Ligand binding to a G protein–coupled receptor captured in a mass spectrometer

Yen, Hsin‐Yung; Hopper, Jonathan T. S.; Liko, Idlir; Allison, Timothy M.; Zhu, Yun; Wang, Dejian; Stegmann, Monika; Mohammed, Shabaz; Wu, Beili; Robinson, Carol V.

doi:10.1126/sciadv.1701016

Cited by 55 publications

(67 citation statements)

References 42 publications

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“…Having established the utility of OGDs for membrane protein research and native MS, we focus on one of the most challenging protein families in structural biology: GPCRs. A recent breakthrough came with the demonstration that native MS has the potential to elucidate how post-translational modifications and lipid interactions affect the structure and function of GPCRs 42,43 . However, research on GPCRs remains generally challenging, partly because of the limited range of detergents that facilitate their purification and analysis [44][45][46] .…”

Section: Resultsmentioning

confidence: 99%

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

et al. 2020

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Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.

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Section: Resultsmentioning

confidence: 99%

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

et al. 2020

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“…To this end, mass spectrometry (MS) has enabled the discovery of several bioactive peptides (Fricker et al, ; Hatcher et al, ), even though it is very difficult to detect the inherently limited temporal and spatial expression of secreted peptides in mixed samples containing large quantities of other proteins. MS has been applied to peptide ligand screening, as it is label‐free and unbiased with respect to signalling pathways (Yen et al, ). Recently, HPLC and MS of bile and cell culture supernatants have led to the discovery of a post‐translationally modified peptide, S‐geranylgeranyl‐L‐GSH as a potent endogenous ligand for the orphan receptor P2RY8 (Lu, Wolfreys, Muppidi, Xu, & Cyster, ).…”

Section: Approaches For Peptide‐gpcr De‐orphanisationmentioning

confidence: 99%

“…To this end, mass spectrometry (MS) has enabled the discovery of several bioactive peptides (Fricker et al, 2000;Hatcher et al, 2008), even though it is very difficult to detect the inherently limited temporal and spatial expression of secreted peptides in mixed samples containing large quantities of other proteins. MS has been applied to peptide ligand screening, as it is label-free and unbiased with respect to signalling pathways (Yen et al, 2017).…”

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confidence: 99%

Novel approaches leading towards peptide GPCR de‐orphanisation

Hauser

Gloriam

Bräuner‐Osborne

et al. 2020

British J Pharmacology

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The discovery of novel ligands for orphan GPCRs has profoundly affected our understanding of human biology, opening new opportunities for research, and ultimately for therapeutic development. Accordingly, much effort has been directed towards the remaining orphan receptors, yet the rate of GPCR de‐orphanisation has slowed in recent years. Here, we briefly review contemporary methodologies of de‐orphanisation and then highlight our recent integrated computational and experimental approach for discovery of novel peptide ligands for orphan GPCRs. We identified putative endogenous peptide ligands and found peptide receptor sequence and structural characteristics present in selected orphan receptors. With comprehensive pharmacological screening using three complementary assays, we discovered novel pairings of 17 peptides with five different orphan GPCRs and revealed potential additional ligands for nine peptide GPCRs. These promising findings lay the foundation for future studies on these peptides and receptors to characterise their roles in human physiology and disease.

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“…Studies of the ABC transporter P-gp [27] revealed that this protein preferentially binds to negatively charged lipids and cardiolipins (over zwitterionic lipids), and that binding to an inhibitor enhances cardiolipin binding, demonstrating that native MS could be used to probe synergistic binding events. Native MS has also been used to capture off-target drug interactions with MPs [131] and, excitingly, to monitor ligand binding to G-protein-coupled receptors (GPCRs), enabling native MS methods to be applied to GPCR drug discovery [132].…”

Section: Stoichiometry Ligand and Lipid Binding And Thermodynamics Rmentioning

confidence: 99%

Mass spectrometry-enabled structural biology of membrane proteins

Calabrese

Radford

2018

Methods

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a b s t r a c tThe last $25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.

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Ligand binding to a G protein–coupled receptor captured in a mass spectrometer

Abstract: Drug binding to the P2Y1 receptor prevents phosphorylation and competes with an endogenous ligand.

Cited by 55 publications

References 42 publications

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

Novel approaches leading towards peptide GPCR de‐orphanisation

Mass spectrometry-enabled structural biology of membrane proteins

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