Disordered Motifs and Domains in Cell Control - October 11-15, 2014

Disordered Motifs and Domains in Cell Control

Poster Session II

41-POS Board 17 Molecular Basis behind the Regulation of Activity of the Novel Kinase Doc by the Antitoxin Phd Steven De Gieter , Abel Garcia-Pino, Nico Van Nuland, Remy Loris. Free University Brussels, Brussel, Belgium. Fic proteins (Filamentation induced by cAMP) are ubiquitous in all domains of life and play a critical role in a myriad processes, such as bacterial pathogenesis. These proteins are defined by a conserved FIC domain and catalyze AMPylation (transfer of AMP) of target proteins. Doc is a Fic-like protein that belongs to the phd/doc toxin-antitoxin module and was shown to inhibit bacterial translation via phosphorylation of Elongation Factor Tu. Here we show how the intrinsically disordered C-terminal domain of the antitoxin Phd regulates the activity of Doc. Doc adopts a rather dynamic ensemble of conformations in its free state, which collapses into a more condensed and rigid structure upon binding of Phd. The intrinsically disordered Doc binding region of Phd (residues 52-73) harbors two distinct segments with distinct functionalities. The hydrophilic segment (Phd65-73) sterically competes with ATP binding and acts as a direct inhibitor. On its own, its affinity is weak, binding occurs with high on and off rates and does not lead to los of dynamics in Doc. On the other hand, the hydrophobic segment encompassing residues 52-64 (Phd52-64) does not overlap with the active site of Doc but binding is tight and induces the same structural changes as observed for the complete binding region of Phd. The collaborative action of both segments results in an intrinsically disordered locking mechanism suggested to prevent binding of the catalytic NTP. Our findings represent a novel structural paradigm for the plasticity of the catalytic mechanisms used by the active centers of enzymes and their cognate neutralization mechanisms. Methods: Isothermal titration Calorimetry, Surface Plasmon Resonance, Small Angle X-ray scattering, X-ray crystallography.

- 93 -