Induced conformational changes in the activation of the Pseudomonas aeruginosa type III toxin, ExoU

Abstract

ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca(2+)-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.

Figure 1

The MTSL spin label (left) reacts with the cysteine sulfhydryl group to yield the R1 side chain (right).

Figure 2

EPR spectra of MTSL-labeled cysteine variants near catalytic residue S142 of ExoU. (Solid lines) Experimental spectra. (Shaded lines) Two-component simulations. Spectra are scaled to approximately equal amplitude of the center line.

Figure 3

Liposome and SOD1-induced conformational changes in S137R1. Samples contained ExoU-S137R1 in 10 mM Tris pH 7.0, 15 mM NaCl, and 20% glycerol. (A) Buffer control; (B) after addition of POPC/POPS (1:1) liposomes; and (C–E) after addition of liposomes and SOD1. The SOD1:ExoU molar ratios were (C) 6.25:1, (D) 12.5:1, and (E) 25:1. The lipid/ExoU molar ratio was held constant at 44:1. Spectra are scaled to equal amplitude of the center line. Positions of the more immobile (i) and mobile (m) motional states of the nitroxide side chain are indicated. Overlays of spectra A and B and of spectra A and E are provided in Fig. S4.

Figure 4

EPR spectra of MTSL-labeled cysteine mutants in the C-terminal domain of ExoU. (Solid lines) Experimental spectra. (Shaded lines) Multicomponent simulations. (Arrows) Positions of relatively mobile (m) and immobile (i) motional states of the R1 side chain.

Figure 5

Liposome and SOD1-induced conformational changes in S643R1. Samples contained ExoU-S643R1 in buffer, after addition of liposomes, after addition of SOD1, and after addition of both liposomes and SOD1. The lipid/ExoU and SOD1/ExoU molar ratios were 44:1 and 15:1, respectively. Positions of the more immobile (i) and mobile (m) motional states of the nitroxide side chain are indicated.

Figure 6

Effect of sucrose on S643R1. Comparison of the EPR spectra of S643R1 in buffer alone and in the presence of various concentrations (w/v) of sucrose. Spectra were normalized to the same integrated intensity.

Figure 7

DEER evidence for a SOD1-induced conformational change in ExoU. (A and C) Dipolar evolution curves (black) and fits (red) obtained using model-free Tikhonov regularization for the S137R1-S643R1 rExoU double cysteine variant in the (A) absence and (C) presence of SOD1. (B and D) Corresponding distance distributions in the (B) absence and (D) presence of SOD1. The regularization parameter (α) was 10 for both fits. L-curves are given in Fig. S8.

Figure 8

Model for the interaction of ExoU (gray) with its activating cofactor (blue) and the membrane bilayer. The observation of multiple distances between S643R1 and S137R1 suggests that the C-terminal domain may be conformationally flexible in the absence of the cofactor. SOD1 is envisioned as causing a realignment of the N- and C-terminal domains that facilitates membrane binding and exposure of the catalytic site to its lipid substrate. The conformational change in S137R1 observed in the presence of liposomes and SOD1 is represented as a change in the shape of the active site.