Intrinsically Disordered p53 and Its Complexes Populate Compact Conformations in the Gas Phase

Pagel, Kevin; Natan, Eviatar; Hall, Zoe; Fersht, Alan R.; Robinson, Carol V.

doi:10.1002/anie.201203047

Cited by 88 publications

(80 citation statements)

References 36 publications

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“…The overall similar CCS distribution patterns of FL- and TM-OmpA indicates that some compact conformers likely originate from a collapse of the loops and that the lower charge states of the extended conformer correspond to native-like populations of the monomer. The compact conformations could also arise from a collapse of the flexible linker bringing the C-terminal and TM domain in contact, as seen in IM-MS experiments of proteins containing intrinsically unstructured regions (Pagel et al, 2013) and in molecular dynamics simulations of monomeric FL-OmpA in a lipid bilayer (Khalid et al, 2008). When manually collapsing the C-terminal region of the FL-OmpA monomer onto the TM domain, and calculating a CCS for this model, we obtain a value that is in good agreement with the one determined experimentally (Figure 6F, blue line).…”

Section: Resultsmentioning

confidence: 99%

Mass Spectrometry Defines the C-Terminal Dimerization Domain and Enables Modeling of the Structure of Full-Length OmpA

et al. 2014

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SUMMARY The transmembrane domain of the Outer membrane protein A (OmpA) from Escherichia coli is an excellent model for structural and folding studies of β-barrel membrane proteins. However, full-length OmpA resists crystallographic efforts and the link between its function and tertiary structure remains controversial. Here we use site directed mutagenesis and mass spectrometry of different constructs of OmpA, released in the gas phase from detergent micelles, to define the minimal region encompassing the C-terminal dimer interface. Combining knowledge of the location of the dimeric interface with molecular modeling and ion mobility data allows us to propose a low-resolution model for the full-length OmpA dimer. Our model of the dimer is in remarkable agreement with experimental ion mobility data, with none of the unfolding or collapse observed for full-length monomeric OmpA, implying that dimer formation stabilises the overall structure and prevents collapse of the flexible linker that connects the two domains.

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Section: Resultsmentioning

confidence: 99%

Mass Spectrometry Defines the C-Terminal Dimerization Domain and Enables Modeling of the Structure of Full-Length OmpA

et al. 2014

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“…Although many reports have demonstrated a strong correlation between experimental CCS measurements and CCS values extracted from solution-phase protein models, the strength of this correlation can depend on the domain structure and globularity of the protein analyte in question. [42,43] Moreover, the magnitude and nature of the errors incorporated into IM-MS multiprotein models through the coarse-graining process are currently unknown. In order to investigate such coarse-graining errors, we extracted a non-redundant set of 191 high-resolution protein complex structures from the 3D complex set database,[44] and developed a method for the rapid generation of CG structures based on these entries where the extent of coarse graining can be treated as a variable.…”

Section: Assessing Coarse-graining Errors In Multiprotein Models Genementioning

confidence: 99%

Coming to Grips with Ambiguity: Ion Mobility-Mass Spectrometry for Protein Quaternary Structure Assignment

Eschweiler

Frank

Ruotolo

2017

J. Am. Soc. Mass Spectrom.

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Multiprotein complexes are central to our understanding of cellular biology, as they play critical roles in nearly every biological process. Despite many impressive advances associated with structural characterization techniques, large and highly-dynamic protein complexes are too often refractory to analyze by conventional, high-resolution approaches. To fill this gap, ion mobility-mass spectrometry (IM-MS) methods have emerged as a promising approach for characterizing the structures of challenging assemblies due in large part to the ability of these methods to characterize the composition, connectivity, and topology of large, labile complexes. In this Critical Insight, we present a series of bioinformatics studies aimed at assessing the information content of IM-MS datasets for building models of multiprotein structure. Our computational data highlights the limits of current coarse-graining approaches, and compelled us to develop an improved workflow for multiprotein topology modeling, which we benchmark against a subset of the multiprotein complexes within the PDB. This improved workflow has allowed us to ascertain both the minimal experimental restraint sets required for generation of high-confidence multiprotein topologies, and quantify the ambiguity in models where insufficient IM-MS information is available. We conclude by projecting the future of IM-MS in the context of protein quaternary structure assignment, where we predict that a more complete knowledge of the ultimate information content and ambiguity within such models will undoubtedly lead to applications for a broader array of challenging biomolecular assemblies.

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“…We used spFRET to study the structure of the protein in solution and compare these results to insights obtained via our free-energy simulations. 33,34 Agreement between the distance distributions obtained from spFRET and the corresponding ensemble-averaged distributions from the free energy landscape demonstrate that the free energy landscape better models the native ensemble of CcdA than the structures derived from the NMR data. These observations highlight that care needs to be taken when interpreting NMR experiments on disordered homodimeric proteins.…”

Section: Specific Ccda Conformations Are Recognized By Lon Proteasementioning

confidence: 83%

Hidden States within Disordered Regions of the CcdA Antitoxin Protein

et al. 2017

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The bacterial toxin-antitoxin system CcdB-CcdA provides a mechanism for the control of cell death and quiescence. The antitoxin CcdA protein is a homodimer composed of two monomers that each contain a folded N-terminal region and an intrinsically disordered C-terminal arm.Binding of the intrinsically disordered C-terminal arm of CcdA to the toxin CcdB prevents CcdB from inhibiting DNA Gyrase and thereby averts cell-death. Accurate models of the unfolded state of the partially disordered CcdA antitoxin can therefore provide insight into general mechanisms whereby protein disorder regulates events that are crucial to cell survival. Previous structural studies were able to model only two of three distinct structural states Ð a closed state and an open state Ð that were adopted by the C-terminal arm of CcdA. Using a combination of free energy simulations, single-pair Fšrster resonance energy transfer experiments, and existing NMR data, we developed structural models for all three states of the protein. Contrary to prior studies, we find that CcdA samples a previously unknown state where only one of the disordered C-terminal arms makes extensive contacts with the folded N-terminal domain. Moreover, our data suggest that previously unobserved conformational states play a role in regulating antitoxin concentrations and the activity of CcdAÕs cognate toxin. These data demonstrate that intrinsic disorder in CcdA provides a mechanism for regulating cell-fate.3

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Intrinsically Disordered p53 and Its Complexes Populate Compact Conformations in the Gas Phase

Cited by 88 publications

References 36 publications

Mass Spectrometry Defines the C-Terminal Dimerization Domain and Enables Modeling of the Structure of Full-Length OmpA

Mass Spectrometry Defines the C-Terminal Dimerization Domain and Enables Modeling of the Structure of Full-Length OmpA

Coming to Grips with Ambiguity: Ion Mobility-Mass Spectrometry for Protein Quaternary Structure Assignment

Hidden States within Disordered Regions of the CcdA Antitoxin Protein

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