Hydrogen-deuterium exchange mass spectrometry reveals folding and allostery in protein-protein interactions

Ramírez‐Sarmiento, César A.; Komives, Elizabeth A.

doi:10.1016/j.ymeth.2018.04.001

Cited by 33 publications

(32 citation statements)

References 85 publications

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“…However, there is increasing evidence that shifts in conformational dynamics occurring outside the core interface can modulate target recognition, binding specificity and even binding affinity in protein–protein interactions. 26 Detecting and characterizing these shifts can therefore reveal additional submolecular targets for new therapeutics, indicators of potential vaccine potency or mechanistic insights for known therapeutics that bind regions outside of the interface. For instance, several neutralizing antibodies for SARS-CoV-2 spike protein isolated from COVID-19 patients ( e.g ., REGN10954, 10986, 1933, and 10964 among others) 27 have HDX-MS measured epitopes that include interface-adjacent regions we identify here as being impacted by hACE2 complexation.…”

Section: Resultsmentioning

confidence: 99%

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization “Hotspots” in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction

Narang

James

Balmer

et al. 2021

J. Am. Soc. Mass Spectrom.

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The novel severe respiratory syndrome-like coronavirus (SARS-CoV-2) causes COVID-19 in humans and is responsible for one of the most destructive pandemics of the last century. At the root of SARS-CoV infection is the interaction between the viral spike protein and the human angiotensin converting enzyme 2 protein, which allows the virus to gain entry into host cells through endocytosis. In this work, we apply hydrogen–deuterium exchange mass spectrometry (HDX-MS) to provide a detailed view of the functional footprint and conformational dynamics associated with this interaction. Our results broadly agree with the binding interface derived from high resolution X-ray crystal structure data but also provide insights into shifts in structure and dynamics that accompany complexation, including some that occur immediately outside of the core binding interface. We propose that dampening of these “binding-site adjacent” dynamic shifts could represent a mechanism for neutralizing activity in a multitude of spike protein-targeted mAbs that have been found to specifically bind these “peripheral” sites. Our results highlight the unique capacity of HDX-MS to detect potential neutralization “hotspots” outside of the core binding interfaces defined by high resolution structural data.

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Section: Resultsmentioning

confidence: 99%

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization “Hotspots” in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction

Narang

James

Balmer

et al. 2021

J. Am. Soc. Mass Spectrom.

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“…Because side-chain and terminal-amine deuterons exchange back relatively rapidly with protons during analysis, HDX-MS data reports exclusively on backbone-amide exchange. This ability to directly probe protein dynamics has led to diverse applications ( 4 ), including studies of allostery ( 5 , 6 , 7 ), epitope mapping for protein-protein or protein-lipid interactions ( 8 , 9 , 10 , 11 ), effects of ligand binding ( 12 , 13 , 14 , 15 ), mechanisms of membrane proteins ( 16 , 17 , 18 , 19 , 20 , 21 , 22 ), and dynamics of large macromolecular complexes ( 23 , 24 , 25 , 26 ). This progress notwithstanding, the interpretation of HDX-MS data in structural and mechanistic terms has been, generally speaking, largely qualitative and lacking objective metrics.…”

Section: Introductionmentioning

confidence: 99%

Interpretation of HDX Data by Maximum-Entropy Reweighting of Simulated Structural Ensembles

Bradshaw

Marinelli

Faraldo‐Gómez

et al. 2020

Biophysical Journal

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Hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS) is a widely applied biophysical technique that probes the structure and dynamics of biomolecules without the need for site-directed modifications or bio-orthogonal labels. The mechanistic interpretation of HDX data, however, is often qualitative and subjective, owing to a lack of quantitative methods to rigorously translate observed deuteration levels into atomistic structural information. To help address this problem, we have developed a methodology to generate structural ensembles that faithfully reproduce HDX-MS measurements. In this approach, an ensemble of protein conformations is first generated, typically using molecular dynamics simulations. A maximum-entropy bias is then applied post hoc to the resulting ensemble such that averaged peptide-deuteration levels, as predicted by an empirical model, agree with target values within a given level of uncertainty. We evaluate this approach, referred to as HDX ensemble reweighting (HDXer), for artificial target data reflecting the two major conformational states of a binding protein. We demonstrate that the information provided by HDX-MS experiments and by the model of exchange are sufficient to recover correctly weighted structural ensembles from simulations, even when the relevant conformations are rarely observed. Degrading the information content of the target data—e.g., by reducing sequence coverage, by averaging exchange levels over longer peptide segments, or by incorporating different sources of uncertainty—reduces the structural accuracy of the reweighted ensemble but still allows for useful insights into the distinctive structural features reflected by the target data. Finally, we describe a quantitative metric to rank candidate structural ensembles according to their correspondence with target data and illustrate the use of HDXer to describe changes in the conformational ensemble of the membrane protein LeuT. In summary, HDXer is designed to facilitate objective structural interpretations of HDX-MS data and to inform experimental approaches and further developments of theoretical exchange models.

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“…Computational and experimental assessment of local stability in the metamorphic protein RfaH. (A) Thermodynamic cycle of the confinement MD approach (25) (26). Both full-length RfaH and the isolated CTD were incubated in deuterated buffer for different reaction times, quenched and pepsin-digested for analyzing the local extent of deuteron incorporation throughout RfaH.…”

Section: Figurementioning

confidence: 99%

Differential local stability governs the metamorphic fold-switch of bacterial virulence factor RfaH

Galaz‐Davison

Molina

Silletti

et al. 2019

Preprint

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A regulatory factor RfaH, present in many Gram-negative bacterial pathogens, is required for transcription and translation of long operons encoding virulence determinants.Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA sequence within these operons. Upon recruitment, the C-terminal domain of this twodomain protein refolds from an α-hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound β-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (α-hairpin) and active (β-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood.Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the Cterminal hairpin (residues 125-145) is stably folded in the autoinhibited state (~20% deuteron incorporation), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the β-folded state. Computationallypredicted ΔGs agree with these results by displaying similar anisotropic stability within the tip of the α-hairpin and on neighboring N-terminal domain residues. Remarkably, the βfolded state shows comparable stability to non-metamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins, and for devising targeted strategies to combat bacterial diseases. Keywordsprotein folding; bacterial virulence; molecular dynamics; hydrogen-deuterium exchange; transcription factor. SignificanceInfections caused by Gram-negative bacteria are a worldwide health threat due to rapid acquisition of antibiotic resistance. RfaH, a protein essential for virulence in several Gramnegative pathogens, undergoes a large-scale structural rearrangement in which one RfaH domain completely refolds. Refolding transforms RfaH from an inactive state that restricts RfaH recruitment to a few target genes into an active state that binds to, and couples, transcription and translation machineries to elicit dramatic activation of gene expression.However, the molecular basis of this unique conformational change is poorly understood.Here, we combine molecular dynamics and structural biology to unveil the hotspots that differentially stabilize both states of RfaH. Our findings provide novel insights that will guide design of inhibitors blocking RfaH action.

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Hydrogen-deuterium exchange mass spectrometry reveals folding and allostery in protein-protein interactions

Cited by 33 publications

References 85 publications

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization “Hotspots” in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization “Hotspots” in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction

Interpretation of HDX Data by Maximum-Entropy Reweighting of Simulated Structural Ensembles

Differential local stability governs the metamorphic fold-switch of bacterial virulence factor RfaH

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