De La Cruz, E. M. | bioRxiv Science

bioRxiv2018

14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface

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, 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{beta}: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, 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 hydrophobic C-terminal segment.\n\nShort summaryCrystal structures of Pseudomonas exotoxins-S and -T identify a novel hydrophobic 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.

biochemistry↗

bioRxiv2018

Insights into the cooperative nature of ATP hydrolysis in actin filaments

Actin filaments continually assemble and disassemble within a cell. Assembled filaments \"age\" as a bound nucleotide ATP within each actin subunit quickly hydrolyzes, followed by a slower release of the phosphate Pi, leaving behind a bound ADP. This subtle change in nucleotide state of actin subunits affects filament rigidity as well as its interactions with binding partners. We present here a systematic multiscale ultra-coarse-graining (UCG) approach that provides a computationally efficient way to simulate a long actin filament undergoing ATP hydrolysis and phosphate release reactions, while systematically taking into account available atomistic details. The slower conformational changes and their dependence on the chemical reactions are simulated with the UCG model by assigning internal states to the coarse-grained sites. Each state is represented by a unique potential surface of a local heterogeneous elastic network. Internal states undergo stochastic transitions that are coupled to conformations of the underlying molecular system. The UCG model reproduces mechanical properties of the filament and allows us to study whether fluctuations in actin subunits produce cooperative aging in the filament. Our model predicts that nucleotide state of neighboring subunit significantly modulates the reaction kinetics, implying cooperativity in ATP hydrolysis and Pi release. We further systematically coarse-grain the system into a Markov state model that incorporates assembly and disassembly, facilitating a direct comparison with previously published models. We find that cooperativity in ATP hydrolysis and Pi release significantly affects the filament growth dynamics only near the critical G-actin monomer concentration, while both cooperative and random mechanisms show similar growth dynamics far from the critical concentration. In contrast, filament composition in terms of the bound nucleotide distribution varies significantly at all monomer concentrations studied. These results provide new insights into the cooperative nature of ATP hydrolysis and Pi release and the implications it has for actin filament properties, providing novel predictions for future experimental studies.

biophysics↗