Identification of the major ubiquitin-binding domain of the Pseudomonas aeruginosa ExoU A2 phospholipase

Abstract

Numerous Gram-negative bacterial pathogens use type III secretion systems to deliver effector molecules into the cytoplasm of a host cell. Many of these effectors have evolved to manipulate the host ubiquitin system to alter host cell physiology or the location, stability, or function of the effector itself. ExoU is a potent A2 phospholipase used by Pseudomonas aeruginosa to destroy membranes of infected cells. The enzyme is held in an inactive state inside of the bacterium due to the absence of a required eukaryotic activator, which was recently identified as ubiquitin. This study sought to identify the region of ExoU required to mediate this interaction and determine the properties of ubiquitin important for binding, ExoU activation, or both. Biochemical and biophysical approaches were used to map the ubiquitin-binding domain to a C-terminal four-helix bundle of ExoU. The hydrophobic patch of ubiquitin is required for full binding affinity and activation. Binding and activation were uncoupled by introducing an L8R substitution in ubiquitin. Purified L8R demonstrated a parental binding phenotype to ExoU but did not activate the phospholipase in vitro. Utilizing these new biochemical data and intermolecular distance measurements by double electron-electron resonance, we propose a model for an ExoU-monoubiquitin complex.

Keywords: Bacterial Toxins; Phospholipase; Protein-Protein Interactions; Pseudomonas aeruginosa; Ubiquitin.

FIGURE 1.

Identification of the ExoU ubiquitin-binding domain. A, solid-phase plate binding assay detecting the binding of ExoU-GST fusion proteins to immobilized linear yeast pentaubiquitin. Reported fluorescence values include the subtraction of nonspecific binding and fit to a theoretical binding curve by non-linear regression analysis using GraphPad Prism 5.0 software (n = 3–5, mean ± S.E. (error bars)). B, Western blot analysis of GST pulldown assays utilizing non-tagged linear yeast pentaubiquitin and different GST-ExoU fusions. C, schematic diagram of the functional domains of ExoU. The catalytic dyad is composed of Ser-142 and Asp-344. MLD, membrane localization domain; RFU, relative fluorescence units; Ub, ubiquitin.

FIGURE 2.

Structure of the ExoU C-terminal ubiquitin-binding region. A, crystal structure of select residues from the ubiquitin-binding region of isopeptidase T (IsoT) in complex with the C terminus of monoubiquitin shown in gray (Protein Data Bank code 2G45). B, analogous structure of an ExoU C-terminal domain modeled from Protein Data Bank code 3TU3. Loop L1 was not observable in the crystal structure and is modeled by the authors. C, various residue substitutions for subsequent testing in cofactor binding assays are indicated. Helix α22 is shown in yellow. D, binding curves of GST-ExoU 480–683 or derivatives generated from solid-phase binding analysis detecting binding to immobilized linear yeast pentaubiquitin. Reported fluorescence values include subtraction of nonspecific binding and were analyzed similarly to Fig. 1. Right panel, bar graph of apparent dissociation constants of each GST fusion (n = 3, mean ± S.E. (error bars); *, p < 0.01). E, far-UV circular dichroism spectra of full-length ExoU with Y619E and R661E derivatives. RFU, relative fluorescence units; deg, degrees.

FIGURE 3.

NMR chemical shift mapping of monoubiquitin residues upon binding to the C-terminal ExoU ubiquitin-binding domain. A, select chemical shifts from a ¹H-¹⁵N heteronuclear single quantum coherence titration of ¹⁵N-labeled monoubiquitin in the presence of purified recombinant ExoU fragment residues 480–683. Ubiquitin chemical shifts are shown in the presence of the ExoU fragment at 0 (black), 0.5-, 1-, and 2-fold (green) molar excess over ubiquitin. B, quantification of the chemical shift changes between labeled ubiquitin only (0.2 mm) and in the presence of equimolar amounts (0.2 mm) of the C-terminal ExoU fragment. Magenta columns signify residues with significant chemical shifting (>0.4 ppm), and blue columns demarcate residues with notable shifting (0.2–0.39 ppm). C, surface map of the binding surface of ubiquitin utilized by the ExoU C terminus color-coded according to B. Site-specific substitutions analyzed in further experiments are shown in orange.

FIGURE 4.

Characterization of functional residues in ubiquitin required for ExoU binding. A, far-UV circular dichroism spectra of ubiquitin and related proteins containing site-specific point mutations. B, solid-phase binding curves (with theoretical non-linear regression line fitting) of the C-terminal region of ExoU to each of the immobilized ubiquitin derivatives. Right, quantification of the apparent affinities of the ExoU C-terminal domains for parental ubiquitin and each derivative (n = 3, mean ± S.E. (error bars); *, p < 0.05). RFU, relative fluorescence units; deg, degrees.

FIGURE 5.

Solution-phase EPR binding analysis of full-length ExoU or ExoU 480–683 to MTSL-labeled monoubiquitin. A, EPR spectra of monoubiquitin A28R1 upon titration with ExoU. The arrow denotes the motionally restricted spectral component due to formation of the ExoU-ubiquitin complex. The final ubiquitin concentration was 50 μm. Scan width, 100 G. B, EPR spectra of bound and free A28R1. C, binding isotherm based on the fraction of bound A28R1 (f_b) as a function of ExoU (open circles, full-length enzyme; closed squares, residues 480–683) concentration. The dashed lines represent the best fit to a single binding site model, and the inset represents the double reciprocal plot of the binding data. Ub, ubiquitin.

FIGURE 6.

Model of ExoU in complex with monoubiquitin. A, spin-labeled monoubiquitin (gray with specific residues highlighted according to Fig. 3 chemical shift data) is positioned onto the structure of ExoU in an orientation based on DEER measurements to S137R1 in the core of the toxin. The ExoU structure N-terminal to the proposed ubiquitin-binding region is colored green with a C-terminal domain corresponding to residues 587–683 marked in red. The catalytic residues in ExoU are shown as black spheres with Ser-137 near to the bound ubiquitin and Asp-344 distal. Several features of ExoU not depicted in the crystal structure are modeled according to the Phyre2 structural prediction program including a helix containing Asp-344* (41). B, ZDOCK complex of the C-terminal domain of ExoU in complex with monoubiquitin. ExoU and ubiquitin residues are color-coded as in A. This model was guided by NMR chemical shift mapping data and represents the only top scoring model to include Tyr-619 in the binding interface (42, 43). Ub, ubiquitin.