Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination
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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 the application of dominant negative G proteins of Gαs and Gαi2 to the purification of stable complexes where this was not possible with wild-type G protein. Active state complexes of adenosine:A1 receptor:Gαi2βγ and calcitonin gene-related peptide (CGRP):CLR:RAMP1:Gαsβγ: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 Gα-proteins are thus high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery.
Liang, Y-L., Zhao, P., Draper-Joyce, C. J., Baltos, J-A., Glukhova, A., Truong, T. T., May, L. T., Christopoulos, A., Wootten, D., Sexton, P., & Furness, S. (2018). Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination. ACS Pharmacology & Translational Science, 1(1), 12-20. https://doi.org/10.1021/acsptsci.8b00017