Publications by authors named "Dmitri I Svergun"

339 Publications

Order from disorder in the sarcomere: FATZ forms a fuzzy but tight complex and phase-separated condensates with α-actinin.

Sci Adv 2021 May 28;7(22). Epub 2021 May 28.

Department of Structural and Computational Biology, Max Perutz Labs, University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria.

In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.
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http://dx.doi.org/10.1126/sciadv.abg7653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163081PMC
May 2021

Molecular model of a sensor of two-component signaling system.

Sci Rep 2021 May 24;11(1):10774. Epub 2021 May 24.

Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141700, Dolgoprudny, Russia.

Two-component systems (TCS) are widespread signaling systems present in all domains of life. TCS typically consist of a signal receptor/transducer and a response regulator. The receptors (histidine kinases, chemoreceptors and photoreceptors) are often embedded in the membrane and have a similar modular structure. Chemoreceptors were shown to function in highly ordered arrays, with trimers of dimers being the smallest functional unit. However, much less is known about photoreceptors. Here, we use small-angle scattering (SAS) to show that detergent-solubilized sensory rhodopsin II in complex with its cognate transducer forms dimers at low salt concentration, which associate into trimers of dimers at higher buffer molarities. We then fit an atomistic model of the whole complex into the SAS data. The obtained results suggest that the trimer of dimers is "tripod"-shaped and that the contacts between the dimers occur only through their cytoplasmic regions, whereas the transmembrane regions remain unconnected.
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http://dx.doi.org/10.1038/s41598-021-89613-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144572PMC
May 2021

Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation.

Commun Biol 2021 May 20;4(1):600. Epub 2021 May 20.

Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany.

The eukaryotic signal recognition particle (SRP) contains an Alu domain, which docks into the factor binding site of translating ribosomes and confers translation retardation. The canonical Alu domain consists of the SRP9/14 protein heterodimer and a tRNA-like folded Alu RNA that adopts a strictly 'closed' conformation involving a loop-loop pseudoknot. Here, we study the structure of the Alu domain from Plasmodium falciparum (PfAlu), a divergent apicomplexan protozoan that causes human malaria. Using NMR, SAXS and cryo-EM analyses, we show that, in contrast to its prokaryotic and eukaryotic counterparts, the PfAlu domain adopts an 'open' Y-shaped conformation. We show that cytoplasmic P. falciparum ribosomes are non-discriminative and recognize both the open PfAlu and closed human Alu domains with nanomolar affinity. In contrast, human ribosomes do not provide high affinity binding sites for either of the Alu domains. Our analyses extend the structural database of Alu domains to the protozoan species and reveal species-specific differences in the recognition of SRP Alu domains by ribosomes.
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http://dx.doi.org/10.1038/s42003-021-02132-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137916PMC
May 2021

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis.

Nat Commun 2021 05 17;12(1):2889. Epub 2021 May 17.

European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany.

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.
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http://dx.doi.org/10.1038/s41467-021-23151-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129110PMC
May 2021

Anomalous SAXS at P12 beamline EMBL Hamburg: instrumentation and applications.

J Synchrotron Radiat 2021 May 14;28(Pt 3):812-823. Epub 2021 Apr 14.

European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Small-angle X-ray scattering (SAXS) is an established method for studying nanostructured systems and in particular biological macromolecules in solution. To obtain element-specific information about the sample, anomalous SAXS (ASAXS) exploits changes of the scattering properties of selected atoms when the energy of the incident X-rays is close to the binding energy of their electrons. While ASAXS is widely applied to condensed matter and inorganic systems, its use for biological macromolecules is challenging because of the weak anomalous effect. Biological objects are often only available in small quantities and are prone to radiation damage, which makes biological ASAXS measurements very challenging. The BioSAXS beamline P12 operated by the European Molecular Biology Laboratory (EMBL) at the PETRA III storage ring (DESY, Hamburg) is dedicated to studies of weakly scattering objects. Here, recent developments at P12 allowing for ASAXS measurements are presented. The beamline control, data acquisition and data reduction pipeline of the beamline were adapted to conduct ASAXS experiments. Modelling tools were developed to compute ASAXS patterns from atomic models, which can be used to analyze the data and to help designing appropriate data collection strategies. These developments are illustrated with ASAXS experiments on different model systems performed at the P12 beamline.
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http://dx.doi.org/10.1107/S1600577521003404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127372PMC
May 2021

Molecular basis of F-actin regulation and sarcomere assembly via myotilin.

PLoS Biol 2021 Apr 12;19(4):e3001148. Epub 2021 Apr 12.

Department of Structural and Computational Biology, Max Perutz Labs, University of Vienna, Vienna, Austria.

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.
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http://dx.doi.org/10.1371/journal.pbio.3001148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062120PMC
April 2021

: expanded functionality and new tools for small-angle scattering data analysis.

J Appl Crystallogr 2021 Feb 1;54(Pt 1):343-355. Epub 2021 Feb 1.

European Molecular Biology Laboratory, Hamburg Site, Notkestrasse 85, Building 25 A, Hamburg, 22607, Germany.

The software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the package are described. They include , for simulating isotropic 2D scattering patterns; , to perform operations on 2D images and masks; , a method for variance estimation of structural invariants through parametric resampling; , which computes the pair distance distribution function by a direct Fourier transform of the scattering data; , to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in for Bayesian consensus-based concentration-independent molecular weight estimation; , an shape analysis method that optimizes the search model directly against the scattering data; , an application to set up the initial search volume for multiphase modelling of membrane proteins; , to perform quasi-atomistic modelling of liposomes with elliptical shapes; , which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; , which reconstructs the shape of an unknown intermediate in an evolving system; and and , for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the graphical interface to Qt5, updating - a plugin to run a subset of tools - to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future releases. All these features are implemented in , freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.
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http://dx.doi.org/10.1107/S1600576720013412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941305PMC
February 2021

Restoring structural parameters of lipid mixtures from small-angle X-ray scattering data.

J Appl Crystallogr 2021 Feb 1;54(Pt 1):169-179. Epub 2021 Feb 1.

Hamburg Outstation, European Molecular Biology Laboratory, Notkestrasse 85, Hamburg, 22607, Germany.

Small-angle X-ray scattering (SAXS) is widely utilized to study soluble macromolecules, including those embedded into lipid carriers and delivery systems such as surfactant micelles, phospho-lipid vesicles and bilayered nanodiscs. To adequately describe the scattering from such systems, one needs to account for both the form factor (overall structure) and long-range-order Bragg reflections emerging from the organization of bilayers, which is a non-trivial task. Presently existing methods separate the analysis of lipid mixtures into distinct procedures using form-factor fitting and the fitting of the Bragg peak regions. This article describes a general approach for the computation and analysis of SAXS data from lipid mixtures over the entire angular range of an experiment. The approach allows one to restore the electron density of a lipid bilayer and simultaneously recover the corresponding size distribution and multilamellar organization of the vesicles. The method is implemented in a computer program, , and its performance is demonstrated on an aqueous solution of layered lipid vesicles undergoing an extrusion process. The approach is expected to be useful for the analysis of various types of lipid-based systems, for the characterization of interactions between target drug molecules and potential carrier/delivery systems.
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http://dx.doi.org/10.1107/S1600576720015368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941313PMC
February 2021

Comment on the Optimal Parameters to Derive Intrinsically Disordered Protein Conformational Ensembles from Small-Angle X-ray Scattering Data Using the Ensemble Optimization Method.

J Chem Theory Comput 2021 Apr 16;17(4):2014-2021. Epub 2021 Mar 16.

Centre de Biologie Structurale (CBS), INSERM, CNRS, Université de Montpellier, 29, rue de Navacelles, 34090 Montpellier, France.

The Ensemble Optimization Method (EOM) is a popular approach to describe small-angle X-ray scattering (SAXS) data from highly disordered proteins. The EOM algorithm selects subensembles of coexisting states from large pools of randomized conformations to fit the SAXS data. Based on the unphysical bimodal radius of gyration () distribution of conformations resulting from the EOM analysis, a recent article (Fagerberg et al. 2019, 15 (12), 6968-6983) concluded that this approach inadequately described the SAXS data measured for human Histatin 5 (Hst5), a peptide with antifungal properties. Using extensive experimental and synthetic data, we explored the origin of this observation. We found that the one-bead-per-residue coarse-grained representation with averaged scattering form factors (provided in the EOM as an add-on to represent disordered missing loops or domains) may not be appropriate for EOM analyses of scattering data from short (below 50 residues) proteins/peptides. The method of choice for these proteins is to employ atomistic models (e.g., from molecular dynamics simulations) to sample the protein conformational landscape. As a convenient alternative, we have also improved the coarse-grained approach by introducing amino acid specific form factors in the calculations. We also found that, for small proteins, the search for relatively large subensembles of 20-50 conformers (as implemented in the original EOM version) more adequately describes the conformational space sampled in solution than the procedures optimizing the ensemble size. Our observations have been added as recommendations into the information for EOM users to promote the proper utilization of the program for ensemble-based modeling of SAXS data for all types of disordered systems.
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http://dx.doi.org/10.1021/acs.jctc.1c00014DOI Listing
April 2021

Limitations of the iterative electron density reconstruction algorithm from solution scattering data.

Nat Methods 2021 03 1;18(3):244-245. Epub 2021 Mar 1.

European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany.

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http://dx.doi.org/10.1038/s41592-021-01082-xDOI Listing
March 2021

Self-assembly and regulation of protein cages from pre-organised coiled-coil modules.

Nat Commun 2021 02 11;12(1):939. Epub 2021 Feb 11.

Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia.

Coiled-coil protein origami (CCPO) is a modular strategy for the de novo design of polypeptide nanostructures. CCPO folds are defined by the sequential order of concatenated orthogonal coiled-coil (CC) dimer-forming peptides, where a single-chain protein is programmed to fold into a polyhedral cage. Self-assembly of CC-based nanostructures from several chains, similarly as in DNA nanotechnology, could facilitate the design of more complex assemblies and the introduction of functionalities. Here, we show the design of a de novo triangular bipyramid fold comprising 18 CC-forming segments and define the strategy for the two-chain self-assembly of the bipyramidal cage from asymmetric and pseudo-symmetric pre-organised structural modules. In addition, by introducing a protease cleavage site and masking the interfacial CC-forming segments in the two-chain bipyramidal cage, we devise a proteolysis-mediated conformational switch. This strategy could be extended to other modular protein folds, facilitating the construction of dynamic multi-chain CC-based complexes.
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http://dx.doi.org/10.1038/s41467-021-21184-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878516PMC
February 2021

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2.

Nat Commun 2020 11 4;11(1):5588. Epub 2020 Nov 4.

Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607, Hamburg, Germany.

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.
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http://dx.doi.org/10.1038/s41467-020-19204-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642358PMC
November 2020

Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering.

Langmuir 2020 11 27;36(44):13331-13341. Epub 2020 Oct 27.

Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University Mainz, Staudingerweg 5, D-55099 Mainz, Germany.

Messenger ribonucleic acid (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clinical practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clinical efficacy. In this study, pH-induced changes in internal molecular organization and overall physicochemical characteristics of lipoplexes comprising ionizable lipids were investigated using small-angle X-ray scattering and supplementary techniques. These changes were determined for different types of ionizable lipids, present at various molar fractions and N/P ratios inside the phospholipid membranes. The investigated systems showed a lamellar organization, allowing an accurate determination of pH-dependent structural changes. The differences in the pH responsiveness of the systems comprising different ionizable lipids and mRNA fractions could be clearly revealed from their structural evolution. Measurements of the degree of ionization and pH-dependent mRNA loading into the systems by fluorescence assays supported the findings from the structural investigation. Our approach allows for direct in situ determination of the structural response of the lipoplex systems to changes of the environmental pH similar to that observed for endosomal uptake. These data therefore provide valuable complementary information for understanding and fine-tuning of tailored mRNA delivery systems toward improved cellular uptake and endosomal processing.
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http://dx.doi.org/10.1021/acs.langmuir.0c02446DOI Listing
November 2020

Capturing the Conformational Ensemble of the Mixed Folded Polyglutamine Protein Ataxin-3.

Structure 2021 01 15;29(1):70-81.e5. Epub 2020 Oct 15.

The Maurice Wohl Clinical Neuroscience Institute, King's College London, 5 Cutcombe Road, SE5 9RT London, UK; UK Dementia Research Institute at King's College London, SE5 9RT London, UK. Electronic address:

Ataxin-3 is a deubiquitinase involved in protein quality control and other essential cellular functions. It preferentially interacts with polyubiquitin chains of four or more units attached to proteins delivered to the ubiquitin-proteasome system. Ataxin-3 is composed of an N-terminal Josephin domain and a flexible C terminus that contains two or three ubiquitin-interacting motifs (UIMs) and a polyglutamine tract, which, when expanded beyond a threshold, leads to protein aggregation and misfolding and causes spinocerebellar ataxia type 3. The high-resolution structure of the Josephin domain is available, but the structural and dynamical heterogeneity of ataxin-3 has so far hindered the structural description of the full-length protein. Here, we characterize non-expanded and expanded variants of ataxin-3 in terms of conformational ensembles adopted by the proteins in solution by jointly using experimental data from nuclear magnetic resonance and small-angle X-ray scattering with coarse-grained simulations. Our results pave the way to a molecular understanding of polyubiquitin recognition.
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http://dx.doi.org/10.1016/j.str.2020.09.010DOI Listing
January 2021

Structural basis for DNA recognition and allosteric control of the retinoic acid receptors RAR-RXR.

Nucleic Acids Res 2020 09;48(17):9969-9985

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.

Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers.
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http://dx.doi.org/10.1093/nar/gkaa697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515732PMC
September 2020

RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression.

PLoS Pathog 2020 09 23;16(9):e1008552. Epub 2020 Sep 23.

Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.
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http://dx.doi.org/10.1371/journal.ppat.1008552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535981PMC
September 2020

Anomeric Selectivity of Trehalose Transferase with Rare l-Sugars.

ACS Catal 2020 Aug 22;10(15):8835-8839. Epub 2020 Jul 22.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.

Retaining LeLoir glycosyltransferases catalyze the formation of glycosidic bonds between nucleotide sugar donors and carbohydrate acceptors. The anomeric selectivity of trehalose transferase from was investigated for both d- and l-glycopyranose acceptors. The enzyme couples a wide range of carbohydrates, yielding trehalose analogues with conversion and enantioselectivity of >98%. The anomeric selectivity inverts from α,α-(1 → 1)-glycosidic bonds for d-glycopyranose acceptors to α,β-(1 → 1)-glycosidic bonds for l-glycopyranose acceptors, while ()-selectivity was retained for both types of sugar acceptors. Comparison of protein crystal structures of trehalose transferase in complex with α,α-trehalose and an unnatural α,β-trehalose analogue highlighted the mechanistic rationale for the observed inversion of anomeric selectivity.
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http://dx.doi.org/10.1021/acscatal.0c02117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493220PMC
August 2020

Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA.

Cells 2020 09 5;9(9). Epub 2020 Sep 5.

Department of Pharmaceutics and Biopharmaceutics, Johannes Gutenberg University Mainz, D-55131 Mainz, Germany.

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.
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http://dx.doi.org/10.3390/cells9092034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563888PMC
September 2020

Structure of a collagen VI α3 chain VWA domain array: adaptability and functional implications of myopathy causing mutations.

J Biol Chem 2020 09 21;295(36):12755-12771. Epub 2020 Jul 21.

Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.
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http://dx.doi.org/10.1074/jbc.RA120.014865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476709PMC
September 2020

Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum.

Cell Rep 2020 06;31(12):107817

Centre for Structural Systems Biology, 22607 Hamburg, Germany; Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany; University of Hamburg, 20146 Hamburg, Germany. Electronic address:

Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers; hence we name it P. falciparum lipocalin (PfLCN). We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress-induced cell damage during multiplication of parasites within erythrocytes.
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http://dx.doi.org/10.1016/j.celrep.2020.107817DOI Listing
June 2020

An automated data processing and analysis pipeline for transmembrane proteins in detergent solutions.

Sci Rep 2020 05 15;10(1):8081. Epub 2020 May 15.

European Molecular Biology Laboratory (EMBL) Hamburg Unit, DESY, Notkestrasse 85, 22607, Hamburg, Germany.

The application of small angle X-ray scattering (SAXS) to the structural characterization of transmembrane proteins (MPs) in detergent solutions has become a routine procedure at synchrotron BioSAXS beamlines around the world. SAXS provides overall parameters and low resolution shapes of solubilized MPs, but is also meaningfully employed in hybrid modeling procedures that combine scattering data with information provided by high-resolution techniques (eg. macromolecular crystallography, nuclear magnetic resonance and cryo-electron microscopy). Structural modeling of MPs from SAXS data is non-trivial, and the necessary computational procedures require further formalization and facilitation. We propose an automated pipeline integrated with the laboratory-information management system ISPyB, aimed at preliminary SAXS analysis and the first-step reconstruction of MPs in detergent solutions, in order to streamline high-throughput studies, especially at synchrotron beamlines. The pipeline queries an ISPyB database for available a priori information via dedicated services, estimates model-free SAXS parameters and generates preliminary models utilizing either ab initio, high-resolution-based, or mixed/hybrid methods. The results of the automated analysis can be inspected online using the standard ISPyB interface and the estimated modeling parameters may be utilized for further in-depth modeling beyond the pipeline. Examples of the pipeline results for the modelling of the tetrameric alpha-helical membrane channel Aquaporin0 and mechanosensitive channel T2, solubilized by n-Dodecyl β-D-maltoside are presented. We demonstrate how increasing the amount of a priori information improves model resolution and enables deeper insights into the molecular structure of protein-detergent complexes.
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http://dx.doi.org/10.1038/s41598-020-64933-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228933PMC
May 2020

Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex.

Elife 2020 05 11;9. Epub 2020 May 11.

Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with -Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III-like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.
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http://dx.doi.org/10.7554/eLife.53789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289601PMC
May 2020

Simulation of small-angle X-ray scattering data of biological macromolecules in solution.

J Appl Crystallogr 2020 Apr 18;53(Pt 2):536-539. Epub 2020 Feb 18.

European Molecular Biology Laboratory, Notkestrasse 85 c/o DESY, Hamburg, 22607, Germany.

This article presents , an application to simulate two-dimensional small-angle X-ray scattering patterns and, further, one-dimensional profiles from biological macromolecules in solution. implements a statistical approach yielding two-dimensional images in TIFF, CBF or EDF format, which may be readily processed by existing data-analysis pipelines. Intensities and error estimates of one-dimensional patterns obtained from the radial average of the two-dimensional images exhibit the same statistical properties as observed with actual experimental data. With initial input on an absolute scale, [cm]/c[mg ml], the simulated data frames may also be scaled to absolute scale such that the forward scattering after subtraction of the background is proportional to the molecular weight of the solute. The effects of changes of concentration, exposure time, flux, wavelength, sample-detector distance, detector dimensions, pixel size, and the mask as well as incident beam position can be considered for the simulation. The simulated data may be used in method development, for educational purposes, and also to determine the most suitable beamline setup for a project prior to the application and use of the actual beamtime. is available as part of the software package (3.0.0) and is freely available for academic use (http://www.embl-hamburg.de/biosaxs/download.html).
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http://dx.doi.org/10.1107/S1600576720000527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7133063PMC
April 2020

Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution.

Biomolecules 2020 04 7;10(4). Epub 2020 Apr 7.

Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics" of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia.

Quaternary structure of CBS-pyrophosphatases (CBS-PPases), which belong to the PPases of family II, plays an important role in their function ensuring cooperative behavior of the enzymes. Despite an intensive research, high resolution structures of the full-length CBS-PPases are not yet available making it difficult to determine the signal transmission path from the regulatory to the active center. In the present work, small-angle X-ray scattering (SAXS) combined with size-exclusion chromatography was applied to determine the solution structures of the full-length wild-type CBS-PPases from three different bacterial species. Previously, in the absence of an experimentally determined full-length CBS-PPase structure, a homodimeric model of the enzyme based on known crystal structures of the CBS domain and family II PPase without this domain has been proposed. Our SAXS analyses demonstrate, for the first time, the existence of stable tetramers in solution for all studied CBS-PPases from different sources. Our findings show that further studies are required to establish the functional properties of these enzymes. This is important not only to enhance our understanding of the relation between CBS-PPases structure and function under normal conditions but also because some human pathogens harbor this class of enzymes.
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http://dx.doi.org/10.3390/biom10040564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226116PMC
April 2020

Author Correction: Octa-repeat domain of the mammalian prion protein mRNA forms stable A-helical hairpin structure rather than G-quadruplexes.

Sci Rep 2020 Mar 4;10(1):4378. Epub 2020 Mar 4.

Institute of Biochemistry and Molecular Biology University of Hamburg, Hamburg, Germany.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-61336-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054425PMC
March 2020

Structural Modeling Using Solution Small-Angle X-ray Scattering (SAXS).

J Mol Biol 2020 04 6;432(9):3078-3092. Epub 2020 Feb 6.

Hamburg Outstation, European Molecular Biology Laboratory, Notkestrasse 85, 22607 Hamburg, Germany. Electronic address:

Small-angle X-ray scattering (SAXS) offers a way to examine the overall shape and oligomerization state of biological macromolecules under quasi native conditions in solution. In the past decades, SAXS has become a standard tool for structure biologists due to the availability of high brilliance X-ray sources and the development of data analysis/interpretation methods. Sample handling robots and software pipelines have significantly reduced the time necessary to conduct SAXS experiments. Presently, most synchrotrons feature beamlines dedicated to biological SAXS, and the SAXS-derived models are deposited into dedicated and accessible databases. The size of macromolecules that may be analyzed ranges from small peptides or snippets of nucleic acids to gigadalton large complexes or even entire viruses. Compared to other structural methods, sample preparation is straightforward, and the risk of inducing preparation artefacts is minimal. Very importantly, SAXS is a method of choice to study flexible systems like unfolded or disordered proteins, providing the structural ensembles compatible with the data. Although it may be utilized stand-alone, SAXS profits a lot from available experimental or predicted high-resolution data and information from complementary biophysical methods. Here, we show the basic principles of SAXS and review latest developments in the fields of hybrid modeling and flexible systems.
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http://dx.doi.org/10.1016/j.jmb.2020.01.030DOI Listing
April 2020

Molecular Mechanisms of the Interactions of -(2-Hydroxypropyl)methacrylamide Copolymers Designed for Cancer Therapy with Blood Plasma Proteins.

Pharmaceutics 2020 Jan 28;12(2). Epub 2020 Jan 28.

Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.

The binding of plasma proteins to a drug carrier alters the circulation of nanoparticles (NPs) in the bloodstream, and, as a consequence, the anticancer efficiency of the entire nanoparticle drug delivery system. We investigate the possible interaction and the interaction mechanism of a polymeric drug delivery system based on -(2-hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) with the most abundant proteins in human blood plasma-namely, human serum albumin (HSA), immunoglobulin G (IgG), fibrinogen (Fbg), and apolipoprotein (Apo) E4 and A1-using a combination of small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), and nuclear magnetic resonance (NMR). Through rigorous investigation, we present evidence of weak interactions between proteins and polymeric nanomedicine. Such interactions do not result in the formation of the protein corona and do not affect the efficiency of the drug delivery.
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http://dx.doi.org/10.3390/pharmaceutics12020106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076460PMC
January 2020

Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering.

Structure 2020 03 30;28(3):348-354.e3. Epub 2019 Dec 30.

The Hamburg Centre for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany; Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany. Electronic address:

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs.
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http://dx.doi.org/10.1016/j.str.2019.12.001DOI Listing
March 2020

Structural properties of [2Fe-2S] ISCA2-IBA57: a complex of the mitochondrial iron-sulfur cluster assembly machinery.

Sci Rep 2019 12 12;9(1):18986. Epub 2019 Dec 12.

Magnetic Resonance Center CERM, University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy.

In mitochondria, a complex protein machinery is devoted to the maturation of iron-sulfur cluster proteins. Structural information on the last steps of the machinery, which involve ISCA1, ISCA2 and IBA57 proteins, needs to be acquired in order to define how these proteins cooperate each other. We report here the use of an integrative approach, utilizing information from small-angle X-ray scattering (SAXS) and bioinformatics-driven docking prediction, to determine a low-resolution structural model of the human mitochondrial [2Fe-2S] ISCA2-IBA57 complex. In the applied experimental conditions, all the data converge to a structural organization of dimer of dimers for the [2Fe-2S] ISCA2-IBA57 complex with ISCA2 providing the homodimerization core interface. The [2Fe-2S] cluster is out of the ISCA2 core while being shared with IBA57 in the dimer. The specific interaction pattern identified from the dimeric [2Fe-2S] ISCA2-IBA57 structural model allowed us to define the molecular grounds of the pathogenic Arg146Trp mutation of IBA57. This finding suggests that the dimeric [2Fe-2S] ISCA2-IBA57 hetero-complex is a physiologically relevant species playing a role in mitochondrial [4Fe-4S] protein biogenesis.
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http://dx.doi.org/10.1038/s41598-019-55313-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908724PMC
December 2019