Activation of ClpP Protease by ADEP Antibiotics: Insights from Hydrogen Exchange Mass Spectrometry

Sowole, Modupeola A.; Alexopoulos, John A.; C, Yi; Ortega, Joaquı́n; Konermann, Lars

doi:10.1016/j.jmb.2013.08.005

Cited by 44 publications

(58 citation statements)

References 67 publications

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“…Our data structurally expand this finding, showing that this conformational control is also present within tetradecameric ClpP. Further indications came from hydrogen-exchange mass spectrometry experiments where ADEPs were used as ClpX mimetics and ADEP binding induced rigidification of ClpP in the equatorial handle region 48 . Moreover, mutations in the N-terminal region were used to propose that substrate access might coordinate with protease active site reactivity 54 .…”

Section: Discussionsupporting

confidence: 75%

“…Biochemical experiments have shown that ClpX interacts with ClpP primarily through docking of its IGF loop into a hydrophobic pocket on the surface of ClpP that is also used by ADEPs to dysregulate ClpP [22][23][24] . The N-termini of ClpP have been shown to gate the entry into the degradation chamber by adopting different conformations, where the 'down' conformation as a hydrophobic plug closes and the 'up' conformation opens the entry portal 47,48 . Although ADEPs have already been shown to regulate these gated pores, further activating influences of the antibiotic had not been considered.…”

Section: Discussionmentioning

confidence: 99%

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AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

et al. 2015

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The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA þ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

et al. 2015

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“…Notably, changes in D-uptake rates at residues remote from the binding sites have been demonstrated previously for protein-carbohydrate complexes [25,27], as well as other protein-ligand interactions [52][53][54]. Both decreases [25,52,53] and increases [25,27,53,54] in the D-uptake outside of binding sites have been observed. These data suggest that changes in the structure or dynamics of the N-terminal region Interestingly, no such ligand-induced structural changes are evident from X-ray crystallography data [38].…”

Section: Tcda-a2 and Its Interaction With Cd-greasesupporting

confidence: 64%

“…It can be seen in Figure 4b that the de-protected regions are mainly located close to the N-terminus, which is the artificial truncation point of the fragment [38]. Notably, changes in D-uptake rates at residues remote from the binding sites have been demonstrated previously for protein-carbohydrate complexes [25,27], as well as other protein-ligand interactions [52][53][54]. Both decreases [25,52,53] and increases [25,27,53,54] in the D-uptake outside of binding sites have been observed.…”

Section: Tcda-a2 and Its Interaction With Cd-greasesupporting

confidence: 61%

Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

Zhang

Kitova

et al. 2015

J. Am. Soc. Mass Spectrom.

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Abstract. The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM 1 pentasaccharides (β-Gal-, respectively, served as model systems for this study.Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

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“…HDX-MS is the technique of choice for assessing the conformational dynamics of a system, identifying interacting interfaces with any type of partner, studying conformational intermediates [33], and characterizing conformational changes due to PTMs [23] or ligand binding [34]. However the potential for back-exchange can lead to a loss of information, although performing the chromatographic separation at À20°C has been proposed to reduce this side-effect [35].…”

Section: Hydrogen/deuterium Exchange and Covalent Labelingmentioning

confidence: 99%

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

Marcoux

Cianférani

2015

Methods

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Activation of ClpP Protease by ADEP Antibiotics: Insights from Hydrogen Exchange Mass Spectrometry

Cited by 44 publications

References 67 publications

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

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