Assembly of a GPCR-G Protein Complex

Du, Yang; Duc, Nguyen Minh; Rasmussen, Søren G. F.; Hilger, Daniel; Kubiak, Xavier; Wang, Liwen; Bohon, Jen; Kim, Hee Ryung; Wegrecki, Marcin; Asuru, Awuri; Jeong, Kyung Min; Lee, Jeongmi; Chance, Mark R.; Lodowski, David T.; Kobilka, Brian K.; Chung, Ka Young

doi:10.1016/j.cell.2019.04.022

Cited by 179 publications

(218 citation statements)

References 56 publications

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“…In this study, they could also observe allosteric coupling upon complex formation, manifested by a decrease in HDX in the catalytic site of the kinase. More recent work from the Chung group used pulsed HDX‐MS experiments and protein footprinting MS to dissect the sequence of events leading to assembly of the GPCR‐protein G complex . They studied assembly formation of protein G with either β2ar or A2A receptors.…”

Section: Going Native—beyond the State Of The Artmentioning

confidence: 99%

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A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Martens

Politis

2020

Protein Science

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Integral membrane proteins (IMPs) control countless fundamental biological processes and constitute the majority of drug targets. For this reason, uncovering their molecular mechanism of action has long been an intense field of research.They are, however, notoriously difficult to work with, mainly due to their localization within the heterogeneous of environment of the biological membrane and the instability once extracted from the lipid bilayer. High-resolution structures have unveiled many mechanistic aspects of IMPs but also revealed that the elucidation of static pictures has limitations. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has recently emerged as a powerful biophysical tool for interrogating the conformational dynamics of proteins and their interactions with ligands. Its versatility has proven particularly useful to reveal mechanistic aspects of challenging classes of proteins such as IMPs. This review recapitulates the accomplishments of HDX-MS as it has matured into an essential tool for membrane protein structural biologists. K E Y W O R D Sallosteric coupling, conformational dynamics, GPCRs, hydrogen-deuterium exchange mass spectrometry, integral membrane proteins, transporters Standard: LC: Reverse-phase chromatography on C18 column with prior trapping for desalting. MS: Often Synapt or Xevo-G2 in MS E mode √ Drift-time aligned MS E (HDMS E ) 17 √ LC column with shorter alkyl chain-for example, BEH C4 or C8 13 √ Gradient optimization (8-30%, 8-50%) 15 √ Saw-tooth gradient after peptides elution to prevent carryover 16 Miscellaneous observationsUse of PEG-based detergents (e.g., Triton X-100) complicates peptides identification. Good practice to start with a benchmark condition-for example, locked protein. 16,18,19 Adding TCEP helps but too much (>1 M) reduces spectral quality. 15Note: The tick indicates parameters that have shown improvement compared with the standard conditions.

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Section: Going Native—beyond the State Of The Artmentioning

confidence: 99%

“…In both cases, the authors could identify the role of α5 helix of the Gα subunit as the starting point of complex formation. They suggest that the initial complex conformation is transient and might differ from the states captured by X‐ray crystallography and Cryo‐EM …”

Section: Going Native—beyond the State Of The Artmentioning

confidence: 99%

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Martens

Politis

2020

Protein Science

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“…4b). However, superimposition of the inactive GDP-bound G s heterotrimer 28 onto the nucleotide-free G s in the complex suggests that the bent α5 helix of the GDP-bound G protein (α5 GDP ) together with the known inherent flexibility of its very C-terminal end 29,30 , might allow the initial engagement of the GDP-bound G protein without outward displacement of TM6 (Fig. 4b).…”

Section: Main Textmentioning

confidence: 99%

Structural insights into ligand efficacy and activation of the glucagon receptor

Hilger

Kumar

et al. 2019

Preprint

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“…Vertebrate olfactory systems have evolved to sense volatile substances through their recognition by olfactory receptors (ORs) located on the membrane of olfactory sensory neurons in the olfactory epithelium [1] and consequent initiation of signaling cascades that transform odorantreceptor chemical interactions into electrochemical signals [2,3]. These receptors belong to the class A G protein-coupled receptors (GPCRs), a major drug target protein family [4] involved in the transduction of extracellular signals through second messenger cascades controlled by different heterotrimeric guanine nucleotide-binding proteins (Golf in the case of ORs) coupled at their intracellular regions [5,6].…”

Section: Introductionmentioning

confidence: 99%

“…ORs are characterized by intronless coding regions of average length of 310 codons (~1kb) and constitute the largest multigene family in humans, with a functional gene repertoire estimated between 322 and 390 genes, divided into two main classes, 17 families and more than 150 subfamilies [7]. This broad array of receptors, like in other terrestrial mammals, is shared with tetrapods (families [1][2][3][4][5][6][7][8][9][10][11][12][13][14] and marine vertebrates (families 51-56) [8], and seems necessary to respond efficiently to the extraordinary chemical diversity of odorants in Earth's ecosystems [9].…”

Section: Introductionmentioning

confidence: 99%

The mutational landscape of human olfactory G protein-coupled receptors

Jiménez

Casajuana-Martín

García-Recio

et al. 2020

Preprint

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Olfactory receptors (ORs) constitute a large family of sensory proteins that enable us to recognize a wide range of chemical volatiles in the environment. By contrast to the extensive information about human olfactory thresholds for thousands of odorants, studies of the genetic influence on olfaction are limited to a few examples. Here, we analyzed a compendium of 118,057 natural variants in human ORs collected from the public domain. OR mutations were categorized depending on their genomic and protein contexts, as well as their frequency of occurrence in several human populations. Functional interpretation of the natural changes was estimated from the increasing knowledge of the structure and function of the G protein-coupled receptor (GPCR) family, to which ORs belong. Our analysis reveals an extraordinary diversity of natural variations in the olfactory gene repertoire between individuals and populations, with a significant number of changes occurring at structural conserved regions. A particular attention is paid to mutations in positions linked to the conserved GPCR activation mechanism that could imply phenotypic variation in the olfactory perception. An interactive web application (available at http://lmc.uab.cat/hORMdb) was developed for the management and visualization of this mutational dataset.

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Assembly of a GPCR-G Protein Complex

Abstract: Highlightsd Temporal assembly of a GPCR-Gs complex revealed by time-resolved mass spectrometry d The sequence of GPCR-mediated G protein activation was elucidated d Key structural elements were found to dictate nucleotide release

Cited by 179 publications

References 56 publications

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Structural insights into ligand efficacy and activation of the glucagon receptor

The mutational landscape of human olfactory G protein-coupled receptors

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