scholarly journals Capturing Peptide–GPCR Interactions and Their Dynamics

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4724
Author(s):  
Anette Kaiser ◽  
Irene Coin
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Structural Changes ◽  
Structural Heterogeneity ◽  
Receptor Interaction ◽  
Spectroscopic Techniques ◽  
Peptide Receptors ◽  
Physiological Processes ◽  
High Dynamics ◽  
G Protein Coupled

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

Influence of Ascorbic Acid on the Structure and Function of Alpha-2- macroglobulin: Investigations using Spectroscopic and Thermodynamic Techniques

2020 ◽  
Vol 27 (3) ◽  
pp. 201-209
Author(s):  
Syed Saqib Ali ◽  
Mohammad Khalid Zia ◽  
Tooba Siddiqui ◽  
Haseeb Ahsan ◽  
Fahim Halim Khan
Keyword(s):  
Ascorbic Acid ◽  
Conformational Changes ◽  
Structural Changes ◽  
The Body ◽  
Titration Calorimetry ◽  
Spectroscopic Techniques ◽  
Human Beings ◽  
Plasma Ascorbic Acid ◽  
Dietary Antioxidant ◽  
And Function

Background: Ascorbic acid is a classic dietary antioxidant which plays an important role in the body of human beings. It is commonly found in various foods as well as taken as dietary supplement. Objective: The plasma ascorbic acid concentration may range from low, as in chronic or acute oxidative stress to high if delivered intravenously during cancer treatment. Sheep alpha-2- macroglobulin (α2M), a human α2M homologue is a large tetrameric glycoprotein of 630 kDa with antiproteinase activity, found in sheep’s blood. Methods: In the present study, the interaction of ascorbic acid with alpha-2-macroglobulin was explored in the presence of visible light by utilizing various spectroscopic techniques and isothermal titration calorimetry (ITC). Results: UV-vis and fluorescence spectroscopy suggests the formation of a complex between ascorbic acid and α2M apparent by increased absorbance and decreased fluorescence. Secondary structural changes in the α2M were investigated by CD and FT-IR spectroscopy. Our findings suggest the induction of subtle conformational changes in α2M induced by ascorbic acid. Thermodynamics signatures of ascorbic acid and α2M interaction indicate that the binding is an enthalpy-driven process. Conclusion: It is possible that ascorbic acid binds and compromises antiproteinase activity of α2M by inducing changes in the secondary structure of the protein.

scholarly journals Dissecting the allosteric networks governing agonist efficacy and potency in G protein-coupled receptors

2021 ◽  
Author(s):  
Franziska Marie Heydenreich ◽  
Maria Marti-Solano ◽  
Manbir Sandhu ◽  
Brian K Kobilka ◽  
Michel Bouvier ◽  
...  
Keyword(s):  
G Protein ◽  
Conformational Changes ◽  
Structural Changes ◽  
Receptor Activation ◽  
Residue Level ◽  
G Protein Coupled Receptors ◽  
Maximal Response ◽  
Pharmacological Properties ◽  
Receptor Ligand ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution of individual amino acids. We use epinephrine and β2-adrenergic receptor as a model to integrate residue-level pharmacology data with intramolecular residue contact data describing receptor activation. This unveils the allosteric networks driving ligand efficacy and potency. We provide detailed insights into how structural rearrangements are linked to fundamental pharmacological properties at single-residue level in a receptor-ligand system. Our approach can be used to determine such pharmacological networks for any receptor-ligand complex.

scholarly journals Spectroscopic characterization of insulin and small molecule ligand binding to the insulin receptor

2002 ◽  
Vol 16 (3-4) ◽  
pp. 147-159 ◽  
Author(s):  
Morten Schlein ◽  
Svend Ludvigsen ◽  
Helle B. Olsen ◽  
Michael F. Dunn ◽  
Niels C. Kaarsholm
Keyword(s):  
Ligand Binding ◽  
Insulin Receptor ◽  
Small Molecule ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Insulin Analogue ◽  
Three Dimensional ◽  
Insulin Binding ◽  
Spectroscopic Characterization ◽  
Spectroscopic Techniques

We have applied spectroscopic techniques to study two kinds of ligand binding to the insulin receptor. First, a fluorescently labelled insulin analogue is used to characterize the mechanism of reversible 1 :1 complex formation with a fragment of the insulin receptor ectodomain. The receptor induced fluorescence enhancement of the labelled insulin analogue provides the basis for stopped flow kinetic experiments. The kinetic data are consistent with a bimolecular binding event followed by a conformational change. This emphasizes the importance of insulin induced conformational changes in the activation of the insulin receptor. Second, the binding of fluorescein derivatives to the insulin receptor is studied. These small molecule ligands displace insulin from its receptor with micromolar affinity. The binding is verified by transferred NOESY NMR experiments. Their chromophoric properties are used to measure the affinity by UV-vis and fluorescence difference spectroscopies and the resulting Kdvalues are similar to those observed in the displacement receptor binding assay. However, these experiments and a stoichiometry determination indicate multiple binding sites, of which one overlaps with the insulin binding site. These two examples illustrate how spectroscopy complements biochemical receptor binding assays and provides information on ligand–insulin receptor interactions in the absence of three dimensional structures.

scholarly journals Ligand Binding to the Androgen Receptor Induces Conformational Changes That Regulate Phosphatase Interactions

2007 ◽  
Vol 27 (9) ◽  
pp. 3390-3404 ◽  
Author(s):  
Chun-Song Yang ◽  
Hong-Wu Xin ◽  
Joshua B. Kelley ◽  
Adam Spencer ◽  
David L. Brautigan ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Structural Changes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Phosphorylation Sites ◽  
A Subunit ◽  
Androgen Binding

ABSTRACT We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen- and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation.

scholarly journals Structural basis for σ1 receptor ligand recognition

2018 ◽  
Author(s):  
Hayden R. Schmidt ◽  
Robin M. Betz ◽  
Ron O. Dror ◽  
Andrew C. Kruse
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Md Simulations ◽  
Integral Membrane Protein ◽  
Structural Basis ◽  
Σ1 Receptor ◽  
Agonist Action ◽  
Slow Kinetics ◽  
Multistep Process ◽  
G Protein Coupled

The σ1 receptor is a poorly understood integral membrane protein expressed in most cells and tissues in the human body. It has been shown to modulate the activity of other membrane proteins such as ion channels and G protein-coupled receptors1–4, and ligands targeting the σ1 receptor are currently in clinical trials for treatment of Alzheimer’s disease5, ischemic stroke6, and neuropathic pain7. Despite its importance, relatively little is known regarding σ1 receptor function at the molecular level. Here, we present crystal structures of the human σ1 receptor bound to the classical antagonists haloperidol and NE-100, as well as the agonist (+)-pentazocine, at crystallographic resolutions of 3.1 Å, 2.9 Å, and 3.1 Å respectively. These structures reveal a unique binding pose for the agonist. The structures and accompanying molecular dynamics (MD) simulations demonstrate that the agonist induces subtle structural rearrangements in the receptor. In addition, we show that ligand binding and dissociation from σ1 is a multistep process, with extraordinarily slow kinetics limited by receptor conformational change. We use MD simulations to reconstruct a ligand binding pathway that requires two major conformational changes. Taken together, these data provide a framework for understanding the molecular basis for agonist action at σ1.

scholarly journals An Improved Free Energy Perturbation FEP+ Sampling Protocol for Flexible Ligand-Binding Domains

2019 ◽  
Author(s):  
Filip Fratev ◽  
suman sirimulla
Keyword(s):  
Free Energy ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Structural Changes ◽  
Binding Mode ◽  
Sampling Time ◽  
Free Energy Perturbation ◽  
Sampling Protocol ◽  
Sampling Schemes ◽  
Energy Perturbation

Recent improvements to free energy perturbation (FEP) calculations, especiallyFEP+, established their utility for pharmaceutical lead optimization. However, to dateFEP has typically been helpful only when (1) high-quality X-ray data is available and(2) the target protein does not undergo significant conformational changes. Also, alack of systematic studies on determining an adequate sampling time is often one ofthe primary limitations of FEP calculations. Herein, we propose a modified versionof the FEP/REST (i.e., replica exchange with solute tempering) sampling protocol,based on systematic studies on several targets by probing a large number of permutations with different sampling schemes. Improved FEP+ binding affinity predictions for regular flexible-loop (F-loop) motions and considerable structural changes can be obtained by extending the pre-REST sampling time from 0.24 ns to 5 ns/λand 2×10 ns/λ, respectively. We obtained much more precise ∆∆G calculations of the individual perturbations, including the sign of the transformations and less error. We extended the REST simulations from 5 ns to 8 ns to achieve reasonable free energy convergence.Implementing REST to the entire ligand as opposed to solely the perturbed region, and also some important flexible protein residues (pREST region) in ligand binding domain (LBD) , also considerably improved the FEP+ results in most of the studied cases. Preliminary molecular dynamics (MD) runs were useful for establishing the correct binding mode of the compounds and thus precise alignment for FEP+.<br>

scholarly journals The Science Behind G Protein-Coupled Receptors (GPCRs) and Their Accurate Visual Representation in Scientific Research

2017 ◽  
Vol 41 (1) ◽  
Author(s):  
Amy Sojka ◽  
Kevin Brennan ◽  
Evelyn Maizels ◽  
Christine Young
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Conformational Changes ◽  
Intracellular Signaling ◽  
Three Dimensional ◽  
G Protein Coupled Receptors ◽  
Membrane Topology ◽  
Extracellular Domains ◽  
Gpcr Structure ◽  
G Protein Coupled

G Protein-Coupled Receptors (GPCRs) are transmembrane (TM) proteins that span the cell membrane seven times, and contain intracellular and extracellular domains, comprised of connecting loops, as well as terminal extension sequences. GPCRs bind ligands within their transmembrane and/or extracellular domains. Ligand binding elicits conformational changes that initiate downstream intracellular signaling events through arrestins and G proteins. GPCRs play central roles in many physiological processes, from sensory to neurological, cardiovascular, endocrine, and reproductive functions. This paper strives to provide an entry point to current GPCR science, and to identify visual approaches to communicate select aspects of GPCR structure and function with clarity and accuracy. The overall GPCR structure, primary sequence and the implications of sequence for membrane topology, ligand binding and helical rearrangements accompanying activation are considered and discussed in the context of visualization strategies, including two-dimensional topological diagrams, three-dimensional representations, and common errors that arise from these representation.

scholarly journals The Molecular Basis of G Protein–Coupled Receptor Activation

2018 ◽  
Vol 87 (1) ◽  
pp. 897-919 ◽  
Author(s):  
William I. Weis ◽  
Brian K. Kobilka
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Conformational Changes ◽  
Cellular Responses ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Active Conformation ◽  
Allosteric Coupling ◽  
External Stimuli ◽  
G Protein Coupled

G protein–coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and βγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

scholarly journals ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Mark F. Mabanglo ◽  
Elisa Leung ◽  
Siavash Vahidi ◽  
Thiago V. Seraphim ◽  
Bryan T. Eger ◽  
...  
Keyword(s):  
Small Molecule ◽  
Conformational Changes ◽  
Electrostatic Interaction ◽  
Structural Changes ◽  
Structural Heterogeneity ◽  
Interaction Networks ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Bacterial Cell Death

Abstract Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

scholarly journals Dimerization of the Trk receptors in the plasma membrane: effects of their cognate ligands

2018 ◽  
Vol 475 (22) ◽  
pp. 3669-3685 ◽  
Author(s):  
Fozia Ahmed ◽  
Kalina Hristova
Keyword(s):  
Plasma Membrane ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Tyrosine Kinases ◽  
Structural Changes ◽  
Control Cell ◽  
Trk Receptors ◽  
Surface Receptors ◽  
Neuronal Tissues ◽  
Membrane Effects

Receptor tyrosine kinases (RTKs) are cell surface receptors which control cell growth and differentiation, and play important roles in tumorigenesis. Despite decades of RTK research, the mechanism of RTK activation in response to their ligands is still under debate. Here, we investigate the interactions that control the activation of the tropomyosin receptor kinase (Trk) family of RTKs in the plasma membrane, using a FRET-based methodology. The Trk receptors are expressed in neuronal tissues, and guide the development of the central and peripheral nervous systems during development. We quantify the dimerization of human Trk-A, Trk-B, and Trk-C in the absence and presence of their cognate ligands: human β-nerve growth factor, human brain-derived neurotrophic factor, and human neurotrophin-3, respectively. We also assess conformational changes in the Trk dimers upon ligand binding. Our data support a model of Trk activation in which (1) Trks have a propensity to interact laterally and to form dimers even in the absence of ligand, (2) different Trk unliganded dimers have different stabilities, (3) ligand binding leads to Trk dimer stabilization, and (4) ligand binding induces structural changes in the Trk dimers which propagate to their transmembrane and intracellular domains. This model, which we call the ‘transition model of RTK activation,’ may hold true for many other RTKs.

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