Structural and functional analysis of the minimal orthomyxovirus-like polymerase of Tilapia Lake Virus from the highly diverged Amnoonviridae family.

Tilapia Lake Virus (TiLV), a recently discovered pathogen of tilapia fish, belongs to the Amnoonviridae family from the Articulavirales order. Its ten genome segments have characteristic conserved ends and encode proteins with no known homologues, apart from the segment 1, which encodes an orthomyxo-like RNA-dependent-RNA polymerase core subunit. Here we show that segments 1-3 encode respectively the PB1, PB2 and PA-like subunits of an active heterotrimeric polymerase that maintains all domains found in the distantly related influenza polymerase, despite an unprecedented overall size reduction of 40%. Multiple high-resolution cryo-EM structures of TiLV polymerase in pre-initiation, initiation and active elongation states, show how it binds the vRNA and cRNA promoters and performs RNA synthesis, with both transcriptase and replicase configurations being characterised. However, the highly truncated endonuclease-like domain appears inactive and the putative cap-binding domain is autoinhibited, emphasising that many functional aspects of TiLV polymerase remain to be elucidated.

Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation.

The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism. Integrating cryo-electron microscopy with molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and experiments in cells, we demonstrate a dynamic, multistep phosphorylation mechanism, identify catalytically relevant interactions, and show that MAP2K-disordered amino termini determine pathway specificity. Our work captures a fundamental step of cell signaling: a kinase phosphorylating its downstream target kinase.

Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus.

CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic ß-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.

Cellular assays identify barriers impeding iron-sulfur enzyme activity in a non-native prokaryotic host.

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.

SynBac: Enhanced Baculovirus Genomes by Iterative Recombineering.

Baculovirus expression vector systems (BEVS) are widely used to produce heterologous proteins for a wide range of applications. Developed more than 30 years ago, BEVS have been constantly modified to improve product quality and ease-of-use. Plasmid reagents were tailored and engineered to facilitate introduction of heterologous genes into baculoviral genomes. At the same time, detrimental modalities such as genes encoding proteases or apoptotic factors were removed to improve protein yield. Advances in DNA synthesis and manipulation now enable the engineering of part or whole synthetic baculovirus genomes, opening up new avenues to redesign and tailor the system to specific applications. Here, we describe a simple protocol for designing and constructing baculovirus genomes comprising segments of synthetic DNA through the use of iterative Red/ET homologous recombination reactions.

AMPfret: synthetic nanosensor for cellular energy states.

Cellular energy is a cornerstone of metabolism and is crucial for human health and disease. Knowledge of the cellular energy states and the underlying regulatory mechanisms is therefore key to understanding cell physiology and to design therapeutic interventions. Cellular energy states are characterised by concentration ratios of adenylates, in particular ATP:ADP and ATP:AMP. We applied synthetic biology approaches to design, engineer and validate a genetically encoded nano-sensor for cellular energy state, AMPfret. It employs the naturally evolved energy sensing of eukaryotic cells provided by the AMP-activated protein kinase (AMPK). Our synthetic nano-sensor relies on fluorescence resonance energy transfer (FRET) to detect changes in ATP:ADP and ATP:AMP ratios both in vitro and in cells in vivo. Construction and iterative optimisation relied on ACEMBL, a parallelised DNA assembly and construct screening technology we developed, facilitated by a method we termed tandem recombineering (TR). Our approach allowed rapid testing of numerous permutations of the AMPfret sensor to identify the most sensitive construct, which we characterised and validated both in the test tube and within cells.

Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism.

AMP-activated protein kinase AMPK senses and regulates cellular energy state. AMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex. This is exploited here for the construction of a synthetic sensor of cellular energetics and allosteric AMPK activation, AMPfret. Based on engineered AMPK fused to fluorescent proteins, the sensor allows direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). AMPfret faithfully and dynamically reports the binding of AMP and ADP to AMPK γ-CBS sites, competed by Mg2+-free ATP. FRET signals correlate with activation of AMPK by allosteric mechanisms and protection from dephosphorylation, attributed here to specific CBS sites, but does not require activation loop phosphorylation. Moreover, AMPfret detects binding of pharmacological compounds to the AMPK α/ß-ADaM site enabling activator screening. Cellular assays demonstrate that AMPfret is applicable in vivo for spatiotemporal analysis of energy state and allosteric AMPK activation.

MultiBac: from protein complex structures to synthetic viral nanosystems.

The MultiBac baculovirus/insect cell expression vector system was conceived as a user-friendly, modular tool-kit for producing multiprotein complexes for structural biology applications. MultiBac has allowed the structure and function of many molecular machines to be elucidated, including previously inaccessible high-value drug targets. More recently, MultiBac developments have shifted to customized baculoviral genomes that are tailored for a range of applications, including synthesizing artificial proteins by genetic code expansion. We review some of these developments, including the ongoing rewiring of the MultiBac system for mammalian applications, notably CRISPR/Cas9-mediated gene editing.

Genetically Encoded Fluorescent Biosensors to Explore AMPK Signaling and Energy Metabolism.

Maintenance of energy homeostasis is a basic requirement for cell survival. Different mechanisms have evolved to cope with spatial and temporal mismatch between energy-providing and -consuming processes. Among these, signaling by AMP-activated protein kinase (AMPK) is one of the key players, regulated by and itself regulating cellular adenylate levels. Further understanding its complex cellular function requires deeper insight into its activation patterns in space and time at a single cell level. This may become possible with an increasing number of genetically encoded fluorescent biosensors, mostly based on fluorescence resonance energy transfer, which have been engineered to monitor metabolic parameters and kinase activities. Here, we review basic principles of biosensor design and function and the advantages and limitations of their use and provide an overview on existing FRET biosensors to monitor AMPK activation, ATP concentration, and ATP/ADP ratios, together with other key metabolites and parameters of energy metabolism.

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts.

Multicomponent biological systems perform a wide variety of functions and are crucially important for a broad range of critical health and disease states. A multitude of applications in contemporary molecular and synthetic biology rely on efficient, robust and flexible methods to assemble multicomponent DNA circuits as a prerequisite to recapitulate such biological systems in vitro and in vivo. Numerous functionalities need to be combined to allow for the controlled realization of information encoded in a defined DNA circuit. Much of biological function in cells is catalyzed by multiprotein machines typically made up of many subunits. Provision of these multiprotein complexes in the test-tube is a vital prerequisite to study their structure and function, to understand biology and to develop intervention strategies to correct malfunction in disease states. ACEMBL is a technology concept that specifically addresses the requirements of multicomponent DNA assembly into multigene constructs, for gene delivery and the production of multiprotein complexes in high-throughput. ACEMBL is applicable to prokaryotic and eukaryotic expression hosts, to accelerate basic and applied research and development. The ACEMBL concept, reagents, protocols and its potential are reviewed in this contribution.

Characterization and production of protein complexes by co-expression in Escherichia coli.

The functional units within cells are often macromolecular complexes rather than single species. Production of these complexes as assembled homogenous samples is a prerequisite for their biophysical and structural characterization and hence an understanding of their function in molecular terms. Co-expression in Escherichia coli has been used routinely to decipher the subunit composition, assembly, and production of whole protein complexes. Such complexes can then be used to reconstitute protein/nucleic acid complexes in vitro. In this chapter we present protocols for the widely utilized ACEMBL and pET-MCN/pET-MCP vector series which enable the rapid and automated co-expression of protein complexes in Escherichia coli.