Global analysis of complex PELDOR time traces

Rein, Stephan; Lewe, Philipp; Andrade, Susana L. A.; Kacprzak, Sylwia; Weber, Stefan

doi:10.1016/j.jmr.2018.07.015

Cited by 17 publications

(21 citation statements)

References 48 publications

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“…PELDOR analyses have been carried out using the analysis tool GloPel (34), which is available free of charge at https://www.radicals.uni-freiburg.de/de/software/glopel-1.…”

Section: Peldormentioning

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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“…PELDOR analyses have been carried out using the analysis tool GloPel (34), which is available free of charge at https://www.radicals.uni-freiburg.de/de/software/glopel-1.…”

Section: Peldormentioning

confidence: 99%

Glycosaminoglycans are specific endosomal receptors forYersinia pseudotuberculosisCytotoxic Necrotizing Factor

Schmidt

Kowarschik

Schöllkopf

et al. 2020

Preprint

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“…Among the computer programs published, the DeerAnalysis is the most popular one for the convenient extraction of distance distributions from DEER experimental data . Several methods have been developed to improve the results obtained by TIKR analysis . The resultant distance distributions provide long distant constraints (as opposed to relatively shorter distances obtained from NMR) that are useful for the determination of both structure (by averages of the measured distances) and conformational dynamics (by distribution widths and peak components) in individual proteins or protein complexes.…”

Section: General Procedures Of Pulsed Dipolar Esr Measurement and Analmentioning

confidence: 99%

“…This method has been proven useful for validating conformational heterogeneity when the distance distributions recovered by TIKR are present with multiple peaks. Recently, several methods have been developed to improve the recovery of distance distributions and to provide an estimate of uncertainty in the TIKR results …”

Section: Study Of Activated Bax Protein Structure Is Extremely Challementioning

confidence: 99%

“…[20] Several methods have been developed to improvet he results obtained by TIKR analysis. [21][22][23][24] The resultant distance distributions provide long distantc onstraints (as opposed to relativelys horter distances obtained from NMR) that are useful for the determination of both structure (by averages of the measured distances) and conformational dynamics (by distribution widths andp eak components) in individual proteins or protein complexes. Below,w ep rovide few recent examples that best reveal the great features of pulsed dipolar ESR technique.…”

Section: General Procedures Of Pulsed Dipolar Esr Measurement and Analmentioning

confidence: 99%

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Identifying Protein Conformational Dynamics Using Spin‐label ESR

Lai

Kuo

Chiang

2019

Chemistry An Asian Journal

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Spin‐label electron spin resonance (ESR) has emerged as a powerful tool to characterize protein dynamics. One recent advance is the development of ESR for resolving dynamical components that occur or coexist during a biological process. It has been applied to study the complex structural and dynamical aspects of membranes and proteins, such as conformational changes in protein during translocation from cytosol to membrane, conformational exchange between equilibria in response to protein‐protein and protein‐ligand interactions in either soluble or membrane environments, protein oligomerization, and temperature‐ or hydration‐dependent protein dynamics. As these topics are challenging but urgent for understanding the function of a protein on the molecular level, the newly developed ESR methods to capture individual dynamical components, even in low‐populated states, have become a great complement to other existing biophysical tools.

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“…As the determination of interspin distance distributions is associated with an ill-posed inverse problem, the solution (i.e., the distance distribution) to the inverse problem depends critically on the SNR of PDS data. [19][20][21] Increasing the intensity of the modulation depth, which directly leads to an enhancement in the SNR of PDS data, is the key to improving the distance resolution.…”

Section: Introductionmentioning

confidence: 99%