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Designer proteins that competitively inhibit Gαq by targeting its effector site

https://doi.org/10.1016/j.jbc.2021.101348Get rights and content
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During signal transduction, the G protein, Gαq, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gαq, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gαq. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gαq with low micromolar affinity and inhibits Gαq by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gαq, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gαq, and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gαq/i with high affinity in vitro (KD = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gαq in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions.

Keywords

heterotrimeric G protein
q
Rosetta molecular modeling program
protein design
cancer
molecular modeling
phospholipase C
peptide interaction

Abbreviations

CD
circular dichroism
FACS
fluorescent-activated cell sorting
GAP
GTPase-activating protein
GPCR
G-protein-coupled receptor
HTH
helix-turn-helix
PLC-β
phospholipase C-β
SRE
serum response element

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Mahmud Hussain is a senior-level scientist and a team leader at Precision BioSciences Inc, USA. His current research involves engineering antibodies aimed at developing chimeric antigen receptor T (CAR-T) cell therapy to treat several forms of cancer. As highlighted in his recent JBC article, Mahmud utilizes nature-inspired and structure-guided combinatorial biology tools to discover novel solutions in protein–protein interaction for developing CAR-T cell therapy.

Matthew C. Cummins is a graduate student in the Department of Pharmacology at the University of North Carolina at Chapel Hill. He studies protein engineering methods and G proteins. Unfortunately, few inhibitors against G proteins exist, and those that do have limitations. Matt is using computational protein design to engineer de novo proteins that are tailored to inhibit G proteins.

Present addresses for Mahmud Hussain: Precision Biosciences, Durham, North Carolina, USA.

These authors contributed equally to this work.