Fig. 3: Extending the GPlad toolbox.
From: De novo designed protein guiding targeted protein degradation
a Schematic representation of antiGPlad. A binder was designed to specifically bind to the activation site of McsB, thereby inhibiting its marking ability. b Binding interface selection (Left) and design models (Right) for antiMcsB complexes. Left: McsB surface showing hydrophobic residues (red) and Patchdock-selected binding sites (yellow). Right: Designed complexes of antiMcsB-McsB, with binding sites (yellow) and targets (gray). c Inhibition of McsB phosphorylation by antiMcsB expression. Corresponding cells were treated with 1% arabinose to induce antiMcsB, leading to the inhibition of arginine phosphorylation, which was detected by western blotting. d Inhibition of protein degradation by antiMcsB induction. Corresponding cells were treated with 0.5% arabinose to induce antiMcsB, inhibiting protein degradation. e Schematic representation of OptoGPlad. Expression of OptoGPlad was inhibited by EL222. Under blue light (450 nm) and arabinose induction, the OptoGPlad system was activated, forming a complex with GPmKate2-pMagFast1 and nMagHigh1-McsB, which induced mKate2 degradation. In the darkness, the complex dissociates, halting OptoGPlad expression and thus stopping degradation. f The plasmid used in OptoGPlad; g OptoGPlad-mediated protein degradation. Corresponding cells were treated with 1% arabinose to induce OptoGPlad. h Schematic representation of GPTAC. In GPTAC, one GP was bound to McsB while the other was bound to the target protein, positioning McsB close to the target protein. i Binding interface selection (Left) and design models (Right) for GP complexes. Left: McsB surface showing hydrophobic residues (red) and Patchdock-selected binding sites (yellow). Right: Designed complexes of GPMcsB-McsB, with binding sites (yellow) and targets (gray). j GPTAC-mediated protein degradation. Corresponding cells were treated with 1% arabinose to induce GPTAC. k Schematic representation of GPTAC-mediated mKate2 and eGFP degradation. The GPTAC for mKate2 and eGFP was under the control of ParaBAD. l Design model of GPeGFP in complex with eGFP, with binding sites (yellow) and targets (gray). m GPTAC-induced mKate2 and eGFP degradation. Corresponding cells were treated with 2% arabinose to induce GPTAC. All experiments are presented as mean ± s.d. from three biologically independent replicates. Source data are provided as a Source Data file. (a, e, h, k) are created in BioRender https://BioRender.com/8h6634z.
