The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin

Belyy, Alexander; Santecchia, Ignacio; Renault, Louis; Bourigault, Blandine; Ladant, Daniel; Mechold, Undine

doi:10.1074/jbc.ra118.003784

Cited by 15 publications

(15 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our data show that ExoY is a monomer in solution, and a previous mutational study suggested that ExoY possesses only one F-actin-binding region, the C-terminal end of ExoY (5). We therefore hypothesize that ExoY likely dimerizes to promote F-actin bundling, a mechanism commonly used by many cellular F-actin-bundling or crosslinking proteins, as discussed above.…”

Section: Exoy Induces F-actin Bundlingsupporting

confidence: 71%

See 1 more Smart Citation

Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments

Mancl

Suarez

Liang

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro, via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin–bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY–F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity.

show abstract

Section: Exoy Induces F-actin Bundlingsupporting

confidence: 71%

“…ExoS and ExoT both exhibit G-protein activating and ADP-ribosylation activities, whereas ExoU is a potent phospholipase (4). ExoY is a promiscuous nucleotidyl cyclase that has recently been demonstrated to be activated by binding to actin filaments (F-actin), but not globular actin (G-actin) (5)(6)(7)(8).…”

mentioning

confidence: 99%

Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments

Mancl

Suarez

Liang

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…After activation, the toxin generates a supraphysiologic amount of 207 cGMP and cAMP that impedes cell signaling. It was previously demonstrated that the 208 mutagenesis of D25 in actin abolishes ExoY binding to F-actin (Belyy et al, 2018). The same 209 actin mutation also prevents Lifeact binding, we therefore hypothesize that Lifeact and ExoY 210 have overlapping binding sites.…”

Section: Lifeact Mutations Increase Its Affinity To F-actin 156mentioning

confidence: 56%

Structure of the Lifeact–F-actin complex

Belyy

Merino

Sitsel

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

9Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) 10 structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe 11 has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The 12 molecular basis for such artefacts is poorly understood. Here, we determined the high-13resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy. The 14 structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and 15 stretches over two adjacent actin subunits, stabilizing the DNase I-binding loop of actin in the 16 closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding 17 proteins, such as cofilin and myosin and actin-binding toxins, such as TccC3HVR from 18Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays 19 and activity measurements demonstrate that Lifeact indeed competes with these proteins, 20providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, 21we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations 22 into the peptide, laying the foundation for designing improved actin probes for live cell 23 imaging. 24

show abstract

“…After activation, the toxin generates a supraphysiologic amount of 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) that impedes cell signaling. The mutagenesis of D25 in actin abolishes ExoY binding to F-actin [ 35 ]. Since the same actin mutation also prevents Lifeact binding, we hypothesize that Lifeact and ExoY have overlapping binding sites.…”

Section: Resultsmentioning

confidence: 99%

Structure of the Lifeact–F-actin complex

et al. 2020

Self Cite

View full text Add to dashboard Cite

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as a hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

show abstract

The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin

Cited by 15 publications

References 46 publications

Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments

Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments

Structure of the Lifeact–F-actin complex

Structure of the Lifeact–F-actin complex

Contact Info

Product

Resources

About