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. 2016 Jul 22;291(30):15767-77.
doi: 10.1074/jbc.M115.708701. Epub 2016 May 17.

Structural and Functional Investigations of the Effector Protein LpiR1 from Legionella pneumophila

Ksenia A Beyrakhova  1 , Karin van Straaten  1 , Lei Li  1 , Michal T Boniecki  1 , Deborah H Anderson  2 , Miroslaw Cygler  3
Affiliations

Structural and Functional Investigations of the Effector Protein LpiR1 from Legionella pneumophila

Ksenia A Beyrakhova et al. J Biol Chem. .

Abstract

Legionella pneumophila is a causative agent of a severe pneumonia, known as Legionnaires' disease. Legionella pathogenicity is mediated by specific virulence factors, called bacterial effectors, which are injected into the invaded host cell by the bacterial type IV secretion system. Bacterial effectors are involved in complex interactions with the components of the host cell immune and signaling pathways, which eventually lead to bacterial survival and replication inside the mammalian cell. Structural and functional studies of bacterial effectors are, therefore, crucial for elucidating the mechanisms of Legionella virulence. Here we describe the crystal structure of the LpiR1 (Lpg0634) effector protein and investigate the effects of its overexpression in mammalian cells. LpiR1 is an α-helical protein that consists of two similar domains aligned in an antiparallel fashion. The hydrophilic cleft between the domains might serve as a binding site for a potential host cell interaction partner. LpiR1 binds the phosphate group at a conserved site and is stabilized by Mn(2+), Ca(2+), or Mg(2+) ions. When overexpressed in mammalian cells, a GFP-LpiR1 fusion protein is localized in the cytoplasm. Intracellular signaling antibody array analysis revealed small changes in the phosphorylation state of several components of the Akt signaling pathway in HEK293T cells overexpressing LpiR1.

Keywords: Legionella infection; bacterial pathogenesis; cell signaling; cellular localization; crystal structure; protein evolution; protein-protein interaction.

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Figures

FIGURE 1.
FIGURE 1.
The overall structure of LpiR1. A, schematic representation of LpiR1. The N-terminal domain is colored wheat, the C-terminal domain is magenta. B, the fold of the N-terminal domain is in rainbow colors, from blue at the N terminus to red at the C terminus. The up-and-down four helix bundle is shown on the left with α5-α6 packing on one side perpendicular to the helix bundle axis. C, superposition of the N- and C-terminal domains of LpiR1. Colors are as in panel A with the exception of the parts that differ in conformation in the N- and C-terminal domains, which are colored green (N-terminal domain) or blue (C-terminal domain). Structural representations were prepared with the program PyMOL.
FIGURE 2.
FIGURE 2.
Residue lining the interdomain groove and residue conservation in LpiR1 homologs. A, a close-up view of the residues lining the interdomain groove. The helices α5–6 and α11–12 are shown in schematic representation. Side chains pointing into the groove are shown in stick representation. The groove has a strongly hydrophilic character. B, surface representation of LpiR1 colored according to the level of conservation of residues, which was determined by the program CONSURF (31). The orientation is similar to that shown in A. The color assignment is indicated on the attached scale. Insets show conserved residues in Cluster 1 and Cluster 2 under a semitransparent surface. Dashed green lines represent hydrogen bonds, the red oval shows the location of Cluster 1, and the dashed white oval indicates the location of Cluster 2 on the underside of molecule as depicted in this view. C, the location of phosphate ion coordinated by residues from the conserved site 2. The difference electron density map is drawn at 7σ level and colored green. The map was calculated after refinement of the LpiR1 model and before the phosphate groups were added. The ion forms seven hydrogen bonds (dashed blue lines) to the protein, one additional through a bridging water molecule and to two other waters. D, docking of Thr(P) (left) or Ala-Tyr(P)-Pro tripeptide to the phosphate binding site. The phosphate groups were superimposed on the observed position of the phosphate. E, stereo view of the Mn2+ binding site. The initial difference electron density map is drawn at 3σ (green) and 7σ (magenta) level with the refined model near the cation binding site. Glu-141, Arg-143, and Asp-322 are from conserved site 1. Asp-320 forms a salt bridge with Arg-143.
FIGURE 3.
FIGURE 3.
Structural comparison of LpiR1 and Lpg1851. A, superposition of Lpg1851 (schematic representation, green) with N- and C-terminal domains of LpiR1 (Cα trace, wheat and magenta, respectively). B, sequence alignment of N and C domains of LpiR1 with Lpg1851 based on structural alignment. C, comparison of the LpiR1 monomer (wheat and magenta) with a dimer of Lpg1851 (light green and dark green), generated by the application of crystallographic symmetry, The superposition is based on the helices α6 and α12 and the equivalent helices in the Lpg1851 dimer, to emphasize small differences in the relative orientation of the two domains in these proteins. D, surface residue conservation of Lpg1851 in similar orientation to LpiR1 viewed from the side showing the interchain depression. Conservation was calculated by the program CONSURF. The inset shows the conserved residues in the largest surface exposed cluster, which is located on the underside of the dimer in the shown view and marked approximately by the white dashed oval.
FIGURE 4.
FIGURE 4.
Phylogenetic analysis of LpiR1 domains and their homologs by the Maximum Likelihood method. The percentage of replicate trees in which the associated proteins clustered together in the bootstrap test (500 replicates) are shown next to the branches. N-terminal and C-terminal domains of LpiR1 orthologs are indicated by N or C in front of the NCBI RefSeq protein accessions, respectively.
FIGURE 5.
FIGURE 5.
Subcellular localization of LpiR1-GFP and Lpg1851-GFP. A, confocal microscopy images of HEK293T cells 24 h after transfection with pEGFP-N1 (upper panel) or LpiR1-GFP (middle panel) or Lpg1851-GFP (lower panel). Green fluorescence indicates the position of GFP or LpiR1-GFP or Lpg1851-GFP, whereas the blue fluorescence (DAPI) indicates the position of the nucleus. B, immunofluorescence staining for HA3 (green, upper panel) or HA3-LpiR1 (green, lower panel) in HEK293T cells. The blue fluorescence (DAPI) indicates the position of the nucleus.
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