Abstract
Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.
Highlights
Pseudomonas are a common cause of hospital-acquired infections that may be lethal
To identify suitable combinations of exotoxin ART domains and 14-3-3 isoforms to study, we evaluated 14-3-3 concentration-dependent exotoxin activities in vitro
While it is well documented that LDLA-box 1 is crucial for exotoxin activity in vivo, we showed here that the exotoxins-S and -T (ExoS) ART domain is intrinsically active, and that LDLA-box 1 is dispensable for 14-3-3-mediated exotoxin activity in vitro
Summary
Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADPribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. We show that 14-3-3 stabilizes ExoS against thermal aggregation Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment. To initiate the infection process, the bacterium uses a type III secretion system to deliver a small set of exotoxins into the host cell.[1,2] Two of these, exotoxins-S and -T (ExoS; ExoT), are homologous enzymes consisting of an N-terminal GTPase-activating protein (GAP) domain (73% identity; 81% similarity) and a C-terminal ADP-ribosyltransferase (ART) domain (78% identity; 87% similarity).[3,4] Their GAP domains target Rho-family GTPases which leads to a remodeling of the host actin cytoskeleton. We present crystal structures of human 14-3-3β in complex with the ART domain of both ExoS and ExoT Our results indicate that 14-3-3 proteins act as exotoxin chaperones rather than activators
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