RelA-Containing NFκB Dimers Have Strikingly Different DNA-Binding Cavities in the Absence of DNA

Narang, Dominic; Chen, Wei; Ricci, Clarisse Gravina; Komives, Elizabeth A.

doi:10.1016/j.jmb.2018.03.020

Cited by 16 publications

(27 citation statements)

References 25 publications

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“…Deuterium uptake for each peptide was calculated by comparing the centroids of the mass envelopes of the deuterated samples with the undeuterated controls. Back-exchange correction factors were applied as previously reported 44 The Y-axis limit for each plot reflects the total number of amides within the peptide that can possible exchange. Each plot includes the peptide MH+ value, sequence, and sequential residue numbering.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of ABC Transporter P-glycoprotein in Three Conformational States

Kopcho

Chang

Komives

2019

Sci Rep

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We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a comprehensive view of transporter dynamics (85.8% sequence coverage) occurring throughout the multidrug efflux transporter P-glycoprotein (P-gp) in three distinct conformational states: predominantly inward-facing apo P-gp, pre-hydrolytic (E552Q/E1197Q) P-gp bound to Mg+2-ATP, and outward-facing P-gp bound to Mg+2-ADP-VO4−3. Nucleotide affinity was measured with bio-layer interferometry (BLI), which yielded kinetics data that fit a two Mg+2-ATP binding-site model. This model has one high affinity site (3.2 ± 0.3 µM) and one low affinity site (209 ± 25 µM). Comparison of deuterium incorporation profiles revealed asymmetry between the changes undergone at the critical interfaces where nucleotide binding domains (NBDs) contact intracellular helices (ICHs). In the pre-hydrolytic state, both interfaces between ICHs and NBDs decreased exchange to similar extents relative to inward-facing P-gp. In the outward-facing state, the ICH-NBD1 interface showed decreased exchange, while the ICH-NBD2 interface showed less of an effect. The extracellular loops (ECLs) showed reduced deuterium uptake in the pre-hydrolytic state, consistent with an occluded conformation. While in the outward-facing state, increased ECL exchange corresponding to EC domain opening was observed. These findings point toward asymmetry between both NBDs, and they suggest that pre-hydrolytic P-gp occupies an occluded conformation.

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Section: Methodsmentioning

confidence: 99%

Dynamics of ABC Transporter P-glycoprotein in Three Conformational States

Kopcho

Chang

Komives

2019

Sci Rep

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“…The resulting peptides were captured at 0 °C on a BEH C18 Vanguard pre-column, separated by analytical chromatography (Acquity UPLC BEH C18, 1.0 × 50 mm, Waters Corporation) using a 7–85% gradient acetonitrile in 0.1% formic acid over 7.5 min, and electrosprayed into the Waters SYNAPT G2Si quadrupole time-of-flight mass spectrometer. The mass spectrometer settings and peptide identification methods have been reported previously 44 . The experiments were performed in triplicate, and independent replicates of the triplicate experiment were performed to verify the results.…”

Section: Methodsmentioning

confidence: 99%

Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold

Perez-Riba

Komives

Main

et al. 2019

Sci Rep

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The simple topology and modular architecture of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats makes them straightforward to dissect and redesign. Repeat-protein stability can be manipulated in a predictable way using site-specific mutations. Here we explore a different type of modification - loop insertion - that will enable a simple route to functionalisation of this versatile scaffold. We previously showed that a single loop insertion has a dramatically different effect on stability depending on its location in the repeat array. Here we dissect this effect by a combination of multiple and alternated loop insertions to understand the origins of the context-dependent loss in stability. We find that the scaffold is remarkably robust in that its overall structure is maintained. However, adjacent repeats are now only weakly coupled, and consequently the increase in solvent protection, and thus stability, with increasing repeat number that defines the tandem-repeat protein class is lost. Our results also provide us with a rulebook with which we can apply these principles to the design of artificial repeat proteins with precisely tuned folding landscapes and functional capabilities, thereby paving the way for their exploitation as a versatile and truly modular platform in synthetic biology.

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“…Conformational Heterogeneity in Free NFĸB. Previous coarse-grained (14) and all-atom MD simulations (5) showed that the two NTDs in a NFĸB dimer can rotate and translate with respect to each other. To experimentally probe the conformational heterogeneity of the NTDs, we carried out TIRF-based smFRET experiments by labeling each NTD with either a donor or acceptor fluorophore.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation

Chen

Wolynes

et al. 2021

Preprint

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The transcription factor NFκB (RelA-p50) is a multidomain protein that binds DNA and its inhibitor, IκBα with apparently different conformations. We used single-molecule FRET to characterize the interdomain motions of the N-terminal DNA-binding domains in the free protein and also in various bound states. Several surprising results emerged from this study. First, the domains moved with respect to each other on several widely different timescales from hundreds of milliseconds to minutes. The free NFκB displayed stochastic motions leading to a broad distribution of states, ranging from very low-FRET states to high-FRET states. Varying the ionic strength altered the slow motions suggesting that they may be due to different weak electrostatic interactions between the domains creating a rugged energy landscape. Third, the DNA-binding domains continued to be mobile even when the protein was bound to its cognate DNA, but in this case the majority of the states were either high-FRET, a state expected from the available x-ray structures, or low-FRET, a state consistent with one of the DNA-binding domains dissociated. The fluctuations of the DNA-bound states were of lower amplitude and slightly faster frequency. Fourth, the inhibitor, IκBα freezes the domains into a low-FRET state, expected to be incapable of binding DNA. Neutralization of five acidic residues in the IκBα PEST sequence, which was previously shown to impair IκBαs ability to strip NFκB from the DNA, also impaired its ability to freeze the domains into a low-FRET state indicating that the freezing of motions of the DNA-binding domains is essential for efficient molecular stripping.

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RelA-Containing NFκB Dimers Have Strikingly Different DNA-Binding Cavities in the Absence of DNA

Cited by 16 publications

References 25 publications

Dynamics of ABC Transporter P-glycoprotein in Three Conformational States

Dynamics of ABC Transporter P-glycoprotein in Three Conformational States

Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold

Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation

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