ORCID Profile
0000-0002-1991-7922
Current Organisations
Deutsches Elektronen-Synchrotron DESY
,
University of Siegen
,
Centre for Structural Systems Biology
,
Leibniz-Institut für Virologie
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Publications
Fucose-Functionalized Precision Glycomacromolecules Targeting Human Norovirus Capsid Protein
Publisher: American Chemical Society (ACS)
Date: 02-08-2018
DOI: 10.1021/ACS.BIOMAC.8B00829
Abstract: Norovirus infection is the major cause of nonbacterial gastroenteritis in humans and has been the subject of numerous studies investigating the virus's biophysical properties and biochemical function with the aim of deriving novel and highly potent entry inhibitors to prevent infection. Recently, it has been shown that the protruding P domain dimer (P-dimer) of a GII.10 Norovirus strain exhibits two new binding sites for l-fucose in addition to the canonical binding sites. Thus, these sites provide a novel target for the design of multivalent fucose ligands as entry inhibitors of norovirus infections. In this current study, a first generation of multivalent fucose-functionalized glycomacromolecules was synthesized and applied as model structures to investigate the potential targeting of fucose binding sites in human norovirus P-dimer. Following previously established solid phase polymer synthesis, eight precision glycomacromolecules varying in number and position of fucose ligands along an oligo(amidoamine) backbone were obtained and then used in a series of binding studies applying native MS, NMR, and X-ray crystallography. We observed only one fucose per glycomacromolecule binding to one P-dimer resulting in similar binding affinities for all fucose-functionalized glycomacromolecules, which based on our current findings we attribute to the overall size of macromolecular ligands and possibly to steric hindrance.
Human norovirus GII.4(MI001) P dimer binds fucosylated and sialylated carbohydrates
Publisher: Oxford University Press (OUP)
Date: 27-09-2017
Abstract: Human noroviruses (HuNoV), members of the family Caliciviridae, are the major cause of acute viral gastroenteritis worldwide. Successful infection is linked to the ability of the protruding (P) domain of the viral capsid to bind histo-blood group antigens (HBGA). Binding to gangliosides plays a major role for many nonhuman calici- and noroviruses. Increasing evidence points to a broader role of sialylated carbohydrates such as gangliosides in norovirus infection. Here, we compare HBGA and ganglioside binding of a GII.4 HuNoV variant (MI001), previously shown to be infectious in a HuNoV mouse model. Saturation transfer difference nuclear magnetic resonance spectroscopy, native mass spectrometry (MS) and surface plasmon resonance spectroscopy were used to characterize binding epitopes, affinities, stoichiometry and dynamics, focusing on 3'-sialyllactose, the GM3 ganglioside saccharide and B antigen. Binding was observed for 3'-sialyllactose and various HBGAs following a multistep binding process. Intrinsic affinities (Kd) of fucose, 3'-sialyllactose and B antigen were determined for the in idual binding steps. Stronger affinities were observed for B antigen over 3'-sialyllactose and fucose, which bound in the mM range. Binding stoichiometry was analyzed by native MS showing the presence of four B antigens or two 3'-sialyllactose in the complex. Epitope mapping of 3'-sialyllactose revealed direct interaction of α2,3-linked sialic acid with the P domain. The ability of HuNoV to engage multiple carbohydrates emphasizes the multivalent nature of norovirus glycan-specificity. Our findings reveal direct binding of a GII.4 HuNoV P dimer to α2,3-linked sialic acid and support a broader role of ganglioside binding in norovirus infection.
Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion
Publisher: Springer Science and Business Media LLC
Date: 24-04-2020
DOI: 10.1038/S41467-020-15738-3
Abstract: The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella , which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.
Norovirus-like VP1 particles exhibit isolate dependent stability profiles
Publisher: IOP Publishing
Date: 17-01-2018
Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product
Publisher: Elsevier BV
Date: 03-2016
Attachment of Norovirus to Histo Blood Group Antigens: A Cooperative Multistep Process
Publisher: Wiley
Date: 19-08-2015
Abstract: Human noroviruses recognize histo blood group antigens (HBGAs) as cellular attachment factors. Recently, it has been discovered that norovirus infection can be significantly enhanced by HBGA binding. Yet the attachment process and how it promotes host-cell entry is only poorly understood. The binding of a norovirus protruding (P) domain of a predominant GII.4 Saga strain to HBGAs at atomic resolution was studied. So far, independent and equivalent multiple binding sites were held responsible for attachment. Using NMR experiments we show that norovirus-HBGA binding is a cooperative multi-step process, and native mass spectrometry reveals four instead of two HBGA binding sites per P-dimer. An accompanying crystallographic study has disclosed four instead of two L-fucose binding sites per P-dimer of a related GII.10 strain1 further supporting our findings. We have uncovered a novel paradigm for norovirus-HBGA recognition that will inspire further studies into norovirus-host interactions.
A data set from flash X-ray imaging of carboxysomes
Publisher: Springer Science and Business Media LLC
Date: 08-2016
Abstract: Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth’s carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus . A new aerosol s le-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows s ling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.
A post-translational modification of human Norovirus capsid protein attenuates glycan binding
Publisher: Springer Science and Business Media LLC
Date: 21-03-2019
DOI: 10.1038/S41467-019-09251-5
Abstract: Attachment of human noroviruses to histo blood group antigens (HBGAs) is essential for infection, but how this binding event promotes the infection of host cells is unknown. Here, we employ protein NMR experiments supported by mass spectrometry and crystallography to study HBGA binding to the P-domain of a prevalent virus strain (GII.4). We report a highly selective transformation of asparagine 373, located in an antigenic loop adjoining the HBGA binding site, into an iso-aspartate residue. This spontaneous post-translational modification (PTM) proceeds with an estimated half-life of a few days at physiological temperatures, independent of the presence of HBGAs but dramatically affecting HBGA recognition. Sequence conservation and the surface-exposed position of this PTM suggest an important role in infection and immune recognition for many norovirus strains.
In‐solution structure and oligomerization of human histone deacetylase 6 – an integrative approach
Publisher: Wiley
Date: 19-09-2022
DOI: 10.1111/FEBS.16616
Abstract: Human histone deacetylase 6 (HDAC6) is a structurally unique, multidomain protein implicated in a variety of physiological processes including cytoskeletal remodelling and the maintenance of cellular homeostasis. Our current understanding of the HDAC6 structure is limited to isolated domains, and a holistic picture of the full‐length protein structure, including possible domain interactions, is missing. Here, we used an integrative structural biology approach to build a solution model of HDAC6 by combining experimental data from several orthogonal biophysical techniques complemented by molecular modelling. We show that HDAC6 is best described as a mosaic of folded and intrinsically disordered domains that in‐solution adopts an ensemble of conformations without any stable interactions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration‐dependent manner, and its oligomerization is mediated via the positively charged N‐terminal microtubule‐binding domain. Our findings provide the first insights into the structure of full‐length human HDAC6 and can be used as a basis for further research into structure function and physiological studies of this unique deacetylase.
Megahertz single-particle imaging at the European XFEL
Publisher: Springer Science and Business Media LLC
Date: 29-05-2020
DOI: 10.1038/S42005-020-0362-Y
Abstract: The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from s le preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.
Structural basis for activation of plasma-membrane Ca2+-ATPase by calmodulin
Publisher: Springer Science and Business Media LLC
Date: 26-11-2018
DOI: 10.1038/S42003-018-0203-7
Abstract: Plasma-membrane Ca 2+ -ATPases expel Ca 2+ from the cytoplasm and are key regulators of Ca 2+ homeostasis in eukaryotes. They are autoinhibited under low Ca 2+ concentrations. Calmodulin (CaM)-binding to a unique regulatory domain releases the autoinhibition and activates the pump. However, the structural basis for this activation, including the overall structure of this calcium pump and its complex with calmodulin, is unknown. We previously determined the high-resolution structure of calmodulin in complex with the regulatory domain of the plasma-membrane Ca 2+ -ATPase ACA8 and revealed a bimodular mechanism of calcium control in eukaryotes. Here we show that activation of ACA8 by CaM involves large conformational changes. Combining advanced modeling of neutron scattering data acquired from stealth nanodiscs and native mass spectrometry with detailed dissection of binding constants, we present a structural model for the full-length ACA8 Ca 2+ pump in its calmodulin-activated state illustrating a displacement of the regulatory domain from the core enzyme.
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Related Funding Activities
No related grants have been discovered for Charlotte Uetrecht.