Conformational analysis of complex protein states by hydrogen/deuterium exchange mass spectrometry (HDX-MS): Challenges and emerging solutions

Trabjerg, Esben; Nazari, Zeinab; Rand, Kasper D.

doi:10.1016/j.trac.2018.06.008

Cited by 91 publications

(118 citation statements)

References 115 publications

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“…HDX-MS is a solution-based method which can provide molecular level information on local protein structure and dynamics 31-33 . HDX occurs when backbone amides are made accessible to D 2 O solvent through structure unfolding and H-bond breakage; HDX is fast within unfolded regions and slow within stably folded regions (i.e.…”

Section: Resultsmentioning

confidence: 99%

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

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Ahdash

Fais

et al. 2020

Preprint

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Resistance-nodulation-division (RND) efflux pumps play a key role in inherent and evolved multidrug-resistance (MDR) in bacteria. AcrB is the prototypical member of the RND family and acts to recognise and export a wide range of chemically distinct molecules out of bacteria, conferring resistance to a variety of antibiotics. Although high resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown, preventing a complete mechanistic understanding of efflux and inhibition. Here, we determine these structural dynamics in the presence of AcrB substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular modelling, drug binding and bacterial susceptibility studies. We show that the well-studied efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within an MDR clinical isolate, AcrBG288D, modifies the plasticity of the transport pathway, which could explain its altered substrate specificity. Our results provide molecular insight into drug export and inhibition of a major MDR-conferring efflux pump and the important directive role of its dynamics.

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Section: Resultsmentioning

confidence: 99%

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

Reading

Ahdash

Fais

et al. 2020

Preprint

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“…[44][45][46][47] HDX occurs primarily on the backbone amide hydrogens of a protein because they are distributed along the polypeptide backbone and form a basic hydrogen bond network of secondary structures. 48,49 In this work, HDX-MS was carried out to probe the binding effect The MD results indicated that the binding sites of the four IAs were located in the pocket around heme in Mb, and the order of the binding free energy values calculated using MD was consistent with F I G U R E 7 HDX-MS spectra of Mb and complexes of Mb with IAs. The Mb concentration is 2.5 μM.…”

Section: Hdx-ms Analysismentioning

confidence: 56%

“…HDX‐MS has become a useful tool for the analysis of protein conformation, which enables mass analysis at intact protein levels to explore global changes in protein structure and stability . HDX occurs primarily on the backbone amide hydrogens of a protein because they are distributed along the polypeptide backbone and form a basic hydrogen bond network of secondary structures …”

Section: Resultsmentioning

confidence: 99%

Interactions of indole alkaloids with myoglobin: A mass spectrometry based spectrometric and computational method

Chi

Feng

et al. 2020

Rapid Comm Mass Spectrometry

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Rationale Interactions of drug molecules and proteins play important roles in physiological and pathological processes in vivo. It is of significance to establish a reliable strategy for studying protein–drug ligand interactions and would be helpful for the design and screening of new drugs in pharmacological research. Methods The interactions between four indole alkaloids (IAs) extracted from Ophiorrhiza japonica (O. japonica) and myoglobin (Mb) protein were investigated using a multi‐spectrometric and computational method of native electrospray ionization mass spectrometry (native ESI‐MS), hydrogen/deuterium exchange mass spectrometry (HDX‐MS), circular dichroism (CD) and molecular docking (MD). Results The IA‐bound Mb complexes were analyzed using native ESI‐MS, with the obtained protein‐to‐ligand stoichiometry at 1:1, 1:2 and 1:3. Binding constants were measured according to the interpretation of MS spectra. MD complemented MS measurements, probing the binding sites and modes of the four IAs to Mb. Analyses involving CD and HDX‐MS demonstrated that exposure to IAs could affect the conformation of Mb by decreasing the α‐helix content and made Mb more susceptible to HDX at the backbone. Conclusions A new MS‐based integrated analysis method has been developed to successfully study the interactions of Mb and IAs extracted from O. japonica. The experimental and calculation results have good consistency, revealing all of the four IA molecules could bind to Mb to form 1:1, 1:2 and 1:3 Mb–IA complexes. The order of binding ability of these IAs to Mb was ophiorrhine B > compound C > ophiorrhine A > compound D. CD and HDX‐MS results indicated that binding with IAs destabilizes Mb. HDX‐MS analysis suggests that Mb becomes more susceptible to HDX, indicating that binding with IAs destabilizes the structure of Mb. In addition, the interaction with IAs affected the overall structure of Mb, ascribed to the decrease of α‐helix content and less folding of the backbone.

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“…Broad applicability and label-free sample preparation have made HDX-MS an increasingly attractive biophysical technique to study global biomolecular structure and dynamics under native conditions, as demonstrated by the variety of reported applications on both globular and membrane proteins and frequently-updated reviews (3,4,62). The major challenge, however, has been how to objectively translate the HDX data into structural information, so as to be able to derive quantitative mechanistic insights.…”

Section: Discussionmentioning

confidence: 99%

Structural Interpretation of Hydrogen-Deuterium Exchange with Maximum-Entropy Simulation Reweighting

Bradshaw

Marinelli

Faraldo‐Gómez

et al. 2019

Preprint

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Statement of significanceHDX-MS experiments are a powerful approach for probing the conformational dynamics and mechanisms of proteins. However, the mechanistic implications of HDX-MS observations are frequently difficult to interpret, due to the limited spatial resolution of the technique as well as the lack of quantitative tools to translate measured data into structural information. To overcome these problems, we have developed a computational approach to construct structural ensembles that are maximally diverse while reproducing target experimental HDX-MS data within a given level of uncertainty. Using artificial test data, we demonstrate that the approach can correctly discern distinct structural ensembles reflected in the target data, and thereby facilitate statistically robust evaluations of competing mechanistic interpretations of HDX-MS experiments.

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Conformational analysis of complex protein states by hydrogen/deuterium exchange mass spectrometry (HDX-MS): Challenges and emerging solutions

Cited by 91 publications

References 115 publications

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

Interactions of indole alkaloids with myoglobin: A mass spectrometry based spectrometric and computational method

Structural Interpretation of Hydrogen-Deuterium Exchange with Maximum-Entropy Simulation Reweighting

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