Dipolar Spectroscopy - Double-Resonance Methods

Jeschke, Gunnar

doi:10.1002/9780470034590.emrstm1518

Cited by 50 publications

(55 citation statements)

References 75 publications

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“…To further understand the MamM CTD metal‐dependent conformational changes, site‐directed spin labelling was applied in combination with PELDOR spectroscopy , which affords the detection of both distances and distance distributions between labelled sites within the dimer. During site‐directed spin labelling, the paramagnetic (1‐oxyl‐2,2,5,5‐tetramethyl‐Δ3‐pyrroline‐3‐methyl) methanethiosulfonate spin label (MTSSL) reacts specifically with cysteine residues of the protein.…”

Section: Resultsmentioning

confidence: 99%

Metal binding to the dynamic cytoplasmic domain of the cation diffusion facilitator (CDF) protein MamM induces a ‘locked‐in’ configuration

et al. 2019

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Cation diffusion facilitator (CDF) proteins are a conserved family of transmembrane transporters that ensure cellular homeostasis of divalent transition metal cations. Metal cations bind to CDF protein's cytoplasmic C‐terminal domain (CTD), leading to closure from its apo open V‐shaped dimer to a tighter packed structure, followed by a conformational change of the transmembrane domain, thus enabling transport of the metal cation. By implementing a comprehensive range of biochemical and biophysical methods, we studied the molecular mechanism of metal binding to the magnetotactic bacterial CDF protein MamM CTD. Our results reveal that the CTD is rather dynamic in its apo form, and that two dependent metal‐binding sites, a single central binding site and two symmetrical, peripheral sites, are available for metal binding. However, only cation binding to the peripheral sites leads to conformational changes that lock the protein in a compact state. Thus, this work reveals how metal binding is regulating the sequential uptakes of metal cations by MamM, and extends our understanding of the complex regulation mechanism of CDF proteins. Database Structural data are available in RCSB Protein Data Bank under the accession numbers: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G64, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G55, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G5E and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G6I (for CS, C267S, CS‐C267S and W247A, respectively).

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Section: Resultsmentioning

confidence: 99%

Metal binding to the dynamic cytoplasmic domain of the cation diffusion facilitator (CDF) protein MamM induces a ‘locked‐in’ configuration

et al. 2019

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“…To answer the question whether the shift to acidic pH or the interaction of HSPG with CNFY induces a conformational change, we measured the distance between the two single cysteine residues (C134 and C866) in CNFY using nitroxide spin labeling in combination with PELDOR spectroscopy (18). The PELDOR method exploits the distance dependence of the interaction between the magnetic dipole moments of two spin labels.…”

Section: Heparin Interaction With Cnfy Does Not Induce a Conformationmentioning

confidence: 99%

Glycosaminoglycans are specific endosomal receptors forYersinia pseudotuberculosisCytotoxic Necrotizing Factor

Schmidt

Kowarschik

Schöllkopf

et al. 2020

Preprint

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The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. To analyze toxin-glucosaminoglycan interaction we utilized a truncated CNFY (CNFY 709-1014 ).Especially this C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to its protein receptor seems to induce a first conformational change supporting the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface. Lastly, cleavage of heparin sulfate chains by heparanase is likely required for efficient uptake of the toxic enzyme into the cytosol of mammalian cells. Author SummaryThe RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) from Yersinia pseudotuberculosis is a crucial virulence factor that is important for successful infection 3 of mammalian cells by the pathogen. The mode of action by which CNFY is able to intoxicate cells can be divided into the following steps: Binding to the cell surface, internalization, translocation from the endosome to the cytosol and deamidation of RhoA. We show, that CNFY uses heparan sulfates to maximize the amount of molecules entering the cytosol. While not being necessary for toxin binding and uptake, the sugars hold a key role in the intoxication process. We show that CNFY undergoes a conformational change at a low endosomal pH, allowing the C-terminal domain to be released from the endosomal membrane by the action of heparanase. This study reveals new insights into the CNFY-host interaction and promotes understanding of the complex intoxication process of bacterial toxins.

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“…The advent of integrative structural biology [5] has increased demand for EPR restraints on structure and requires better understanding of their accuracy and precision. Two types of EPR restraints are suitable for model building: accessibility restraints [6] and distance distribution restraints in the nanometre range [7,8]. Accessibility of a site to water or oxygen and spin label mobility are periodic in a site scan along a secondary structure element [9].…”

Section: Introductionmentioning

confidence: 99%

The contribution of modern EPR to structural biology

Jeschke

2018

Emerging Topics in Life Sciences

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Electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labelling is applicable to biomolecules and their complexes irrespective of system size and in a broad range of environments. Neither short-range nor long-range order is required to obtain structural restraints on accessibility of sites to water or oxygen, on secondary structure, and on distances between sites. Many of the experiments characterize a static ensemble obtained by shock-freezing. Compared with characterizing the dynamic ensemble at ambient temperature, analysis is simplified and information loss due to overlapping timescales of measurement and system dynamics is avoided. The necessity for labelling leads to sparse restraint sets that require integration with data from other methodologies for building models. The double electron-electron resonance experiment provides distance distributions in the nanometre range that carry information not only on the mean conformation but also on the width of the native ensemble. The distribution widths are often inconsistent with Anfinsen's concept that a sequence encodes a single native conformation defined at atomic resolution under physiological conditions.

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Dipolar Spectroscopy - Double-Resonance Methods

Cited by 50 publications

References 75 publications

Metal binding to the dynamic cytoplasmic domain of the cation diffusion facilitator (CDF) protein MamM induces a ‘locked‐in’ configuration

Metal binding to the dynamic cytoplasmic domain of the cation diffusion facilitator (CDF) protein MamM induces a ‘locked‐in’ configuration

Glycosaminoglycans are specific endosomal receptors forYersinia pseudotuberculosisCytotoxic Necrotizing Factor

The contribution of modern EPR to structural biology

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