Solution Structure, Self-Assembly, and Membrane Interactions of the Matrix Protein from Newcastle Disease Virus at Neutral and Acidic pH.

Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration.IMPORTANCE The matrix protein of the Newcastle disease virus (NDV) is one of the most abundant viral proteins that regulates the formation of progeny virions. NDV is an avian pathogen that impacts the economics of bird husbandry due to its resulting morbidity and high mortality rates. Moreover, it belongs to the Avulavirus subfamily of the Paramyxoviridae family of Mononegavirales that include dangerous representatives such as respiratory syncytial virus, human parainfluenza virus, and measles virus. Here, we investigate the solution structure and membrane binding properties of this protein at both acidic and neutral pH to distinguish between possible virus entry pathways and propose a mechanism of assembly of the viral matrix scaffold. This work is fundamental for understanding the mechanisms of viral entry as well as to inform subsequent proposals for the possible use of the virus as an adequate template for future drug or vaccine delivery.

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing.

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

MPBuilder: A PyMOL Plugin for Building and Refinement of Solubilized Membrane Proteins Against Small Angle X-ray Scattering Data.

Membrane proteins (MPs) are the target of numerous structural and functional studies in biological and medical/pharmaceutical sciences. Strategies for the high-throughput structural analysis of MPs and of their perturbations driven by ligands having potential therapeutic applications are uncommon, often requiring scaled up crystallization, electron microscopy, and nuclear magnetic resonance (NMR) efforts. Small-angle X-ray scattering (SAXS) provides a rapid means to study low resolution structures and conformational changes of native MPs in solution without cumbersome sample preparations/treatment. The method requires the MPs solubilized in an appropriate medium (eg. detergents, mixed micelles and nanodiscs) and reliable and robust models are needed to describe the relevant complexes. Here we present MPBuilder, a simple and versatile tool for the generation and refinement of all-atom MP systems in the popular software PyMOL, an environment familiar to most biologists. MPBuilder provides building capability for protein-detergent, bicelle, and lipid-scaffold (saposin nanoparticles, nanodiscs) complexes and links this to the ATSAS software package modules for model refinement and validation against the SAXS data.

ASAXS measurements on ferritin and apoferritin at the bioSAXS beamline P12 (PETRA III, DESY).

Small-angle X-ray scattering is widely utilized to study biological macromol-ecules in solution. For samples containing specific (e.g. metal) atoms, additional information can be obtained using anomalous scattering. Here, measuring samples at different energies close to the absorption edges of relevant elements provides specific structural details. However, anomalous small-angle X-ray scattering (ASAXS) applications to dilute macromolecular solutions are challenging owing to the overall low anomalous scattering effect. Here, pilot ASAXS experiments from dilute solutions of ferritin and cobalt-loaded apoferritin are reported. These samples were investigated near the resonance X-ray K edges of Fe and Co, respectively, at the EMBL P12 bioSAXS beamline at PETRA III, DESY. Thanks to the high brilliance of the P12 beamline, ASAXS experiments are feasible on dilute protein solutions, allowing one to extract the Fe- or Co-specific anomalous dispersion terms from the ASAXS data. The data were subsequently used to determine the spatial distribution of either iron or cobalt atoms incorporated into the ferritin/apoferritin protein cages.

A THz transparent 3D printed microfluidic cell for small angle x-ray scattering.

Excitation frequencies in the terahertz (THz) range are expected to lead to functionally relevant domain movements within the biological macromolecules such as proteins. The possibility of examining such movements in an aqueous environment is particularly valuable since here proteins are not deprived of any motional degrees of freedom. Small angle x-ray scattering (SAXS) is a powerful method to study the structure and domain movements of proteins in solution. Here, we present a microfluidic cell for SAXS experiments, which is also transparent for THz radiation. Specifically, cell dimensions and material were optimized for both radiation sources. In addition, the polystyrene cell can be 3D printed and easily assembled. We demonstrate the practicality of our design for SAXS measurements on several proteins in solution.

Investigation of charge ratio variation in mRNA - DEAE-dextran polyplex delivery systems.

mRNA pharmaceuticals represent a new class of therapeutics, with applications, in cancer vaccination, tumour therapy and protein substitution. Formulations are required to deliver messenger RNA (mRNA) to the target sites where induction of genetic transfection following receptor mediated cell uptake & translation is required. In the current study, the cationic polysaccharide diethylaminoethylen (DEAE) - Dextran was selected as a model system carrier for the investigation of polyplex nanoparticle formation together with mRNA as a function of the molar ratio of the components. The structure of the mRNA/Dextran colloids was investigated as a function of the polymer-to-mRNA ratio and correlated with the biological activity determined by cellular transfection with luciferase coding mRNA. Dynamic light scattering (DLS), small angle x-ray scattering (SAXS), and small angle neutron scattering (SANS) with deuterium contrast variation were used to achieve structural insight into the systems. Similarly to previously investigated lipid based systems, colloidally stable particles with confined size were obtained with either excess of positive or negative charge. Highest activity was obtained with positive charge excess. From the scattering experiments information on the internal organization inside the polymer/mRNA systems was derived. Indication for the presence of structural elements in the length scale of ten to 20 nm were found in the excess of dextran, which could be due to either excess or particulate polymer. Information on the molecular organization of the mRNA nanoparticle products may provide a valuable basis for defining critical quality attributes of drug products for pharmaceutical application.

Structural features of the single-stranded DNA-binding protein of Epstein-Barr virus.

We report the structural features of a C-terminal deletion construct of the Epstein-Barr virus single-stranded DNA-binding protein, Balf2 (Balf2DeltaC), which like the herpes simplex virus I encoded protein, infected cell protein 8 (ICP8), binds non-sequence specifically to single-stranded DNA (ssDNA). ICP8, in the absence of ssDNA, assembles into long filamentous structures. Removal of the 60 C-terminal amino acids of ICP8 (ICP8DeltaC) prevents the formation of such filaments, whereas addition of circular ssDNA to ICP8DeltaC induces formation of "super helical" filaments. Balf2DeltaC, which we show is a zinc-binding protein, does not form these filaments under the same conditions but does bind ssDNA in a weakly cooperative manner. Further structural comparison of both proteins in solution by small-angle X-ray scattering shows proteins with similar molecular envelopes. One major difference is the tendency of Balf2DeltaC to dimerize on different surfaces to that used for oligomerization when binding to ssDNA, and this may have implications for the mechanism of replication initiation.

Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. METHODS: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. RESULTS: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an alpha-helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X-ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients' IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti-Der p 21 IgG antibodies inhibited mite-allergic patients' IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross-inhibition studies identified it as a new mite allergen. CONCLUSIONS: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.

ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions.

ATSAS is a comprehensive software suite for the analysis of small-angle scattering data from dilute solutions of biological macromolecules or nanoparticles. It contains applications for primary data processing and assessment, ab initio bead modelling, and model validation, as well as methods for the analysis of flexibility and mixtures. In addition, approaches are supported that utilize information from X-ray crystallography, nuclear magnetic resonance spectroscopy or atomistic homology modelling to construct hybrid models based on the scattering data. This article summarizes the progress made during the 2.5-2.8 ATSAS release series and highlights the latest developments. These include AMBIMETER, an assessment of the reconstruction ambiguity of experimental data; DATCLASS, a multiclass shape classification based on experimental data; SASRES, for estimating the resolution of ab initio model reconstructions; CHROMIXS, a convenient interface to analyse in-line size exclusion chromatography data; SHANUM, to evaluate the useful angular range in measured data; SREFLEX, to refine available high-resolution models using normal mode analysis; SUPALM for a rapid superposition of low- and high-resolution models; and SASPy, the ATSAS plugin for interactive modelling in PyMOL. All these features and other improvements are included in the ATSAS release 2.8, freely available for academic users from https://www.embl-hamburg.de/biosaxs/software.html.

Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering. I. X-ray synchrotron radiation study.

The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50 S ribosomal subunit of Escherichia coli in solution is described. The experimental X-ray data from contrast variation with sucrose are analysed in terms of the basic functions in real space and the scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins are obtained. From these curves models of the shape of the 50 S subunit and its RNA-rich core are evaluated. These two shapes are positioned so that their difference, which approximates the volume occupied by the proteins, produces a scattering curve which is in good agreement with the scattering from the protein moiety.

Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering. II. Neutron scattering study.

The X-ray and neutron contrast variation data of the 50 S ribosomal subunit of Escherichia coli in solution are interpreted in the frame of a two-phase model described by the shapes of the 50 S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50 S subunit and of the RNA-rich core are evaluated with a resolution of about 4 nm. The shape of the envelope is in good agreement with the models of the 50 S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. II. A model of the ribosome and its RNA at 3.5 nm resolution.

Selectively deuterated 70 S E. coli ribosomes and isolated 30 S and 50 S subunits were analyzed by X-ray and neutron solution scattering. The resulting contrast variation data set (42 curves in total) was proven to be consistent in describing the ribosome as a four-phase system composed of the protein and rRNA moieties of both subunits. This data set thus provides ten times more information than a single scattering curve. A solid body four-phase model of the 70 S ribosome at low resolution was built from the envelope functions of the 30 S and 50 S subunits and of those of the corresponding RNA moieties. The four envelopes were parameterized at a resolution of 3.5 nm using spherical harmonics and taking into account interface layers between the phases. The initial approximation for the envelopes of the subunits was taken from electron microscopic data presented recently by J. Frank and co-workers (Albany); the rRNA envelopes were initially approximated by spheres. The optimization and the refinement of the model proceeded by non-linear least squares minimization fitting the available experimental data. The refined envelopes of the subunits differ by about 10% from the starting approximation and the shape of the final 70 S model lies between the outer envelopes of the models by Frank and by M. von Heel & R. Brimacombe (Berlin). The rRNA moiety in the 30 S subunit is more anisometric than the subunit itself, whereas the rRNA of the 50 S subunit forms a compact core. The rRNAs protrude to the surfaces of the subunits and occupy approximately 30 to 40% of the corresponding surface areas. X-ray scattering curves of the two main functional elongation 70 S complexes (pre- and post-translocational) differ only marginally from those of the non-programmed ribosomes, suggesting that the low resolution four-phase model is also valid for the elongating 70 S ribosome.

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. I. Invariants and validation of electron microscopy models.

Solutions of selectively deuterated 70 S Escherichia coli ribosomes and of free 30 S and 50 S subunits were studied by neutron scattering using contrast variation. The integrity of the partially deuterated particles was controlled by parallel X-ray measurements. Integral parameters of the entire ribosome, of its subunits and of the protein and rRNA moieties were evaluated. The data allow an experimental validation of the two most recent electron microscopy reconstructions of the 70 S ribosome presented by the groups of J. Frank (Albany) and of M. van Heel & R. Brimacombe (Berlin). For each reconstruction, integral parameters and theoretical scattering curves from the 70 S and its subunits were calculated and compared with the experimental data. Although neither of the two models yields a comprehensive agreement with the experimental data, Frank's model provides a better fit. For the 50 S subunit of van Heel & Brimacombe's model the fit with the experimental data improves significantly when the internal channels and tunnels are filled up. The poorer fit of the latter model is thus caused by its "sponge"-like structure which may partly be due to an enhancement of high frequency contributions in some of the steps of the three-dimensional image reconstruction. It seems therefore unlikely that the ribosome has a "sponge"-like structure with a pronounced network of channels.

Escherichia coli SecA shape and dimensions.

SecA shape and conformational flexibility in solution were studied by small angle X-ray scattering. Dimeric SecA is a very elongated molecule, 15 nm long and 8 nm wide. SecA is therefore four times as long as the membrane is wide. The two globular protomers are distinctly separated and share limited surface of intermolecular contacts. ATP, ADP or adenylyl-imidodiphosphate (AMP-PNP) binding does not alter the SecA radius of gyration. A SecA mutant that catalyzes multiple rounds of ATP hydrolysis does not undergo conformational changes detectable by small angle X-ray scattering (SAXS). We conclude that SecA conformational alterations observed biochemically during nucleotide interaction are only small-scale and localized. The ramifications of these findings on SecA/SecYEG interaction are discussed.

Automated sample-changing robot for solution scattering experiments at the EMBL Hamburg SAXS station X33.

There is a rapidly increasing interest in the use of synchrotron small-angle X-ray scattering (SAXS) for large-scale studies of biological macromolecules in solution, and this requires an adequate means of automating the experiment. A prototype has been developed of an automated sample changer for solution SAXS, where the solutions are kept in thermostatically controlled well plates allowing for operation with up to 192 samples. The measuring protocol involves controlled loading of protein solutions and matching buffers, followed by cleaning and drying of the cell between measurements. The system was installed and tested at the X33 beamline of the EMBL, at the storage ring DORIS-III (DESY, Hamburg), where it was used by over 50 external groups during 2007. At X33, a throughput of approximately 12 samples per hour, with a failure rate of sample loading of less than 0.5%, was observed. The feedback from users indicates that the ease of use and reliability of the user operation at the beamline were greatly improved compared with the manual filling mode. The changer is controlled by a client-server-based network protocol, locally and remotely. During the testing phase, the changer was operated in an attended mode to assess its reliability and convenience. Full integration with the beamline control software, allowing for automated data collection of all samples loaded into the machine with remote control from the user, is presently being implemented. The approach reported is not limited to synchrotron-based SAXS but can also be used on laboratory and neutron sources.

Isolation and oligomeric composition of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens.

A new procedure for isolation of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens increasing significantly the yield of the purified enzyme is presented. The enzyme is isolated from the soluble fraction of the cell extract as a hexamer, as shown by gel filtration chromatography and small angle X-ray scattering analysis. Thermostability of the hexameric form of the nitrite reductase is characterized in terms of thermoinactivation and thermodenaturation.

Structural and functional properties of mouse proNGF.

The unprocessed pro-form of the NGF (nerve growth factor), proNGF (NGF precursor, without signal peptide), has been suggested to have additional functions distinct from its role as a promoter of protein folding, i.e. apoptosis and/or neurotrophic activity. Aiming to gain insights into the specific molecular interactions that mediate proNGF biological activity and into the structural determinants stabilizing its pro-region, rm-proNGF (recombinant mouse proNGF) was expressed in Escherichia coli, refolded in vitro and characterized by physicochemical methods. X-ray solution scattering measurements (small angle X-ray scattering) revealed that rm-proNGF is dimeric in solution and appears to be anisometric when compared with the compact structure of the NGF dimer. Two structural models, a globular crab-like shape and an elongated rod-like shape, equally fit to the experimental results, pointing to an intrinsically structural disordered pro-region of NGF. The models obtained allowed the interpretation of TrkA (tropomyosin receptor kinase A) binding and activation assays in cell cultures, shedding new light on the key role of proNGF in neuronal survival and neurodegeneration.

The impact of protein characterization in structural proteomics.

Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.