Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry—A review

Percy, Andrew J.; Rey, Martial; Burns, Kyle M.; Schriemer, David C.

doi:10.1016/j.aca.2012.01.037

Cited by 149 publications

(186 citation statements)

References 123 publications

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“…5). Changes in the labeling pattern can be related to changes in the protein structure or dynamics resulting from a ligandbinding event (Englander et al, 2003;Percy et al, 2012). By mapping the observed labeling onto the two cocrystal structures, we observed three areas where most of the structural changes occurred.…”

Section: Resultsmentioning

confidence: 96%

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1αEndoribonuclease Activity

et al. 2015

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Activation of the inositol-requiring enzyme-1 alpha (IRE1a) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1a dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1a-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1a. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1a RNase activity appears to be caused by a conformational change, whereby the aC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1a, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1a RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.

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Section: Resultsmentioning

confidence: 96%

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1αEndoribonuclease Activity

et al. 2015

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“…HDX‐MS has also been used as a powerful tool to map out protein‐protein interfaces both in solution and on membrane surfaces 15, 16, 17, 18, 19, 20. Herein we describe an experimental approach to use HDX‐MS to define dynamic regions within a protein complex, in this case the type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11, and use this information to generate optimized constructs for X‐ray crystallography.…”

Section: Introductionmentioning

confidence: 99%

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

et al. 2016

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The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.

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“…Native electrospray ionization (ESI)-MS and ion mobility spectrometry yield information on quaternary structure and subunit connectivities [2][3][4]. Hydrogen/deuterium exchange reports on secondary structure and dynamics [5][6][7][8]. Cross-linking provides distance constraints that can be essential for structure determination efforts [9][10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

Vahidi

Konermann

2016

J. Am. Soc. Mass Spectrom.

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Time (s)0Bleaching of the reporter dye cyanine-5 (Cy5) served as readout of the timedependent radical milieu. Surprisingly, Cy5 oxidation extends over tens of milliseconds. This time range is four orders of magnitude longer than expected from the FPOP literature. We demonstrate that the glutamine scavenger generates metastable secondary radicals in the FPOP solution, and that these radicals lengthen the time frame of Cy5 oxidation. Cy5 and similar dyes are widely used for monitoring the radical dose experienced by proteins in solution. The measured Cy5 kinetics thus strongly suggest that protein oxidation in FPOP extends over a much longer time window than previously thought (i.e., many milliseconds instead of one microsecond). The optical approach developed here should be suitable for assessing the performance of future FPOP-like techniques with improved temporal labeling characteristics.

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Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry—A review

Cited by 149 publications

References 123 publications

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1αEndoribonuclease Activity

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1αEndoribonuclease Activity

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

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