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Dominant negative G proteins enhance formation and purification of agonist-GPCR-G protein complexes for structure determination Yi-Lynn Liang, Peishen Zhao, Christopher J. Draper-Joyce, Jo-Anne Baltos, Alisa Glukhova, Tin T Truong, Lauren T. May, Arthur Christopoulos, Denise Wootten, Patrick M. Sexton, and Sebastian G. B. Furness ACS Pharmacol. Transl. Sci., Just Accepted Manuscript • DOI: 10.1021/acsptsci.8b00017 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018
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ACS Pharmacology & Translational Science
ACS Pharmacology & Translational Science Letter Dominant negative G proteins enhance formation and purification of agonist-GPCR-G protein complexes for structure determination Yi-Lynn Liang1^, Peishen Zhao1^, Christopher Draper-Joyce1^, Jo-anne Baltos1, Alisa Glukhova1, Tin T. Truong1, Lauren T. May1, Arthur Christopoulos1, Denise Wootten1,2*, Patrick M. Sexton1,2*, Sebastian G. B. Furness1* 1. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Australia 2. School of Pharmacy, Fudan University, Shanghai 201203, China ^These authors contributed equally *Corresponding authors: Dr. Sebastian Furness;
[email protected] Prof. Patrick Sexton;
[email protected] Dr. Denise Wootten;
[email protected] 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-stabilised GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilisation via engineering G proteins with reduced nucleotide affinity, limiting Gα:Gβγ dissociation. We illustrate application of dominant negative G proteins of Gαs and Gαi2 to purification of stable complexes where this wasn’t 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 Å and 3.3 Å resolution, respectively. The DN Gα-proteins are thus high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery.
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GPCRs are premier drug targets and there is substantial interest in structural understanding of these proteins to determine mechanisms of drug interaction and activation. Recent methodological developments in GPCR engineering, including introduction of thermostabilising mutations, replacement of flexible loops with small stable fusion proteins and specialised binding partners, have enabled determination of structures of 53 unique receptors1. Of these structures only 5 receptors are in a fully active, transducer-complexed, state. This state is only achieved when both agonist and trimeric G protein (or other transducer) are present, commonly referred to as the ternary complex2. Thus, understanding the structural basis of this state is critical to understanding mechanisms of GPCR signal transduction and for utilising this information for structure-based drug design. G protein bound active state receptor structures have been achieved using several approaches; of the 5 published Gαs containing structures, 4 (3 unique receptors) utilized a camilid nanobody, Nb35, to stabilise the Gα – Gβγ interface3-6, while the structure of Adenosine A2A receptor utilised a highly engineered mini-Gs partner7. The active state of opsin was determined using a c-terminal peptide fragment of Gt8. The agonist-bound GPCR acts as a guanine-nucleotide exchange factor (GEF) for the Gα subunit, promoting GDP release and subsequent GTP binding. This occurs while the Gα subunit is bound to the Gβγ heterodimer. Physiologically, where GDP and GTP concentrations are relatively high, the ternary complex is unstable, the G protein heterotrimer dissociates from the receptor, and into its component parts, Gα and Gβγ that engage downstream signalling effectors9. This inherent instability makes it extremely challenging to trap and purify complexes of GPCRs bound to heterotrimeric G proteins for structural studies. Using the extensive literature on G protein mutagenesis, mutations within Gαi2 and Gαs (highlighted in Figure 1) predicted to stabilise the ternary complex state were selected. These reduce nucleotide-binding affinities and enhance the stability of agonist:GPCR:G protein heterotrimer complexes; achieving the latter is critical for structural studies. Mutations included 4 residues conserved across all G protein subclasses. SerH1.02 (CGN numbering system10), involved in coordinating Mg2+ and contacting GTP’s β-phosphate, whose mutation to Asn in Gαs11 and Cys in Gαi212, Gαo12 and Gαt13 generates a dominant negative G protein. This inhibits signalling by formation of a stabilised (non-dissociating) ternary complex. Glys3h2.02 of Gα forms a backbone amide hydrogen bond with the γphosphate of GTP14 with Ala substitution increasing the GDP dissociation rate and blocking GTP induced dissociation from the β2 receptor15. A combination of Glys3h2.02Ala with GluH3.04Ala generates a dominant negative16 presumably through disruption of the conserved salt-bridge between residues at s3h2.04 and H3.0414, which is also seen during the conformational rearrangement in the nucleotide free state3. Alas6h5.03 to Ser substitution increases GDP dissociation rate. Although, with these mutations, the free Gαβγ heterotrimer is thermolabile on its own, in combination with Glys3h2.02Ala, GluH3.04Ala and Alas6h5.03Ser, it appears more stable, presumably because of constitutive interactions with GPCRs17. For the Gαs mutant, five additional mutations substituting residues of Gαi2 into Gαs within the α3 helix or α3 helix - β5 strand linker were introduced, improving the dominant negative effect of Gαs17.
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ACS Pharmacology & Translational Science
Here we present data showing the utility of these engineered dominant negative (DN) Gα subunits to enhance formation of stable complexes for structural studies by cryo-electron microscopy (cryo-EM). These Gαs and Gαi2 constructs have enabled solution of fully-active structures of glucagon-like peptide 1 receptor (GLP-1R)6, adenosine A1 receptor (A1-AR)18 and calcitonin gene-related peptide receptor (CGRP-R)19 at resolutions that allow reliable placement of side-chain rotomers for most amino acids in the receptor core, supporting their broad applicability for purification of stable GPCR:heterotrimeric G protein complexes. In the case of GLP-1R, purification of GLP-1R:Gs complexes were attempted using exendinP5 (ExP5), a biased agonist with lower cAMP efficacy than the native GLP-1 that had been used by others to determine a 4.1 Å structure in complex with WT Gs5. The ExP5 complexes were initially formed with ExP5:GLP-1R:WT-Gαs:βγ in the absence of Nb35. These were benchmarked against the same complex containing the DN-Gαs as well as against sCT:CTR containing either WT or DN-Gαs as shown in Figure 2a. The ExP5:GLP-1R:WT-Gαs:βγ could not be purified to homogeneity, however, the inclusion of the DN-Gαs allowed both GLP-1R and CTR containing complexes to be purified to homogeneity (Figure 2a), yielding around 200 µgL-1. These Nb35 free complexes were subjected to negative stain EM yielded ~28% full complex out of the total unfiltered particles (Figure 2b). Inclusion of Nb35 with either WT or DN-Gαs generated a complex with similar yield and stoichiometry to Nb35 free complexes as assessed by western blot (Figure 2c), slightly improved purity as assessed by coomassie stain (compare Figure 2a with 2d, 2e), and a monodispersed peak by SEC. When either was subjected to negative stain EM, the full complex represented ~70% of total unfiltered particles (data not shown) and we chose to image the DN complex. This allowed solution of the structure at 3.3 Å6. We further assessed the utility of DN Gs for formation and purification of complexes with low efficacy agonists, using the endogenous GLP-1R agonist, oxyntomodulin that has 100-fold lower cAMP potency than GLP-1. All preparations included Nb35 to maximize complex stability and yields. Initial formation of complexes was attempted using 1µM peptide, as per previous studies. Complexes were weakly formed with both WT (Figure 3a) and DN Gs (Figure 3b), with the latter providing higher yields. Increasing the agonist concentration to 50 µM during initial formation of the complex substantially improved yields with the DN Gs preparation (Figure 3c), enabling recovery of a monodisperse peak following a second round of purification by SEC (Figure 3d). This peak exhibited apparent stoichiometric levels of component proteins by coomassie stain (Figure 3e) and large numbers of 2D classes of the full complex by negative stain EM (Figure 3f).
Subsequently, we applied this methodology to structure determination of the CGRP receptor. This receptor is a heterodimeric complex containing the calcitonin receptor-like receptor (CLR) and the single pass transmembrane protein, receptor activity-modifying protein 1 (RAMP1); requiring a more complex purification protocol (see methods). Formation of this complex using the agonist αCGRP, WT-Gαs, Gβγ and Nb35 enabled its purification, as shown in Figure 4a, however the yield was extremely poor (
