scholarly journals Conformational rearrangement of the NMDA receptor amino-terminal domain during activation and allosteric modulation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vojtech Vyklicky ◽  
Cherise Stanley ◽  
Chris Habrian ◽  
Ehud Y. Isacoff
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Detectable Effect ◽  
Open Probability ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Binding Domains ◽  
Amino Terminal Domain

AbstractN-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.

scholarly journals Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker.

1993 ◽  
Vol 120 (1) ◽  
pp. 129-139 ◽  
Author(s):  
M Algrain ◽  
O Turunen ◽  
A Vaheri ◽  
D Louvard ◽  
M Arpin
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Actin Filaments ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Binding Domains ◽  
Carboxy Terminal Domain ◽  
Cell Surface Structures ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlström, and A. Vaheri. 1989. J. Biol. Chem. 264:16727-16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy-terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin-containing structures and is partially detergent insoluble. The amino-terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.

scholarly journals CD95 receptor activation by ligand-induced trimerization is independent of its partial pre-ligand assembly

2018 ◽  
Author(s):  
C. Liesche ◽  
J. Berndt ◽  
F. Fricke ◽  
S. Aschenbrenner ◽  
M. Heilemann ◽  
...  
Keyword(s):  
Cell Death ◽  
Ligand Binding ◽  
Single Molecule ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Caspase 8 ◽  
List Type ◽  
Death Domain

AbstractCD95 (Fas, APO-1, TNFRSF6) is a widely expressed single-pass transmembrane protein that is implicated in cell death, inflammatory response, proliferation and cell migration. CD95 ligand (CD95L, FasL, TNFSF6), is a potent apoptotic inducer in the membrane form but not when cleaved into soluble CD95L (sCD95L). Here, we aimed at understanding the relation between ligand-receptor multimerization and receptor activation by correlating the kinetics of ligand binding, receptor oligomerization, FADD (FAS-Associated via Death Domain) recruitment and caspase-8 activation inside living cells. Using single molecule localization microscopy and Förster resonance energy transfer imaging we show that the majority of CD95 receptors on the plasma membrane are monomeric at rest. This was confirmed functionally as the wild-type receptor is not blocked by a receptor mutant that cannot bind ligand. Moreover, using time-resolved fluorescence imaging approaches we demonstrated that receptor multimerization follows instantaneously ligand binding, whereas FADD recruitment is delayed. This process can explain the typical delay time seen with caspase-8 activity reporters. Finally, the low activity of sCD95L, which was caused by inefficient FADD recruitment, was not explained by the low avidity for the receptor but by a receptor clustering mechanism that was different from the one induced by the strong apoptosis inducer IZ-sCD95L. Our results reveal that receptor activation is modulated by the capacity of its ligand to trimerize it.HighlightsAt a density of less than 10 receptors per µm2CD95 exists as monomer (58%) and dimer (42%)Pre-formed dimers do not contribute to ligand-induced CD95 apoptotic signalingThe PLAD of CD95 attenuates overexpression-induced, ligand-independent cell deathsoluble CD95L can rapidly multimerize CD95 after binding but it is still a poor inducer of apoptosis through inefficient FADD recruitmentFADD recruitment kinetics but not ligand binding kinetics correlates with caspase-8 onset of activity

scholarly journals Structure and dynamics underlying elementary ligand binding events in human pacemaking channels

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Marcel P Goldschen-Ohm ◽  
Vadim A Klenchin ◽  
David S White ◽  
John B Cowgill ◽  
Qiang Cui ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cyclic Nucleotide ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Domains ◽  
Pacemaker Channels ◽  
Weak Binding ◽  
Nucleotide Regulation ◽  
Ligand Regulation ◽  
Initial Binding

Although molecular recognition is crucial for cellular signaling, mechanistic studies have relied primarily on ensemble measures that average over and thereby obscure underlying steps. Single-molecule observations that resolve these steps are lacking due to diffraction-limited resolution of single fluorophores at relevant concentrations. Here, we combined zero-mode waveguides with fluorescence resonance energy transfer (FRET) to directly observe binding at individual cyclic nucleotide-binding domains (CNBDs) from human pacemaker ion channels critical for heart and brain function. Our observations resolve the dynamics of multiple distinct steps underlying cyclic nucleotide regulation: a slow initial binding step that must select a 'receptive' conformation followed by a ligand-induced isomerization of the CNBD. X-ray structure of the apo CNBD and atomistic simulations reveal that the isomerization involves both local and global transitions. Our approach reveals fundamental mechanisms underpinning ligand regulation of pacemaker channels, and is generally applicable to weak-binding interactions governing a broad spectrum of signaling processes.

scholarly journals A de novo GRIN1 Variant Associated With Myoclonus and Developmental Delay: From Molecular Mechanism to Rescue Pharmacology

2021 ◽  
Vol 12 ◽  
Author(s):  
Jin Zhang ◽  
Weiting Tang ◽  
Nidhi K. Bhatia ◽  
Yuchen Xu ◽  
Nabina Paudyal ◽  
...  
Keyword(s):  
Single Molecule ◽  
Time Course ◽  
De Novo ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Domain ◽  
Cell Surface Expression ◽  
Agonist Binding ◽  
Open Probability ◽  
Zinc Inhibition

N-Methyl-D-aspartate receptors (NMDARs) are highly expressed in brain and play important roles in neurodevelopment and various neuropathologic conditions. Here, we describe a new phenotype in an individual associated with a novel de novo deleterious variant in GRIN1 (c.1595C>A, p.Pro532His). The clinical phenotype is characterized with developmental encephalopathy, striking stimulus-sensitive myoclonus, and frontal lobe and frontal white matter hypoplasia, with no apparent seizures detected. NMDARs that contained the P532H within the glycine-binding domain of GluN1 with either the GluN2A or GluN2B subunits were evaluated for changes in their pharmacological and biophysical properties, which surprisingly revealed only modest changes in glycine potency but a significant decrease in glutamate potency, an increase in sensitivity to endogenous zinc inhibition, a decrease in response to maximally effective concentrations of agonists, a shortened synaptic-like response time course, a decreased channel open probability, and a reduced receptor cell surface expression. Molecule dynamics simulations suggested that the variant can lead to additional interactions across the dimer interface in the agonist-binding domains, resulting in a more open GluN2 agonist-binding domain cleft, which was also confirmed by single-molecule fluorescence resonance energy transfer measurements. Based on the functional deficits identified, several positive modulators were evaluated to explore potential rescue pharmacology.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

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