Title | Rational design of a heterotrimeric G protein subunit with artificial inhibitor sensitivity |
Publication Type | Journal Article |
Year of Publication | 2019 |
Authors | Malfacini D., Patt J., Annala S., Harpsoe K., Eryilmaz F., Reher R., Crusemann M., Hanke W., Zhang H, Tietze D., Gloriam D.E, Brauner-Osborne H., Stromgaard K., Konig G.M, Inoue A., Gomeza J., Kostenis E. |
Journal | Journal of Biological Chemistry |
Volume | 294 |
Pagination | 5747-5758 |
Date Published | 2019/04 |
Type of Article | Article |
Accession Number | WOS:000465077500002 |
Keywords | activation; Biochemistry & Molecular Biology; Cas; CRISPR; crystal-structure; cyclase; DMR; FR900359; G protein; G protein-coupled receptor (GPCR); g-alpha(q); insights; label-free; mechanism; pharmacology; receptor; signal transduction; structural basis; transducers; YM-254890 |
Abstract | 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. |
DOI | 10.1074/jbc.RA118.007250 |
Short Title | J. Biol. Chem. |