MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets

Hourdel, Véronique; Volant, Stevenn; O’Brien, Darragh P.; Chenal, Alexandre; Chamot‐Rooke, Julia; Dillies, Marie‐Agnès; Brier, Sébastien

doi:10.1093/bioinformatics/btw420

Cited by 59 publications

(58 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Analysis of the tail of Cascade revealed a peptide in the C-terminal domain (CTD) of Cse1 (amino acid residues 381–398) with significant protection (p<0.01) in the dsDNA bound form of the complex compared to the unbound complex (Fig 3A and Supplemental Fig 3) 24 . This is consistent with the path of the displaced strand in the dsDNA bound structure from Hayes et al 8 .…”

Section: Resultsmentioning

confidence: 99%

“…Statistical analysis on deuterium uptake was performed using MEMHDX 24 . P-values less than 0.05 were considered statistically significant.…”

Section: Methodsmentioning

confidence: 99%

“…HDX measures the stability of hydrogen bond networks and solvent accessibility on global and local scales 23, 24 , enabling a mechanistic analysis of coordinated changes associated with target binding. To our knowledge, this is the first application of HDX-MS to a ribonucleoprotein of this complexity (i.e., 5 unique proteins totaling >1,450 amino acid residues).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Erp¹,

Patterson²,

Kant³

et al. 2017

ACS Chem. Biol.

View full text Add to dashboard Cite

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here we use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Statistical analysis on deuterium uptake was performed using MEMHDX 24 . P-values less than 0.05 were considered statistically significant.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Erp¹,

Patterson²,

Kant³

et al. 2017

ACS Chem. Biol.

View full text Add to dashboard Cite

show abstract

“…The reduced sequence coverage witnessed here was due to the experimental design necessitating a lower protein load on column at the 1.6-M urea concentration (down to 3 pmol, in contrast to 10 pmol for all other states), resulting in a knock-on decrease in MS sensitivity. D 2 O uptake at the peptide level was extracted and visualized in uptake charts, difference plots, and heat maps performed in both DynamX and MEMHDX (50). Statistical analysis of HDX-MS results was performed with MEMHDX (Wald test, false discovery rate set to 1%).…”

Section: Hdx-ms Data Processingmentioning

confidence: 99%

Post‐translational acylation controls the folding and functions of the CyaA RTX toxin

et al. 2019

Self Cite

View full text Add to dashboard Cite

The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro‐toxin, pro‐CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro‐CyaA and CyaA proteins. HDX‐MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C‐terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro‐CyaA. Once refolded into monomers, CyaA is more compact and stable than pro‐CyaA, due to a complex set of interactions between domains. Our HDX‐MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation‐dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post‐translational modifications, structural disorder and protein folding. Coupling calcium‐binding and acylation‐driven folding is likely pertinent for other repeat‐in‐toxin cytolysins produced by many Gram‐negative bacterial pathogens.—O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux‐Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post‐translational acylation controls the folding and functions of the CyaA RTX toxin. FASEB J. 33, 10065–10076 (2019). http://www.fasebj.org

show abstract

“…Deuterium uptake for all identified peptides was manually checked and validated. The results were also statistically validated with the open source MEMHDX software 59 with a p-value fixed at 0.05 (see Supplementary Information S5).…”

Section: Hydrogen/deuterium Exchange Mass Spectrometrymentioning

confidence: 99%

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Terral

Champion

Debaene

et al. 2017

mAbs

View full text Add to dashboard Cite

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.

show abstract

MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets

Cited by 59 publications

References 35 publications

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Post‐translational acylation controls the folding and functions of the CyaA RTX toxin

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Contact Info

Product

Resources

About