Rational design of a heterotrimeric G protein α 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

doi:10.1074/jbc.RA118.007250

Rational design of a heterotrimeric G protein α 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

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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.

Original language	English
Journal	The Journal of Biological Chemistry
Volume	294
Issue number	15
Pages (from-to)	5747-5758
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.RA118.007250
Publication status	Published - 12 Apr 2019

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10.1074/jbc.RA118.007250Licence: CC BY

J. Biol. Chem.-2019-Malfacini-5747-58Licence: CC BY

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Malfacini, D., Patt, J., Annala, S., Harpsøe, K., Eryilmaz, F., Reher, R., Crüsemann, M., Hanke, W., Zhang, H., Tietze, D., Gloriam, D. E., Bräuner-Osborne, H., Strømgaard, K., König, G. M., Inoue, A., Gomeza, J., & Kostenis, E. (2019). Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity. The Journal of Biological Chemistry, 294(15), 5747-5758. https://doi.org/10.1074/jbc.RA118.007250

Malfacini, D, Patt, J, Annala, S, Harpsøe, K, Eryilmaz, F, Reher, R, Crüsemann, M, Hanke, W, Zhang, H, Tietze, D, Gloriam, DE , Bräuner-Osborne, H , Strømgaard, K, König, GM, Inoue, A, Gomeza, J & Kostenis, E 2019, 'Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity', The Journal of Biological Chemistry, vol. 294, no. 15, pp. 5747-5758. https://doi.org/10.1074/jbc.RA118.007250

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title = "Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity",

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.",

author = "Davide Malfacini and Julian Patt and Suvi Annala and Kasper Harps{\o}e and Funda Eryilmaz and Raphael Reher and Max Cr{\"u}semann and Wiebke Hanke and Hang Zhang and Daniel Tietze and Gloriam, {David E} and Hans Br{\"a}uner-Osborne and Kristian Str{\o}mgaard and K{\"o}nig, {Gabriele M} and Asuka Inoue and Jesus Gomeza and Evi Kostenis",

note = "Published under license by The American Society for Biochemistry and Molecular Biology, Inc.",

year = "2019",

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day = "12",

doi = "10.1074/jbc.RA118.007250",

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T1 - Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity

AU - Malfacini, Davide

AU - Patt, Julian

AU - Annala, Suvi

AU - Harpsøe, Kasper

AU - Eryilmaz, Funda

AU - Reher, Raphael

AU - Crüsemann, Max

AU - Hanke, Wiebke

AU - Zhang, Hang

AU - Tietze, Daniel

AU - Gloriam, David E

AU - Bräuner-Osborne, Hans

AU - Strømgaard, Kristian

AU - König, Gabriele M

AU - Inoue, Asuka

AU - Gomeza, Jesus

AU - Kostenis, Evi

N1 - Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

PY - 2019/4/12

Y1 - 2019/4/12

N2 - 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.

AB - 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.

U2 - 10.1074/jbc.RA118.007250

DO - 10.1074/jbc.RA118.007250

M3 - Journal article

C2 - 30745359

SN - 0021-9258

VL - 294

SP - 5747

EP - 5758

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 15

ER -

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

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