Hydrogen-Deuterium Exchange Mass Spectrometry to Study Protein Complexes

Kochert, Brent A.; Iacob, Roxana E.; Wales, Thomas E.; Makriyannis, Alexandros; Engen, John R.

doi:10.1007/978-1-4939-7759-8_10

Cited by 36 publications

(34 citation statements)

References 35 publications

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“…To understand the structural basis for the regulation of Clr4 by H3K14ub we performed hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) analysis on the free Clr4 KMT domain and on Clr4KMT in complex with H3-and H3K14ub peptides. HDX-MS measures protein dynamics based on the rate of exchange of protein amide protons with the solvent 35 . Changes in HDX rates upon complex formation identify regions of the protein that are affected by the formation of the complex.…”

Section: H3k14 Ubiquitin Mark Affects the Structural Dynamics Of Clr4mentioning

confidence: 99%

SUV39 SET domains mediate crosstalk of heterochromatic histone marks

Stirpe

Guidotti

Northall

et al. 2020

Preprint

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AbstractThe SUV39 class of methyltransferase enzymes deposits histone lysine 9 di- and trimethylation (H3K9me2/3), the epigenetic hallmark of constitutive heterochromatin, which serves as the central recruitment platform for the heterochromatic silencing machinery. How these enzymes are regulated to mark specific genomic regions as heterochromatic is not well understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast S.pombe and recent evidence suggests that ubiquitination of lysine 14 on H3 tail (H3K14) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remains to be determined. Here we present a structure-function approach to understanding how the H3K14ub mark stimulates Clr4 activity in cis. These results show that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby activates the enzyme by 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to a clr4 deletion. Our work reveals a sensor for H3K14 ubiquitylation in the SET domain of Clr4, which mediates the licensing of heterochromatin formation by an epigenetic cross-talk. This sensor is also active in the human SUV39H2 enzyme.

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Section: H3k14 Ubiquitin Mark Affects the Structural Dynamics Of Clr4mentioning

confidence: 99%

SUV39 SET domains mediate crosstalk of heterochromatic histone marks

Stirpe

Guidotti

Northall

et al. 2020

Preprint

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“…Deuterium exchange of the pre-incubated nanobody-antigen complex was initiated by dilution with 67.5 µL PBS (150 mM NaCl, pH 7.4) prepared with D2O and incubation for 5 and 50 min respectively at 25 °C. To ensure a minimum of 90% of complex formation, the molar ratio of antigen to Nbs was calculated as previously described33 , using the affinity constants of 1.37 nM (NM1228), 3.66 nM (NM1226), 3.82 nM (NM1223), 8.23 nM (NM1230) and 8.34 nM (NM1224) (pre-determined by BLI analysis). The final D2O concentration was 90%.…”

mentioning

confidence: 99%

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

Wagner

Kaiser

Gramlich

et al. 2020

Preprint

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Facing the worldwide disease progression of COVID-19 caused by the SARS-CoV-2 virus, the situation is highly critical and there is an unmet need for effective vaccination, reliable diagnosis and therapeutic intervention. Neutralizing binding molecules such as antibodies or derivatives thereof have become important tools for acute treatment of COVID-19. Additionally, such binders provide the unique possibility to monitor the emergence and presence of a neutralizing immune response in infected or vaccinated individuals. Here we describe a set of 11 unique nanobodies (Nbs), originated from an immunized alpaca which bind with high affinities to the glycosylated SARS-CoV-2 Spike receptor domain (RBD). Using a multiplex in vitro binding assay we showed that eight of the selected Nbs effectively block the interaction between RBD, S1-domain and homotrimeric Spike protein with the angiotensin converting enzyme 2 (ACE2) as the viral docking site on human cells. According to competitive binding analysis and detailed epitope mapping, we grouped all Nbs blocking the RBD:ACE2 interaction in three distinct Nb-Sets and demonstrated their neutralizing effect with IC50 values in the low nanomolar range in a cell-based SARS-CoV-2 neutralization assay. Tested Nb combinations from different sets showed substantially lower IC50 values in both functional assays indicating a profound synergistic effect of Nbs simultaneously targeting different epitopes within the RBD. Finally, we applied the most potent Nb combinations in a competitive multiplex binding assay which we termed NeutrobodyPlex and detected a neutralizing immune response in plasma samples of infected individuals. We envisage that our Nbs have a high potential for prophylactic as well as therapeutic options and provide a novel approach to screen for a neutralizing immune response in infected or vaccinated individuals thus helping to monitor the immune status or to guide vaccine design.

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“…In order to identify more precisely HrpB sites involved in RNA recognition in an unbiased manner, we performed hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) experiments of purified PaHrpB in presence and absence of poly(A) and MS2 RNAs. HDX-MS is a powerful method that looks at protein dynamics in solution based on the rate of exchange of protein amide protons with the solvent [28,29]. Poly(A) and MS2 do not share sequence similarities and have divergent structures, therefore they represent good candidates to assess whether and how HrpB discriminates among different types of RNA molecules.…”

Section: Hdx-ms Reveals the Hrpb Regions Involved In Rna Recognitionmentioning

confidence: 99%

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences

et al. 2020

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RNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

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Hydrogen-Deuterium Exchange Mass Spectrometry to Study Protein Complexes

Cited by 36 publications

References 35 publications

SUV39 SET domains mediate crosstalk of heterochromatic histone marks

SUV39 SET domains mediate crosstalk of heterochromatic histone marks

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences

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