positively charged EF1A loop binds to a negatively charged conserved groove on the LpGT structure, and that two asparagine residues are essential for catalysis. Furthermore, we showed that two further L. pneumophila glycosyltransferases possessed the conserved UDP-glucose-binding sites and EF1A-binding grooves, and are, like LpGT, translocated into the macrophage through the Icm/Dot (intracellular multiplication/defect in organelle trafficking) system.

Figure 1. Structure of LpGT

(A) Ribbon diagram of LppGT crystal structure in complex with UDP, glucose and manganese. (B) Comparison of LppGT crystal structure with TcdB (PDB codes 2BVL and 2BVM [34]). Secondary structures are represented in red and green for α-helixes of LppGT and toxin B N-terminal domain, brown and blue for α-helixes and β-strands in central domain of LppGT and toxin B, and pink and olive colour for α-helixes and β-strands of the C-terminal protrusion domain of LppGT and toxin B respectively. UDP and glucose are shown in green sticks and manganese as a pink sphere. The two aspartic acid residues (Asp²⁴⁶ and Asp²⁴⁸ in LppGT) are shown as cyan sticks. Arrows indicate flexible regions in both crystal structures. (C) Superposition of LppGT (grey) and TcdB (brown). UDP and glucose are shown in green and blue sticks in LppGT and TcdB respectively. (D) Surface representation of the LppGT enzyme, coloured by sequence conservation with Lgt2 and Lgt3 (from red, 100% identity, to grey, <50% identity).

Figure 2. Multiple sequence alignment of the GT88 family members and TcdB

GT88 members are LppGT, LppaGT (LpGT from strain Paris), LplGT, Lgt2 and Lgt3. Secondary structure elements from the LppGT structure are dark grey for the N-terminal domain, black for the central domain and light grey for the protrusion domain. Conserved catalytic glutamine residues are indicated with a black circle, the aspartic residues of the DxD motif are marked with a black star, amino acids interacting with UDP-glucose by direct hydrogen bonds or hydrophobic stacking interactions are highlighted with a black triangle, and amino acids interacting with UDP-glucose by indirect hydrogen bonds through water molecules are highlighted with a grey triangle.

Figure 3. Electrostatic surface representation of LppGT and ScEF1A

Left-hand panel: LppGT has a negatively charged binding groove, which may interact with the positively charged loop on ScEF1A (PDB code 2B7B [37]) that carries the acceptor serine. Tyr⁴⁵⁴, in the putative binding groove on LppGT, and the acceptor serine (Ser⁵³) on ScEF1A are indicated by arrows. Right-hand panel: higher magnification representation of the putative interaction site between LppGT and ScEF1A. The surface of LppGT and ScEF1A are represented in brown and green respectively. Tyr⁴⁵⁴ and Ser⁵³ are shown as sticks in pink; charged residues are in yellow (including some other residues forming the loop in which Ser⁵³ is localized, such as Gly⁵² and Phe⁵⁴).

Figure 4. Active site of LpGT bound to UDP-glucose and metals

Stereo images of the LplGT, LppGT and TcdB active sites. LplGT is shown in complex with UDP-glucose and Mg²⁺. LppGT and TcdB crystal structures are shown in complex with UDP, glucose and Mn²⁺. The amino acids are shown as grey sticks. The ligands and metals are shown as green sticks and pink spheres respectively. Protein–ligand hydrogen bonds are shown as broken black lines. The distances between Asn²⁹³ and Asn⁴⁹⁹ to the anomeric carbon are shown as thin lines. Unbiased (i.e. before inclusion of any inhibitor model) F_o–F_c, ϕ_calc electron density maps are shown at 2.5 σ.

Figure 5. Site-directed mutagenesis, microinjection, translocation and localization studies

(A) The upper panel shows autoradiography of wild-type and mutant enzymes incubated with HEK-293 lysates and UDP-[³H]glucose. The lower panel shows quenching of intrinsic LppGT tryptophan fluorescence measured at increasing concentrations of UDP-glucose. All data points represent the means ± S.D. for three measurements. The K_d for UDP-glucose was determined by fitting fluorescence intensity data against free UDP-glucose concentration (insert). See Table 2 for the K_d values for wild-type and mutant enzymes. (B) HeLa cells microinjected with wild-type LppGT, D246A/D248A double-mutant LppGT (D246A-D248A), LppGT-N293A and GST, as a control, at a protein concentration of 30 μM in the injection needle. Cells were co-injected with Texas Red-conjugated dextran and incubated for 48 h and are counterstained with DAPI. Representative images are shown for the cells after 48 h incubation. After injection of wild-type LppGT, there were few surviving red (Texas-Red–dextran-postive) cells and, of these, most had a rounded-up morphology when compared with cells injected with GST protein and the double mutant LppGT. LppGT-N293A was slightly protective compared with the wild-type enzyme. (C) Translocation experiments. All three enzymes were translocated through the Icm/Dot machinery (grey bars) compared with a mutated ΔicmT Legionella strain (white bars) as measured by cAMP concentrations. (D) Microinjected FLAG-tagged LppGT protein was injected into HeLa cells and incubated for 24 h. The FLAG-tagged protein (green) was diffusely distributed throughout the cell and showed rare co-localization with the counterstain against ribosomal L26 protein (red). The lower panels show a higher magnification of the image in the upper panels. Scale bar, 10 μm (upper panels) or 5 μm (lower panels). The proteins are FLAG-tagged at the C-terminus.