scholarly journals CALX-CBD1 Ca2+-binding cooperativity studied by NMR spectroscopy and ITC with Bayesian statistics

2019 ◽  
Author(s):  
M. V. C. Cardoso ◽  
J. D. Rivera ◽  
P. M. Vitale ◽  
M. F. S. Degenhardt ◽  
L.A. Abiko ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Nmr Spectroscopy ◽  
Binding Sites ◽  
Titration Calorimetry ◽  
Binding Region ◽  
Nmr Titration ◽  
Titration Curves ◽  
Novel Approach ◽  
Intracellular Ca ◽  
Exchange Regime

ABSTRACTThe Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologues, CALX is inhibited by Ca2+-binding to CALX-CBD1, while CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of a β-sandwich and displays four Ca2+ binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+-binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift time scale. Ca2+-binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N CEST and 15N R2 dispersion allowed the determination of Ca2+ dissociation rates (≈ 20 s−1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal due to the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill = 2.9 − 3.1, near maximum cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is at least three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+-binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+-concentration in sensory neuronal cells.SIGNIFICANCECALX-CBD1 is the Ca2+-sensor domain of the Na+/Ca2+ exchanger of Drosophila melanogaster. It consists of a β-sandwich, and contains four Ca2+ binding sites at the distal loops. In this study, we examined the cooperative binding of four Ca2+ ions to CALX-CBD1 using NMR spectroscopy and isothermal titration calorimetry (ITC) experiments. NMR and ITC data were analyzed using the framework of the binding polynomial formalism and Bayesian statistics. A novel approach to analyze NMR titration data in the slow exchange regime was proposed. These results support the view that CALX-CBD1 binds four Ca2+ with high cooperativity. The significant ligand binding cooperativity exhibited by this domain is determinant for the efficient allosteric regulation of this exchanger by intracellular Ca2+.

scholarly journals Design of novel CV-N variants: Modulation of binding dynamics through distal mutations

2020 ◽  
Author(s):  
I. Can Kazan ◽  
Prerna Sharma ◽  
Andrey Bobkov ◽  
Raimund Fromme ◽  
Giovanna Ghirlanda ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Binding Sites ◽  
Computational Method ◽  
Experimental Characterization ◽  
Dynamic Coupling ◽  
Binding Region ◽  
Novel Approach ◽  
Allosteric Sites ◽  
Docking Tool

AbstractWe develop a computational approach to identify distal residues that allosterically modulate the dynamics of binding sites by combining dynamic coupling with statistical analysis of co-evolution. Putative mutants of these predicted allosteric sites are subjected to Adaptive BP-Dock docking tool for binding analysis. Here, we apply this method to a small lectin, Cyanovirin-N (CV-N), that selectively binds to dimannose. Our computational method points out mutations on I34, that is 16Å away from binding site can modulate binding. Experimental characterization of I34 mutants confirms that I34Y increases affinity towards dimannose, while I34K completely abolish binding. The increased affinity is not due to changes in the binding region, which are conserved in the crystal structure. However, ITC analysis reveals an opposite contribution of TΔS (negative in WT, and positive in I34Y) and suggests that modulation of dynamics (i.e., dynamic allostery) is responsible for the change in binding affinity. Our results point to a novel approach to identify and substitute distal sites, guiding the mutational landscape in glycan-binding proteins to improve binding affinity.

scholarly journals K2P2.1 (TREK-1) potassium channel activation protects against hyperoxia-induced lung injury

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tatiana Zyrianova ◽  
Benjamin Lopez ◽  
Riccardo Olcese ◽  
John Belperio ◽  
Christopher M. Waters ◽  
...  
Keyword(s):  
Lung Injury ◽  
Lung Compliance ◽  
Alveolar Epithelial Cells ◽  
Pharmacological Intervention ◽  
Alveolar Epithelial ◽  
Protein Levels ◽  
Cell Counts ◽  
Novel Approach ◽  
Intracellular Ca ◽  
Cell Depolarization

AbstractNo targeted therapies exist to counteract Hyperoxia (HO)-induced Acute Lung Injury (HALI). We previously found that HO downregulates alveolar K2P2.1 (TREK-1) K+ channels, which results in worsening lung injury. This decrease in TREK-1 levels leaves a subset of channels amendable to pharmacological intervention. Therefore, we hypothesized that TREK-1 activation protects against HALI. We treated HO-exposed mice and primary alveolar epithelial cells (AECs) with the novel TREK-1 activators ML335 and BL1249, and quantified physiological, histological, and biochemical lung injury markers. We determined the effects of these drugs on epithelial TREK-1 currents, plasma membrane potential (Em), and intracellular Ca2+ (iCa) concentrations using fluorometric assays, and blocked voltage-gated Ca2+ channels (CaV) as a downstream mechanism of cytokine secretion. Once-daily, intra-tracheal injections of HO-exposed mice with ML335 or BL1249 improved lung compliance, histological lung injury scores, broncho-alveolar lavage protein levels and cell counts, and IL-6 and IP-10 concentrations. TREK-1 activation also decreased IL-6, IP-10, and CCL-2 secretion from primary AECs. Mechanistically, ML335 and BL1249 induced TREK-1 currents in AECs, counteracted HO-induced cell depolarization, and lowered iCa2+ concentrations. In addition, CCL-2 secretion was decreased after L-type CaV inhibition. Therefore, Em stabilization with TREK-1 activators may represent a novel approach to counteract HALI.

scholarly journals Integrated impedance sensing of liquid sample plug flow enables automated high throughput NMR spectroscopy

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Omar Nassar ◽  
Mazin Jouda ◽  
Michael Rapp ◽  
Dario Mager ◽  
Jan G. Korvink ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
High Throughput ◽  
Plug Flow ◽  
Microfluidic System ◽  
High Resolution Spectroscopy ◽  
Dead Volume ◽  
Absolute Difference ◽  
Immiscible Fluid ◽  
Flow Sensors ◽  
Novel Approach

AbstractA novel approach for automated high throughput NMR spectroscopy with improved mass-sensitivity is accomplished by integrating microfluidic technologies and micro-NMR resonators. A flow system is utilized to transport a sample of interest from outside the NMR magnet through the NMR detector, circumventing the relatively vast dead volume in the supplying tube by loading a series of individual sample plugs separated by an immiscible fluid. This dual-phase flow demands a real-time robust sensing system to track the sample position and velocities and synchronize the NMR acquisition. In this contribution, we describe an NMR probe head that possesses a microfluidic system featuring: (i) a micro saddle coil for NMR spectroscopy and (ii) a pair of interdigitated capacitive sensors flanking the NMR detector for continuous position and velocity monitoring of the plugs with respect to the NMR detector. The system was successfully tested for automating flow-based measurement in a 500 MHz NMR system, enabling high resolution spectroscopy and NMR sensitivity of 2.18 nmol s1/2 with the flow sensors in operation. The flow sensors featured sensitivity to an absolute difference of 0.2 in relative permittivity, enabling distinction between most common solvents. It was demonstrated that a fully automated NMR measurement of nine individual 120 μL samples could be done within 3.6 min or effectively 15.3 s per sample.

scholarly journals Binding Sites for Oligosaccharide Repeats from Lactic Acid Bacteria Exopolysaccharides on Bovine β-Lactoglobulin Identified by NMR Spectroscopy

ACS Omega ◽  
2021 ◽  
Author(s):  
Johnny Birch ◽  
Sanaullah Khan ◽  
Mikkel Madsen ◽  
Christian Kjeldsen ◽  
Marie Sofie Møller ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Nmr Spectroscopy ◽  
Lactic Acid Bacteria ◽  
Binding Sites ◽  
Β Lactoglobulin

scholarly journals First Characterization of an Archaeal GTP-Dependent Phosphoenolpyruvate Carboxykinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

2004 ◽  
Vol 186 (14) ◽  
pp. 4620-4627 ◽  
Author(s):  
Wakao Fukuda ◽  
Toshiaki Fukui ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Binding Sites ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Cation Binding ◽  
Activity Levels ◽  
Binding Region ◽  
Hyperthermophilic Archaeon ◽  
Dependent Activity ◽  
Additional Role

ABSTRACT Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C3 and C4 metabolites. This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (Pck Tk ). Pck Tk showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp. Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in Pck Tk . However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs. The recombinant Pck Tk actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+. The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the Km value for oxaloacetate was much lower than that for phosphoenolpyruvate. The transcription and activity levels in T. kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions. These results agreed with the role of Pck Tk in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon. Additionally, under gluconeogenic conditions, we observed higher expression levels of Pck Tk on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon.

scholarly journals In vivo high-resolution magic angle spinning proton NMR spectroscopy of Drosophila melanogaster flies as a model system to investigate mitochondrial dysfunction in Drosophila GST2 mutants

2014 ◽  
Vol 34 (1) ◽  
pp. 327-333 ◽  
Author(s):  
VALERIA RIGHI ◽  
YIORGOS APIDIANAKIS ◽  
NIKOLAOS PSYCHOGIOS ◽  
LAURENCE G. RAHME ◽  
RONALD G. TOMPKINS ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Nmr Spectroscopy ◽  
High Resolution ◽  
Mitochondrial Dysfunction ◽  
Magic Angle Spinning ◽  
Model System ◽  
Magic Angle ◽  
Proton Nmr Spectroscopy ◽  
Angle Spinning

scholarly journals Dust from hog confinement facilities impairs Ca2+ mobilization from sarco(endo)plasmic reticulum by inhibiting ryanodine receptors

2013 ◽  
Vol 114 (5) ◽  
pp. 665-674
Author(s):  
Chengju Tian ◽  
Caronda J. Moore ◽  
Puttappa Dodmane ◽  
Chun Hong Shao ◽  
Debra J. Romberger ◽  
...  
Keyword(s):  
Binding Sites ◽  
Molecular Mechanisms ◽  
Ryanodine Receptors ◽  
Cell Types ◽  
C2c12 Cells ◽  
Common Element ◽  
Plasmic Reticulum ◽  
Skeletal Muscle Weakness ◽  
Intracellular Ca ◽  
Phosphate Buffered Saline

Individuals working in commercial hog confinement facilities have elevated incidences of headaches, depression, nausea, skeletal muscle weakness, fatigue, gastrointestinal disorders, and cardiovascular diseases, and the molecular mechanisms for these nonrespiratory ailments remain incompletely undefined. A common element underlying these diverse pathophysiologies is perturbation of intracellular Ca2+ homeostasis. This study assessed whether the dust generated inside hog confinement facilities contains compounds that alter Ca2+ mobilization via ryanodine receptors (RyRs), key intracellular channels responsible for mobilizing Ca2+ from internal stores to elicit an array of physiologic functions. Hog barn dust (HBD) was extracted with phosphate-buffered saline, sterile-filtered (0.22 μm), and size-separated using Sephadex G-100 resin. Fractions (F) 1 through 9 (Mw >10,000 Da) had no measurable effects on RyR isoforms. However, F10 through F17, which contained compounds of Mw ≤2,000 Da, modulated the [3H]ryanodine binding to RyR1, RyR2, and RyR3 in a biphasic (Gaussian) manner. The Ki values for F13, the most potent fraction, were 3.8 ± 0.2 μg/ml for RyR1, 0.2 ± 0.01 μg/ml and 19.1 ± 2.8 μg/ml for RyR2 (two binding sites), and 44.9 ± 2.8 μg/ml and 501.6 ± 9.2 μg/ml for RyR3 (two binding sites). In lipid bilayer assays, F13 dose-dependently decreased the open probabilities of RyR1, RyR2, and RyR3. Pretreating differentiated mouse skeletal myotubes (C2C12 cells) with F13 blunted the amplitudes of ryanodine- and K+-induced Ca2+ transients. Because RyRs are present in many cell types, impairment in Ca2+ mobilization from internal stores via these channels is a possible mechanism by which HBD may trigger these seemingly unrelated pathophysiologies.

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