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.
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