14-3-3 proteins activatePseudomonasexotoxins-S and –T by chaperoning a hydrophobic surface

Abstract

Short summary -Crystal structures of Pseudomonas exotoxins-S and -T identify a novelhydrophobic interface with 14-3-3 proteins, and we show that 14-3-3 activates these toxins independent of their phosphopeptide groove binding C-termini, by preventing their aggregation.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/342659 doi: bioRxiv preprint first posted online Jun. 8, 2018; AbstractPseudomonas 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. By binding in the 14-3-3 phosphopeptide binding groove, a hydrophobic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3:ExoS and -ExoT complexes presented here reveal an extensive novel binding interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the hydrophobic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the hydrophobic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, weshow 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 hydrophobic C-terminal segment.Pseudomonas aeruginosa is an opportunistic pathogen that is infamous for causing hospital acquired airway and wound infections. 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 ADPribosyltransferase (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. The activities of the ART domains are directed toward a more diverse set of proteins. ExoS targets Rasand Rho-family GTPases, 5,6 ezrin/radixin/moesin (ERM) proteins, 7 and the intermediate All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/342659 doi: bioRxiv preprint first posted online Jun. 8, 2018; filament protein, vimentin.8 ADP-ribosylation disturbs these targets presumably by placing the bulky ADP-ribose moiety in a protein-protein interaction site, and has multiple consequences including and disruption of actin polym...