scholarly journals Structure and dynamics of the CGRP receptor in apo and peptide-bound forms

Science ◽  
2021 ◽  
Vol 372 (6538) ◽  
pp. eabf7258 ◽  
Author(s):  
Tracy M. Josephs ◽  
Matthew J. Belousoff ◽  
Yi-Lynn Liang ◽  
Sarah J. Piper ◽  
Jianjun Cao ◽  
...  
Keyword(s):  
G Protein ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cgrp Receptor ◽  
Exchange Mass Spectrometry ◽  
Gpcr Activation ◽  
Calcitonin Gene ◽  
Hydrogen Deuterium ◽  
Protein Recruitment

G protein–coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene–related peptide (CGRP) receptors from insect cells to determine their cryo–electron microscopy (cryo-EM) structures, and we complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry and three-dimensional variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound CGRP receptor complex, our work provides insight into the mechanisms of class B1 GPCR activation.

scholarly journals Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of β-arrestin 2 coupling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kaushik Sarkar ◽  
Lisa Joedicke ◽  
Marta Westwood ◽  
Rebecca Burnley ◽  
Michael Wright ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Parathyroid Hormone Receptor ◽  
Exchange Mass Spectrometry ◽  
Gpcr Activation ◽  
Hydrogen Deuterium ◽  
Binding Antibody ◽  
Hdx Ms

Abstract Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

Multiple affinity and guanine nucleotide sensitive forms of the calcitonin gene related peptide (CGRP) receptor

1995 ◽  
Vol 73 (7) ◽  
pp. 968-973 ◽  
Author(s):  
Tapan K. Chatterjee ◽  
Rory A. Fisher
Keyword(s):  
G Protein ◽  
Conformational Changes ◽  
Ternary Complex ◽  
Protein S ◽  
Calcitonin Gene Related Peptide ◽  
Cgrp Receptor ◽  
Peripheral Tissues ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
G Protein Coupled

Calcitonin gene related peptide (CGRP) is a novel neuropeptide with an impressive array of biological actions consistent with its diverse tissue distribution and suggested role as neurotransmitter or neuromodulator. Binding sites for CGRP with properties consistent with those of receptors are present in both central and peripheral tissues. Radioligand binding studies were performed to investigate the fundamental processes underlying CGRP receptor activation and signaling following agonist occupancy of the receptor. These studies documented the existence of a selective, high affinity, and homogeneous population of binding sites for CGRP in membranes prepared from central and various peripheral tissues. The affinity of [125I]CGRP for these sites was regulated by GTP or its stable analog GTPγS, indicating coupling of CGRP receptors to G-protein(s). Kinetic studies documented the existence of the CGRP receptor in multiple affinity states when both coupled to and uncoupled from G-proteins(s). These findings suggest that CGRP occupancy of its receptor induces conformational changes in the receptor that may be involved in its coupling to G-proteins and that the resulting ligand–receptor–G-protein ternary complex exists in multiple affinity conformational states. It seems likely that the multiple affinity states of the CGRP receptor ternary complex are involved differentially in signaling by and desensitization of the receptor. This evidence for agonist-induced conformational changes in a G-protein-coupled receptor prior to its coupling with G-protein(s) and for the existence of the ligand–receptor–G-protein ternary complex in multiple affinity conformational states is novel and extends our current understanding of the nature of the processes involved in agonist-dependent activation of G-protein-coupled receptors.Key words: calcitonin gene related peptide receptor, G-protein, multiple affinity.

scholarly journals Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

2021 ◽  
Author(s):  
Valentine V. Courouble ◽  
Sanjay Kumar Dey ◽  
Ruchi Yadav ◽  
Jennifer Timm ◽  
Jerry Joe E. K. Harrison ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Full Length ◽  
Structural Proteomics ◽  
Exchange Mass Spectrometry ◽  
Dimeric Unit ◽  
Hydrogen Deuterium ◽  
Hdx Ms

ABSTRACTCoronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.

scholarly journals Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruyu Jia ◽  
Chloe Martens ◽  
Mrinal Shekhar ◽  
Shashank Pant ◽  
Grant A. Pellowe ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Transition ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Allosteric Coupling ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

scholarly journals Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

2020 ◽  
Author(s):  
Ruyu Jia ◽  
Chloe Martens ◽  
Mrinal Shekhar ◽  
Shashank Pant ◽  
Grant A. Pellowe ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Transition ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Allosteric Coupling ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

scholarly journals Structural Characterization of Receptor–Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers

2021 ◽  
Vol 22 (6) ◽  
pp. 3241
Author(s):  
Raudah Lazim ◽  
Donghyuk Suh ◽  
Jai Woo Lee ◽  
Thi Ngoc Lan Vu ◽  
Sanghee Yoon ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Metabotropic Glutamate Receptor ◽  
Three Dimensional ◽  
Allosteric Modulation ◽  
Experimental Investigations ◽  
G Protein Coupled Receptor ◽  
Metabotropic Glutamate ◽  
Gpcr Activation ◽  
G Protein Coupled

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

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