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Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination.

ABSTRACT: Advances in structural biology have yielded exponential growth in G protein-coupled receptor (GPCR) structure solution. Nonetheless, the instability of fully active GPCR complexes with cognate heterotrimeric G proteins has made them elusive. Existing structures have been limited to nanobody-stabilized GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilization via engineering G proteins with reduced nucleotide affinity, limiting G?:G?? dissociation. We illustrate the application of dominant negative G proteins of G?s and G?i2 to the purification of stable complexes where this was not possible with wild-type G protein. Active state complexes of adenosine:A1 receptor:G?i2?? and calcitonin gene-related peptide (CGRP):CLR:RAMP1:G?s??:Nb35 were purified to homogeneity and were stable in negative stain electron microscopy. These were suitable for structure determination by cryo-electron microscopy at 3.6 and 3.3 Å resolution, respectively. The dominant negative G?-proteins are thus high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery.

SUBMITTER: Liang YL

PROVIDER: S-EPMC7089020 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination.

Liang Yi-Lynn YL Zhao Peishen P Draper-Joyce Christopher C Baltos Jo-Anne JA Glukhova Alisa A Truong Tin T TT May Lauren T LT Christopoulos Arthur A Wootten Denise D Sexton Patrick M PM Furness Sebastian G B SGB

ACS pharmacology & translational science 20180726 1

Advances in structural biology have yielded exponential growth in G protein-coupled receptor (GPCR) structure solution. Nonetheless, the instability of fully active GPCR complexes with cognate heterotrimeric G proteins has made them elusive. Existing structures have been limited to nanobody-stabilized GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilization via engineering G proteins with reduced nucleotide affinity, limiting Gα:Gβγ dissociation. We illustrate ...[more]

PMID: 32219201

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Project description:A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.

Dataset Information

Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination.

Publications

Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination.

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