Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.

A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis.

Temperature controls plant growth and development, and climate change has already altered the phenology of wild plants and crops1. However, the mechanisms by which plants sense temperature are not well understood. The evening complex is a major signalling hub and a core component of the plant circadian clock2,3. The evening complex acts as a temperature-responsive transcriptional repressor, providing rhythmicity and temperature responsiveness to growth through unknown mechanisms2,4-6. The evening complex consists of EARLY FLOWERING 3 (ELF3)4,7, a large scaffold protein and key component of temperature sensing; ELF4, a small α-helical protein; and LUX ARRYTHMO (LUX), a DNA-binding protein required to recruit the evening complex to transcriptional targets. ELF3 contains a polyglutamine (polyQ) repeat8-10, embedded within a predicted prion domain (PrD). Here we find that the length of the polyQ repeat correlates with thermal responsiveness. We show that ELF3 proteins in plants from hotter climates, with no detectable PrD, are active at high temperatures, and lack thermal responsiveness. The temperature sensitivity of ELF3 is also modulated by the levels of ELF4, indicating that ELF4 can stabilize the function of ELF3. In both Arabidopsis and a heterologous system, ELF3 fused with green fluorescent protein forms speckles within minutes in response to higher temperatures, in a PrD-dependent manner. A purified fragment encompassing the ELF3 PrD reversibly forms liquid droplets in response to increasing temperatures in vitro, indicating that these properties reflect a direct biophysical response conferred by the PrD. The ability of temperature to rapidly shift ELF3 between active and inactive states via phase transition represents a previously unknown thermosensory mechanism.

Phase separation and molecular ordering of the prion-like domain of the Arabidopsis thermosensory protein EARLY FLOWERING 3.

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

Small-Angle X-Ray Scattering for Macromolecular Complexes.

Small angle X-ray scattering (SAXS) is a versatile technique that can provide unique insights in the solution structure of macromolecules and their complexes, covering the size range from small peptides to complete viral assemblies. Technological and conceptual advances in the last two decades have tremendously improved the accessibility of the technique and transformed it into an indispensable tool for structural biology. In this chapter we introduce and discuss several approaches to collecting SAXS data on macromolecular complexes, including several approaches to online chromatography. We include practical advice on experimental design and point out common pitfalls of the technique.

BioSAXS at European Synchrotron Radiation Facility - Extremely Brilliant Source: BM29 with an upgraded source, detector, robot, sample environment, data collection and analysis software.

As part of its Extremely Brilliant Source (EBS) upgrade project, the ESRF's BM29 BioSAXS beamline was subject to a significant upgrade and refurbishment. In addition to the replacement of the beamline's original bending magnet source by a two-pole wiggler, leading to an increase in brilliance by a factor of 60, the sample environment of the beamline was almost completely refurbished: a vacuum-compatible Pilatus3 X 2M with a sensitive area of 253.7 mm × 288 mm and frame rates up to 250 Hz was installed, increasing the active area available and thus the q-scaling of scattering images taken; the sample changer was replaced with an upgraded version, allowing more space for customizable sample environments and the installation of two new sample exposure units; the software associated with the beamline was also renewed. In addition, the layout and functionality of the BSXCuBE3 (BioSAXS Customized Beamline Environment) data acquisition software was redesigned, providing an intuitive `user first' approach for inexperienced users, while at the same time maintaining more powerful options for experienced users and beamline staff. Additional features of BSXCuBE3 are queuing of samples; either consecutive sample changer and/or SEC-SAXS (size-exclusion chromatography small-angle X-ray scattering) experiments, including column equilibration were also implemented. Automatic data processing and analysis are now managed via Dahu, an online server with upstream data reduction, data scaling and azimuthal integration built around PyFAI (Python Fast Azimuthal Integration), and data analysis performed using the open source FreeSAS. The results of this automated data analysis pipeline are displayed in ISPyB/ExiSAXS. The upgraded BM29 has been in operation since the post-EBS restart in September 2020, and here a full description of its new hardware and software characteristics together with examples of data obtained are provided.

Molecular mechanisms of Evening Complex activity in Arabidopsis.

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

Domain Organization of Vaccinia Virus Helicase-Primase D5.

UNLABELLED: Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single- and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins. IMPORTANCE: Since the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design.

A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases.

During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.

Production and characterisation of Epstein-Barr virus helicase-primase complex and its accessory protein BBLF2/3.

The helicase-primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein-Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase-primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase-primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases.

An RNA element in human interleukin 6 confers escape from degradation by the gammaherpesvirus SOX protein.

Several viruses express factors to silence host gene expression via widespread mRNA degradation. This phenotype is the result of the coordinated activity of the viral endonuclease SOX and the cellular RNA degradation enzyme Xrn1 during lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection. While most cellular transcripts are highly downregulated, a subset of host mRNA escapes turnover via unknown mechanisms. One of the most prominent escapees is the interleukin 6 (IL-6) mRNA, which accumulates robustly during KSHV lytic infection and is not subjected to SOX-induced degradation. Here we reveal that the IL-6 mRNA contains a dominant, cis-acting ∼100-nucleotide element within its 3' untranslated region (UTR) that renders it directly refractory to cleavage by SOX. This element specifically interacts with a cellular protein complex both in SOX-transfected cells and in KSHV-infected B cells. Using a directed RNA pulldown approach, we identified two components of this complex to be the AU-rich element (ARE) binding proteins AUF1 and HuR. Depletion of these proteins significantly reduced the protective capacity of the IL-6 RNA element in SOX-expressing cells. These findings suggest that SOX activity may be directly counteracted by select RNA regulatory complexes and reveal a novel mechanism contributing to the robust expression of IL-6 during KSHV replication.

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

Analysis of Phase Separation of EARLY FLOWERING 3.

Phase separation is an important mechanism for regulating various cellular functions. The EARLY FLOWERING 3 (ELF3) protein, an essential element of the EVENING COMPLEX (EC) involved in circadian clock regulation, has been shown to undergo phase separation. ELF3 is known to significantly influence elongation growth and flowering time regulation, and this is postulated to be due to whether the protein is in the dilute or phase-separated state. Here, we present a brief overview of methods for analyzing ELF3 phase separation in vitro, including the generation of phase diagrams as a function of pH and salt versus protein concentrations, optical microscopy, fluorescence recovery after photobleaching (FRAP), and turbidity assays.

Structural characterization of protein-DNA complexes using small angle X-ray scattering (SAXS) with contrast variation.

Molecular and atomic level characterization of transcription factor (TF)-DNA complexes is critical for understanding DNA-binding specificity and potentially structural changes that may occur in protein and/or DNA upon complex formation. Often TFs are large, multidomain proteins or contain disordered regions that contribute to DNA recognition and/or binding affinity but are difficult to structurally characterize due to their high molecular weight and intrinsic flexibility. This results in challenges to obtaining high resolution structural information using Nuclear Magnetic Resonance (NMR) spectroscopy due to the relatively large size of the protein-DNA complexes of interest or macromolecular crystallography due to the difficulty in obtaining crystals of flexible proteins. Small angle X-ray scattering (SAXS) offers a complementary method to NMR and X-ray crystallography that allows for low-resolution structural characterization of protein, DNA, and protein-DNA complexes in solution over a greater size range and irrespective of interdomain flexibility and/or disordered regions. One important caveat to SAXS data interpretation, however, has been the inability to distinguish between scattering coming from the protein versus DNA component of the complex of interest. Here, we present a protocol using contrast variation via increasing sucrose concentrations to distinguish between protein and DNA using the model protein bovine serum albumin (BSA) and DNA and the LUX ARRYTHMO TF-DNA complex. Examination of the scattering curves of the components individually and in combination with contrast variation allows the differentiation of protein and DNA density in the derived models. This protocol is designed for use on high flux SAXS beamlines with temperature-controlled sample storage and sample exposure units.

Identification of Plant Transcription Factor DNA-Binding Sites Using seq-DAP-seq.

The identification of genome-wide transcription factor binding sites (TFBS) is a critical step in deciphering gene and transcriptional regulatory networks. However, determining the genome-wide binding of specific TFs or TF complexes remains a technical challenge. DNA affinity purification sequencing (DAP-seq) and modifications such as sequential DAP-seq (seq-DAP-seq) are robust in vitro methods for mapping individual TF or TF complex binding sites in a genome-wide manner. DAP-seq protocols use a genomic DNA (gDNA) library from any target organism with or without amplification, allowing the determination of TF binding on naked or endogenously modified DNA, respectively. As a first step, the gDNA is fragmented to ~200 bp, end-repaired, and sequencing adaptors are added. This gDNA library can be used directly or an amplification step may be performed to remove DNA modifications such as cytosine methylation. DNA libraries are then incubated with an affinity-tagged TF or TF- complex immobilized on magnetic beads. The TF or TF complex of interest is generally produced using recombinant protein expression and purified prior to DNA affinity purification. After incubation of the DNA library with the immobilized TF of interest, multiple wash steps are performed to reduce non-specific DNA binding and the TF-DNA complexes eluted. The eluted DNA is PCR-amplified and sequenced using next-generation sequencing. The resulting sequence reads are mapped to the corresponding reference genome, identifying direct potential bound regions and binding sites of the TF or TF complex of interest. Predictive TFBS models are generated from the bound regions using downstream bioinformatics analysis pipelines. Here, we present a detailed protocol outlining the steps required for seq-DAP-seq of a heterooligomeric TF complex (Fig. 1) and briefly describe the downstream bioinformatics pipeline used to develop a robust TFBS model from sequencing data generated from a DAP-seq experiment.

The Vaccinia Virus DNA Helicase Structure from Combined Single-Particle Cryo-Electron Microscopy and AlphaFold2 Prediction.

Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323-785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.

Analysis of SEC-SAXS data via EFA deconvolution and Scatter.

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

Intermediate-resolution crystal structure of the human adenovirus B serotype 3 fibre knob in complex with the EC2-EC3 fragment of desmoglein 2.

The cryo-electron microscopy (cryo-EM) structure of the complex between the trimeric human adenovirus B serotype 3 fibre knob and human desmoglein 2 fragments containing cadherin domains EC2 and EC3 has been published, showing 3:1 and 3:2 complexes. Here, the crystal structure determined at 4.5 Šresolution is presented with one EC2-EC3 desmoglein fragment bound per fibre knob monomer in the asymmetric unit, leading to an apparent 3:3 stoichiometry. However, in concentrated solution the 3:2 complex is predominant, as shown by small-angle X-ray scattering (SAXS), while cryo-EM at lower concentrations showed a majority of the 3:1 complex. Substitution of the calcium ions bound to the desmoglein domains by terbium ions allowed confirmation of the X-ray model using their anomalous scattering and shows that at least one binding site per cluster of calcium ions is intact and exchangeable and, combined with SAXS data, that the cadherin domains are folded even in the distal part that is invisible in the cryo-EM reconstruction.

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect.

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules.

High-brilliance X-ray beams coupled with automation have led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.