Rational design of a heterotrimeric G protein subunit with artificial inhibitor sensitivity

Davide Malfacini, Julian Patt, Suvi Annala, Kasper Harpsøe, Funda Eryilmaz, Raphael Reher, Max Crüsemann, Wiebke Hanke, Hang Zhang, Daniel Tietze, David E. Gloriam, Hans Bräuner-Osborne, Kristian Strømgaard, Gabriele M. König, Asuka Inoue, Jesus Gomeza, Evi Kostenis

研究成果: Article査読

19 被引用数 (Scopus)

抄録

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid– dependent and –independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other G subunits, we here set out to rationally design G16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gq/G11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold– based inhibitors, provided that these inhibitors mimic the interaction of FR with G proteins harboring engineered FR-binding sites.

本文言語English
ページ(範囲)5747-5758
ページ数12
ジャーナルJournal of Biological Chemistry
294
15
DOI
出版ステータスPublished - 2019 4月 12

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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